Research Article

Split Viewer

J Plant Biotechnol 2016; 43(3): 317-331

Published online September 30, 2016

https://doi.org/10.5010/JPB.2016.43.3.317

© The Korean Society of Plant Biotechnology

Application and utilization of marker assisted selection for biotic stress resistance in hybrid rice (Oryza sativa L.)

Jae-Young Song, Sothea Ouk, Franz Marielle Nogoy, Marjohn C. Ni?o, Soon Wook Kwon, Woongoo Ha, Kwon-Kyoo Kang, and Yong-Gu Cho*

Department of Crop Science, Chungbuk National University, Cheongju 28644, Korea,
Department of Plant Bioscience, Pusan National University, Busan 50463, Korea,
National Institute of Crop Science, Suwon 16429, Korea,
Department of Horticultural Life Science, Hankyong National University, Anseong 17579, Korea

Correspondence to : e-mail: ygcho@cbnu.ac.kr

Received: 13 September 2016; Revised: 13 September 2016; Accepted: 21 September 2016

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Development of disease resistant plant is one of the important objectives in rice breeding programs because biotic stresses can adversely affect rice growth and yield losses. This study was conducted to identify lines with multiple-resistance genes to biotic stress among 173 hybrid rice breeding lines and germplasms using DNA-based markers. Our results showed that one hybrid rice line [IR98161-2- 1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66)] possessed 5 bacterial blight resistance genes (Xa4, xa5, Xa7, Xa13 and Xa21) while two hybrid rice lines [IR98161-2-1-1-k1-2 (IR86409- 3-1-1-1-1-1/IRBB66) and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] possessed 3 bacterial blight resistance genes (Xa4, Xa7 and Xa21, and Xa3, Xa4 and xa5). Molecular survey on rice blast disease revealed that most of these lines had two different resistant genes. Only 11 lines possessed Pib, Pi-5, and Pi-ta. In addition, we further surveyed the distribution of insect resistant genes, such as Bph1, Bph18(t), and Wbph. Three hybrid breeding lines [IR98161-2-1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66), IR98161-2-1-1-k1-2 (IR86409- 3-1-1-1-1-1/IRBB66), and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] contained all three resistance genes. Finally, we obtained four hybrid rice breeding lines and germplasms [IR98161-2-1-1-k1-2 (IR86409-3-1-1-1-1-1/IRBB66), Damm- Noeub Khmau, 7290s, and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] possessing six-gene combination. They are expected to provide higher level of multiple resistance to biotic stress. This study is important for genotyping hybrid rice with resistance to diverse diseases and pests. Results obtained in this study suggest that identification of pyramided resistance genes is very important for screening hybrid rice breeding lines and germplasms accurately for disease and pest resistance. We will expand their cultivation safely through bioassays against diseases, pests, and disaster in its main export countries.

Keywords Hybrid, Rice, MAS, MAB, biotic stress, resistance

Rice (Oryza sativa L.) is one of the most important and essential source of food crops for many people in the world. Maintaining stable rice production is extremely important to feed the constantly growing human population (Maclean et al. 2002; Sasaki and Burr 2000) in this ever changing climate. Improvement of disease resistance is an important objective in rice breeding programs because rice is exposed to various pests and several diseases including bacterial leaf blight (BB), rice blast (BL), sheath blight (ShB), tungro, and brown planthopper (BPH). Among diseases, bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most destructive bacterial disease of rice that limits rice yield in Asia (Mew 1987) and most of the rice growing countries. Rice blast disease caused by the ascomycete fungus, Magnaporthe oryzae (Couch and Kohn 2002), is also one of the leading causes of great yield loss of rice worldwide and is the most devastating fungal disease on cultivated rice as well as other species of the Poaceae (Zeigler et al. 1994; Talbot and Foster 2001; Talbot 2003; Niño et al. 2015). Rice tungro disease (RTD) is caused by two viruses, Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV), and is one of the most serious diseases of rice in South and Southeast Asia (Lee et al. 2010). Rice damaged by RTD show symptoms, such as small growth and yellow-orange discoloration of leaves (Hibino 1983). Among the rice-feeding insects, Nilaparvata lugens, the rice brown planthopper (BPH) is the most damaging pest (Jiang et al. 2012). In addition, a rice phloem sap-sucking insect, whitebacked planthopper (WBPH) (Sogatella furcifera Horvath), can cause serious reductions in plant height, number of productive tillers, filled grains, and yield (Yamasaki et al. 2003).

Breeding for resistant varieties is the most effective control approach to prevent rice production losses as well as reduce pesticides usage. However, development of rice resistant cultivars by conventional breeding without checking the incorporation of resistant genes may lead to the breakdown of the disease resistance after new resistant cultivar is released, due to complexity of genetic control with complementary or additive effects as well as their environment interactions (Yaegashi 1994; Han et al. 2001). Therefore, it is necessary to develop durable resistance cultivars by incorporating several resistant genes against highly variable pathogen or pests (Bonman et al. 1986; Hittalmani et al. 2000) using marker- assisted selection (MAS). Molecular marker techniques such as MAS may provide new ways for identifying and pyramiding valuable genes to enhance the disease resistance and overcome the breakdown of resistance that frequently occurs in rice breeding programs (Song et al. 2014; Ghulam et al. 2013). Conventional breeding approaches are difficult due to dominance and epistatic effects of genes governing disease resistance by gene pyramiding (Joseph et al. 2004; Rajpurohit et al. 2011). However, DNA-based genetic markers identified in accordance with the phenotypic traits or closed linked to each of the resistance genes are recently expected to play an important role in marker-associated breeding (MAB) for assessment of stress-tolerance and disease-resistance in hybrid breeding plants (Nogoy et al. 2016). According to Wang et al. (2005), developing super hybrid rice depends largely on the genetic resources of the parental lines and the conventional breeding technology. However, improving rice varieties based on this breeding technology is complicated, cumbersome and time- consuming. Uncovering the molecular genetic control of rice heterosis would further improve hybrid rice technology.

To develop rice varieties that can withstand the current insect pest and disease problem in the rice growing regions, we initiate to establish rice breeding programs based on conventional breeding and advanced molecular breeding techniques.

Plant materials

We used a rice panel comprised of 173 hybrid rice breeding lines and germplasms (Table 1) and control varieties (Table 2), which can be compared by size and presence/absence of amplicon between resistant and susceptible lines.

Table 1 . List of 173 hybrid rice breeding lines and germplasms used in disease resistance analysis in this study

No Designation Cross/Origin Remark
1Damm-Noeub SarkCambodiaAromatic rice (Jasmine)
2Srau Damm-Noeub Banh-Chras KcharlCambodiaAromatic rice (Jasmine)
3SaigonVietnamRestorer
4Saigon DaratVietnamRestorer
5ReningSeoul University germplasmRestorer
6Jasmin 85ThailandAromatic rice (Jasmine)
7OM 341VietnamLocal variety
8OM 6312-1VietnamLocal variety
9OM 6312-2VietnamLocal variety
10OM 7347VietnamLocal variety
11OM 9538VietnamLocal variety
12HHZ 12-DT10-SAL1-DT1IRRIBreeding line
13VD 20VietnamRestorer
14Anhui collection 1Anhui province, ChinaRestorer
15Anhui collection 2 PTGMSJilin Province, ChinaRestorer
16Anhui collection 3 RAnhui province, ChinaRestorer
17Anhui collection 4 BulkAnhui province, ChinaP/TGMS
18Aromatic rice 10-1IndiaAromatic rice (Basmati)
19Aromatic rice 10-2IndiaAromatic rice (Basmati)
20IR101861-7-1-k1-2MingHui63/IR03A550Restorer
21IR101861-7-1-k1-3MingHui63/IR03A550Restorer
22IR101861-28-1-k1-2MingHui63/IR03A550Restorer
23IR101861-28-1-k1-3MingHui63/IR03A550Restorer
24IR101870-12-1-k1-2MingHui63/IR08N103Restorer
25IR101870-12-1-k1-3MingHui63/IR08N103Restorer
26IR101870-25-1-k1-2MingHui63/IR08N103Restorer
27IR101870-25-1-k1-3MingHui63/IR08N103Restorer
28IR101872-46-1-k1-2MingHui63/IR86590-22-2-2-1-3-1-1-1Restorer
29IR101907-27-2-k1-2IR85593-20-1-1-1-2-3-1-1-1-1/IR86522-29-4-2-1-1-1-1-1Restorer
30IR101907-27-2-k1-3IR85593-20-1-1-1-2-3-1-1-1-1/IR86522-29-4-2-1-1-1-1-1Restorer
31IR98073-3-1-1-k1-3IR72903-131-1-2-3R/IR85485-106-B-B-1-1-1-1Restorer
32IR98116-7-2-1-k1-2SACG7(GID: 2643634)/IRBB23Restorer, BB
33IR98116-7-2-1-k1-3SACG7(GID: 2643634)/IRBB23Restorer, BB
34IR98139-9-1-1-k1-2IR86505-6-15-2-1-1-1-1/IRBB60Restorer, BB
35IR98139-9-1-1-k1-3IR86505-6-15-2-1-1-1-1/IRBB60Restorer, BB
36IR98141-25-1-1-k1-2IR86526-8-8-1-1-1-1-1/IR86555-3-3-1-1-1-1-1-1-1-1Restorer
37IR98141-25-1-1-k1-3IR86526-8-8-1-1-1-1-1/IR86555-3-3-1-1-1-1-1-1-1-1Restorer
38IR98161-2-1-1-k1-2IR86409-3-1-1-1-1-1/IRBB66Restorer, BB
40IR98178-8-2-1-k1-2IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
41IR98178-18-1-1-k1-2IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
42IR98178-18-1-1-k1-3IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
43IR98187-30-1-1-k1-2IR85508-6-1-1-1-1-1-1-1/IR86409-3-1-1-1-1-1Restorer
44IR98187-30-1-1-k1-3IR85508-6-1-1-1-1-1-1-1/IR86409-3-1-1-1-1-1Restorer
45IR98194-9-2-1-k1-2IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
46IR98194-9-2-1-k1-3IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
47IR98200-25-1-1-k1-3IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
48IR98200-25-1-1-k1-4IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
49IR98212-18-2-1-k1-2IR06A169/IR86612-26-9-5-1-1-1-1-1Restorer
50IR98229-2-2-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
51IR98229-2-2-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
52IR98229-9-2-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
53IR98229-9-2-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
54IR98229-24-1-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
55IR98229-24-1-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
56IR98241-24-2-1-k1-2IR06N172/IR86612-38-2-2-1-1-1-1-1Restorer
57IR98241-24-2-1-k1-3IR06N172/IR86612-38-2-2-1-1-1-1-1Restorer
58IR98272-5-1-1-k1-39311/FL478(from Dr. Ismail)Restorer
59IR98272-5-1-1-k1-49311/FL478(from Dr. Ismail)Restorer
60IR98305-10-1-1-k1-3IR68897B/IRBB23maintainer, BB
61Com collection 2-1CambodiaHigh yielding ability
62IR96632-1-1-2-1-k1-2IR85592-7-1-1-1-5-1-1-1/WeedTolerantRice1Maintainer
63IR96632-1-1-2-1-k1-3IR85592-7-1-1-1-5-1-1-1/WeedTolerantRice1Maintainer
64IR96701-28-5-2-1-k1-2IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
65IR96701-28-5-2-1-k1-3IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
66IR96701-28-5-2-1-k1-2IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
67IR96701-28-5-2-1-k1-3IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
68IR 97727-11-1-2-2HANAREUM/NSIC RC 158//HANAREUM
69IR 97727-82-1-2-2HANAREUM/NSIC RC 158//HANAREUM
70IR 97730-27-2-3-1SAEGAEJINMI/IR 6 (PAKISTAN)//SAEGAEJINMI
71BK7 6089-74-40Seoul University germplasmRestorer
72IR2768-11-133-eIRRIRestorer
73RPW6-13Seoul University germplasmRestorer
74GIIB-si 213Seoul University germplasmRestorer
75IR20905-11-3-3IRRIRestorer
76J.P.5-IR946-2-2-2/IR1635-1FIRRIRestorer
77Oksusu byeoChinaRestorer
78IR 68IRRIRestorer
79IR5105-113-3IRRIRestorer
80IR2006IRRIRestorer
81IR2003-97-4-2IRRIRestorer
82IR2042-175-3-2-2IRRIRestorer
83IR1529-430-3IRRIRestorer
84IR1539-823-1-4IRRIRestorer
85Kianse-WuanChinaRestorer
86LaxmiIndiaRestorer
87Quella-IniaSeoul University germplasmRestorer
88IR9859-5-3-3IRRIRestorer
89CB435Seoul University germplasmRestorer
90IR23325-R-R-B-7-2-2IRRIRestorer
91IR4457-5-3-6IRRIRestorer
92VRH624Seoul University germplasmRestorer
939019ChinaRestorer
94Y4037ChinaRestorer
95K100520Seoul University germplasmRestorer
96CondeSeoul University germplasmRestorer
97N22IndiaWide compatibility
98GaruSeoul University germplasmRestorer
99Restorer 1ChinaRestorer
100Restorer 2ChinaRestorer
101Restorer 3ChinaRestorer
102Pare panjangSeoul University germplasmRestorer
103ESMET126Seoul University germplasmRestorer
104BD 43BangladeshRestorer
105BR 26BangladeshRestorer
106IR OM CS 2102IRRI- VietnamRestorer
107IR7760-4-8-2IRRIRestorer
108TGMSChinaTGMS
109Aromatic rice 1-1IndiaAromatic rice (Basmati)
110Aromatic rice 1-2IndiaAromatic rice (Basmati)
111Aromatic rice 1-3IndiaAromatic rice (Basmati)
112Aromatic rice 3-1IndiaAromatic rice (Basmati)
113Aromatic rice 3-2IndiaAromatic rice (Basmati)
114Aromatic rice 3-3IndiaAromatic rice (Basmati)
115Aromatic rice 5-1IndiaAromatic rice (Basmati)
116Aromatic rice 5-2IndiaAromatic rice (Basmati)
117Aromatic rice 5-3IndiaAromatic rice (Basmati)
118Aromatic rice 6-1IndiaAromatic rice (Basmati)
119Aromatic rice 6-2IndiaAromatic rice (Basmati)
120Aromatic rice 6-3IndiaAromatic rice (Basmati)
121Aromatic rice 9-1IndiaAromatic rice (Basmati)
122Aromatic rice 9-2IndiaAromatic rice (Basmati)
123Aromatic rice 9-3IndiaAromatic rice (Basmati)
124Aromatic rice 10-1IndiaAromatic rice (Basmati)
125Aromatic rice 10-2IndiaAromatic rice (Basmati)
127Aromatic rice 11-1IndiaAromatic rice (Basmati)
128Aromatic rice 11-2IndiaAromatic rice (Basmati)
129Aromatic rice 11-3IndiaAromatic rice (Basmati)
130Jasponica H-B-B-8-BSelected lineRestorer, Aromatic rice
131Jasponica H-B-B-7-BSelected lineRestorer, Aromatic rice
132Jasponica H-B-B-16-BSelected lineRestorer, Aromatic rice
133Jasponica H-B-B-21-BSelected lineRestorer, Aromatic rice
134Jasponica H-B-B-26-BSelected lineRestorer, Aromatic rice
135Jasponica H-B-B-7-1Selected lineRestorer, Aromatic rice
136Jasponica H-B-B-9-1Selected lineRestorer, Aromatic rice
137Jasponica H-B-B-12-1Selected lineRestorer, Aromatic rice
138Jasponica H-B-B-17-1Selected lineRestorer, Aromatic rice
139Jasponica H-B-B-18-1Selected lineRestorer, Aromatic rice
140Jasponica H-B-B-19-1Selected lineRestorer, Aromatic rice
141Jasponica H-B-B-21-1Selected lineRestorer, Aromatic rice
142Jasponica H-B-B-27-1Selected lineRestorer, Aromatic rice
143IRRI-A29 B12IRRI maintainerMaintainer, High natural crossing
144IRRI-A29 B13IRRI maintainerMaintainer, High natural crossing
145IR1487-327-1-1-3IRRIRestorer
146DF-1Seoul University germplasmRestorer
147Utri RajapanIRRI_IndonesiaRestorer
148IR4547-2-1-2IRRIRestorer
149IR 01W105IRRIRestorer
150IR 70IRRIRestorer
151Tjempo BrondolIndonesiaRestorer
152Burung PutarIndonesiaRestorer
153Phkar KhgneiCambodiaAromatic rice (Jasmine)
154Damm-Noeub Phka RoluohCambodiaAromatic rice (Jasmine)
155Karn Dal KbalCambodiaAromatic rice (Jasmine)
156Thmar Ror MealCambodiaAromatic rice (Jasmine)
157KaseKamCambodiaAromatic rice (Jasmine)
158Damm-Noeub Trar-sarkCambodiaAromatic rice (Jasmine)
159Damm-Noeub Krar MuonCambodiaAromatic rice (Jasmine)
160Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
161Damm-Noeub Trar-sarkCambodiaAromatic rice (Jasmine)
162Damm-Noeub Chherng MeannCambodiaAromatic rice (Jasmine)
163Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
164Damm-Noeub younCambodiaAromatic rice (Jasmine)
165Damm-Noeub younCambodiaAromatic rice (Jasmine)
166Krar HarmCambodiaAromatic rice (Jasmine)
167Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
1687289sIR75589-31-27-8-33S(S1)/IR102561BTGMS
1697290sIR75589-31-27-8-33S(S1)/IR102568BTGMS
1707292sIR75589-31-27-8-33S(S1)/IR102758BTGMS
1717293sIR75589-31-27-8-33S(S1)/IR58025BTGMS
1727306sIR75589-31-27-8-33S/IR105687BTGMS
173TGMS_bulksIRRITGMS

Table 2 . Information of control varieties used in several disease resistance analyses in this study

GeneResistant Controls Susceptible Controls 
Xa3 IRBB3 IR24
Xa4 IRBB4 IR24
xa5 IRBB5 IR24
Xa7 IRBB7 IR24
xa13 IRBB13 IR24
Xa21 IRBB21 IR24
Pi-b IRBL-b LTH
Pi-ta IRBL-ta(K1), IRBL-ta(CT2), IRBL-ta2(Pi)  LTH
Pi-5 IRBL_3(CP4), IRBL_5(M), IRBL_i(F5) LTH
Bph1 Hangangchal1, IR26 Dongjin, IR24
Bph18(t) Anmi, Anda IR24, Ilpum
WBPH Utri merah, N22 Nipponbare, TN1

DNA extraction

Total genomic DNA was extracted from fresh leaves of two-week-old rice seedlings using modified Cetyl Trimethyl Ammonium Bromide (CTAB) method as previously described by Kump and Javornik (1996). DNA concentration was quantified using a spectrophotometer (NanoDrop One, Thermo Scientific). The DNA solution was then diluted to a working concentration with distilled water and stored at -20°C until use.

Genotyping

Polymerase chain reaction (PCR) was performed using resistant and susceptible gene-specific primers reported in previous studies and developed in this study (Table 3). Approximately 40 ng of genomic DNA was used in a 20 ul PCR reaction containing 2 ul of primer pairs (10 pmol/ul), 2.0 ul of 10 x PCR buffer, 1.6 ul of dNTP (2.5 mM), and 0.2 ul of Taq polymerase (5 unit/ul; Promega. USA). The reaction mixture was subjected to the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s and extension at 72°C for 45-60 s, and final extension at 72°C for 10 min. The PCR amplified products of bacterial blight (Xa3, Xa7, xa13 and Xa21), blast (Pi-b, Pi-ta and Pi-5), and brown planthopper (Bph1) resistant genes were separated on 1.5-2.0 % agarose gel and stained with ethidium bromide. The amplified products of Xa4, xa5, and Wbph were run on a fragment capillary gel electrophoresis system (Fragment analyzer, USA) and fragments were sized and scored using PROSize 2.0 software (Fragment analyzer, USA). The amplification of Bph18(t) is performed on an Eco™ Real-Time PCR System (Illumina, San Diego, California, USA) according to the user guide manual (Illumina). Allele calling of amplified fragments of hybrid lines and control varieties were based on their respective resistance and susceptible controls.

Table 3 . List of markers used for analysis of various diseases resistance

Disease/InsectGeneMarkerTypePrimer sequence (5’ → 3’)Expected Size (bp)Reference
Bacterial blight (BB)Xa3BB3-SuFwCGGAGCGACACAGCTATCAT743Hur et al. 2013
RvCGTGAGGTTCCCTATGGCGATT
BB3-ReFwCCACAATGCCATGTCAGGTGGCATCCCTGCA255
RvAGGTGTTGGAGGATTGGCAT
Xa4RM 224FwATCGATCGATCTTCACGAGG150/120Chen et al. 1997;
RvTGCTATAAAAGGCATTCGGGMcCouch et al. 2002
xa5RM122FwGCACTGCAACCATCAATGAATC236/232Chen et al. 1997
RvCCTAGGAGAAACTAGCCGTCCA
Xa7M5FwCGATCTTACTGGCTCTGCAACTCTGT294/1170Porter et al. 2003
RvGCATGTCTGTGTCGATTCGTCCGTACGA
xa13xa13 promFwGGCCATGGCTCAGTGTTTAT1000/520Zhang et al. 1996;
RvGAGCTCCAGCTCTCCAAATGSingh et al. 2011
Xa21pTA248FwAGACGCGGAAGGGTGGTTCCCGGA1000/750Huang et al. 1997
RvAGACGCGGTAATCGAAAGATGAAA
BlastPibNSbFwATCAACTCTGCCACAAAATCC629Kwon et al. 2008
RvCCCATATCACCACTTGTTCCCC
Pi5JJ817FwGATATGGTTGAAAAGCTAATCTCA1450Kwon et al. 2008
RvATCATTGTCCTTCATATTCAGAGT
Pi-taYL155/87FwAGCAGGTTATAAGCTAGGCC1042Jia et al. 2002, 2004
RvCTACCAACAAGTTCATCAAA
BPHBph1BeP18-3FwCGCTGCGAGAGTGTGACACT523Kim and Sohn, 2005
RvTTGGGTTACACGGGTTTGAC
Bph18(t)SNP23FwCGATGGATTACCCTATCACCTCAA110Developed in this study
SNP24RvAACCCTCTGCACACCATCGG
WBPHWbphRM8213FwAGCCCAGTGATACAAAGATG177Sun et al. 2005
RvGCGAGGAGATACCAAGAAAG

Developing Bph18(t) primer for HRM

Bph18(t) (SNP23/SNP24) was designed based on a single nucleotide variation (G/C) found on the 18,379,251 region of the gene sequences in susceptible (HR 20654-39-3-5, IR 10 K153, IR 10K150, IR 10K152, Jinmi, Kanto 51 and Lemont) and resistant (IR65482, Anda, AG04208, SR14694-57-4- 2-1-3-2-2, SR21733-48-1-12-3-2, Backunchal, Backyang and Chupung) cultivars. The forward and reverse primer sequences that flank the SNP region were generated using Primer 3 (v.0.4.0) (Untergasser et al. 2012; Koressaar and Remm 2007) which were set to amplify 110 bp of product size. To test the primer, we used it to genotype a panel of 10 resistant and 31 susceptible varieties, as shown in Fig. 1.

Fig. 1.

HRM curve profiles of 10 resistant and 31 susceptible varieties using developed specific SNP marker for Bph18(t) in this study


Estimation of Genotypes for BB Resistance Genes

A total of 173 hybrid breeding lines genotyped using PCR- based markers related to Xa3, Xa4, xa5, Xa7, xa13, and Xa21 genes were analyzed for fragment size differences and presence/absence of genes (Fig. 2). The percentage of rice panel identified to contain the gene was 7.5% with Xa7, 22.5% with xa5, 0.6% with xa13, and 1.2% with Xa21 (Fig. 5A and Table 4). Among the hybrid breeding lines, IR98161- 2-1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66) possessed five bacterial blight resistance genes (Xa4, xa5, Xa7, Xa13 and Xa21) while two hybrid rice lines [IR98161-2-1-1-k1-2 (IR86409-3- 1-1-1-1-1/IRBB66) and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] possessed three bacterial blight resistance genes (Xa4, Xa7 and Xa21, and Xa3, Xa4 and xa5). Eighty-one lines did not contain any of the BB resistance genes (Fig. 2 and Table 4). The three multiple gene containing lines were selected for developing resistant varieties using MAS.

Fig. 2.

PCR amplification patterns of markers that discriminate each of the six bacterial blight resistance genes in 173 hybrid rice breeding lines. A, Xa3; B, Xa7; C, xa13; D, Xa21 shows the band patterns for BB resistance specific primer on gel electrophoresis; E, Xa4 and F, xa5 indicates the size differentiation by fragment analyzer gel electrophoresis. R and S in first and second lanes indicate resistant and susceptible control variety


Fig. 3.

PCR amplification patterns of markers that discriminate each of the three blast resistance genes in 173 hybrid breeding lines. A, Pib; B, Pi-ta; C, Pi-5 indicates the amplification patterns for each blast resistance genes in gel electrophoresis


Fig. 4.

PCR amplification patterns for Bph1 and Wbph using specific primers on agarose and fragment analyzer gel electrophoresis. A and G indicates Bph1 and Wbph, respectively. HRM curve profiles of Bph18(t) specific primer in 173 rice hybrid breeding lines. B, control variety; C, 1-44; D, 45-88; E, 89-132; F, 133-173 hybrid breeding lines. R1 and R2 in G indicate resistant control variety such as Utri merah and N22, respectively


Fig. 5.

A, frequency distribution of disease/insect resistant genes among 173 hybrid rice lines and B, the frequency of rice lines having different number of resistant genes


Table 4 . Distribution patterns of disease and insect resistant genes in 173 hybrid rice breeding lines and germplasms

 NoEntryXa3 (Su)Xa3 (Re)Xa4xa5Xa7xa13Xa21No. of R genesPi-bPi-taPi-5No. of R genesBPH1BPH18WBPHNo. of R genesTotal no. of pyramiding R genes
1611504---+---10--01
2611506---+---10--01
3611507---+---10--01
4611509+--+--10--01
5611511---+--10--01
6611513+--+--10--01
7611517---+---10--01
8611531+--+---10--01
9611533+--+--10--01
10611535+--+---10--01
11611571+-----00++22
12611589+-----0+1--01
13611590-+----10-++23
14611611+-----00--00
15611626+--+---10--01
16611631+-+----10--+12
17611632+--+---10--01
18611639+-----0+1--01
19611650+-+---10--01
20611674+------0++2--02
21611675+-----0+1--01
22611677-------0+1--01
23611678-------0++2--02
24611680+---+--1++2--03
25611681+-----0+1++23
26611683+---+--1+1--02
27611684+-----00-+11
28611686-+----10--01
29611689+--+---10--01
30611690+--+---1+1--02
31611693------0++2--02
32611697+------0+1--01
33611698-------0+1--01
34611700+-----0++2--02
35611701+-----0++2--02
36611703+-----0+1--+12
37611704+-----0+1--01
38611707+-++-+3+1--04
39611708+-+++++5+1--06
40611710+-----0++2--02
41611712+-+---1+1--02
42611713+-+---1+1--02
43611715+--+--10--01
44611716+--+--10--01
45611719+-----00--00
46611720+-----00--00
47611722-+----10--01
48611723-+----1+1--02
49611725+-+---1+1++24
50611728+-----0++2-+13
51611729+-----0++2-+13
52611731++----1++2--03
53611732+-----0++2--02
54611734+-----0+++3--03
55611735+-----0+++3-+14
56611737+-----0+++3--03
57611738+-----0++2--02
58611740+-----0++2++24
59611741+--+--1+1--02
60611743-+----1+1--02
61611744+-+---1++2+-+25
62611747+-+---1++2++25
63611748++----1++2++25
64611750+-----0++2++24
65611751+-----0++2++24
66611525+-----00++22
67611527+--+--10--01
68611809+--+--1++2-+14
69611810+--+--1+++3-+15
70611811+-----0+1--01
71611815+-----0++2--02
72611529+--+--10--01
73611817+-----0+1-+12
74611818+--+--1+++3-+15
75611819+-++--20-+13
76611820+--+--10--01
77611821------0+++3-+14
78611822+-----0+1+++34
79611823+-+---1+1-+13
80611824+-++--2+1-++25
81611825+-----0+1-+12
82611826+-----0+1--01
83611827-+----1+1-+13
84611828+-----00--00
85611829-++---2+1-++25
86611830+-----0+1+++34
87611831+-----00++22
88611832+-----0+1--01
89611833---+--1++2-+14
90611834+-+---1++2-+14
91611835+-----00--00
92611836+-+---10--+12
93611837+-+---1+1--02
94611838------0++2--02
95611839+--+--1++2-+14
96611840+-----00--00
97611841+-----0+1-+12
98611842+--+--1+1--02
99611843-++---2+1--03
100611844-+----1+1--02
101611845+-----0+1-+12
102611846+-+---1+++3+-15
103611847+-----0++2--02
104611848+-----0+1++23
105611849+--+--1++2--+14
106611850+-+---1++2-+14
107611851+-+---1++2++25
108611852+-----0+1--+12
109611853+--++--2++2--04
110611854+--++--2++2--04
111611855+--++--2+1+-14
112611856+--+--1+1+-13
113611857+--+--1+1--02
114611858+--+--1+1--02
115611859------0+1--+12
116611860+-----0+1--01
117611861+-----0+1--01
118611862------0+1--+12
119611863------0+1+-+23
120611864+-----0+1+++34
121611865------0+1--+12
122611866------0+1--+12
123611867------0+1--+12
124611868+-----0++2--02
125611869+-+--1++2--03
126611870+-+---1++2+-14
127611871+-----0++2++24
128611872+-+---1+1--02
129611873+-----0++2--02
130611874-+----1++2--03
131611875-+----1+1--02
132611876++----1+1--02
133611877+-----0+1--01
134611878+-----0+1++23
135611879+-----0+1++23
136611880+-----0+1--01
137611881+-----0+1--01
138611882+-----0++2--02
139611883-+----1+1--02
140611884+-----0+1--01
141611885+-----0++2--02
142611886+-----0+1--01
143611887------0+1--01
144611888+---+--1+1--02
145611889+-----0+1--01
146611890+--+---1++2--03
147611891++-+---2+1--03
148611892+-+----1++2-+14
149611893+------0+1--01
150611894+--+--1++2-+14
151611895------0+++3+-+25
152611896+-----0++2--02
153611897----+-1+++3+-15
154611898-+--+-2+1--03
155611899++--+-2+1+-14
156611900-+---1+1--02
157611901-+----1+1--02
158611902-------0++2+-13
159611903-+----1++2+-14
160611904-+----1++2+-+25
161611905-----0++2--+13
162611906-+--+--2++2+-15
163611907-+--+-2++2+-+26
164611908+-----0+1+-12
165611909-+----1+1--02
166611910------0++2+-+24
167611911+------0+++3+-14
168611912+-+----1++2-+14
169611913++-+--2++2++26
170611914++++--3+++3--06
171611915+--+--1++2-+14
172611916+-++--2+1-+14
173611917+--+ --1+  1-+ 13

+: Positive response, -: negative response


Estimation of Genotypes for Rice Blast Resistance Genes

We examined the amplification patterns of three major rice blast resistant genes (Pib, Pi-ta, and Pi-5) in 173 hybrid lines (Fig. 3). The tightly linked Pi-ta on chromosome 12 and Pi-5 genes on chromosome 9 were screened with respective primers, YL155/87 and JJ817 primers. Pi-ta gene was detected in 58 (33.5%) hybrid lines and Pi-5 gene was amplified in 35 (20.2%) hybrid lines, while most of the lines (68.8%) were found to carry Pib (Table 4 and Fig. 5A). According to Ramkumar et al. (2015), Pib gene is one of the major resistance genes located on chromosome 2 which confers resistance to wide range of isolates of rice blast pathogen. Of the total hybrid lines, only 11 were found to contain the three genes, Pib, Pi-5 and Pi-ta genes (Table 4) while none was found to possess at least two genes combination. The three-gene containing lines may provide stable resistance to rice blast isolates.

Estimation of Genotypes for BPH and WBPH Genes

We further surveyed the distribution of resistant genes to brown planthopper (BPH) and whitebacked planthopper (WBPH) in 173 hybrid rice breeding lines and germplasms (Fig. 4). Bph1 was present in 20.8%, Bph18 in 27.2%, and Wbph in 13.3% of the total hybrid rice breeding lines and germplasms (Fig. 5A and Table 4). Among these lines, 75 lines showed the amplification of more than one of BPH and WBPH resistance genes, ranged from 1 (27.2%) to 3 (1.7%) genes. Three hybrid breeding lines, IR68, Laxmi, and Aromatic rice 6-3 contained all three resistance genes, which later can be used to improve the insect resistance in rice.

Estimation of Genotypes for Gene Pyramiding

Distribution of different gene combination varied greatly among the lines. Number of lines with at least two genes was found highest (30.63%) followed by four gene containing lines (17.34%) and five gene containing lines (7.51%). Interestingly in this study, we were able to identify lines that contain up to six different resistance genes. These include IR98161- 2-1-1-k1-3, Damm-Noeub Khmau, 7290s, and 7292s (Table 5). These results prove that it is possible to develop hybrid lines with gene pyramiding through marker-assisted breeding approach.

Table 5 . List of hybrid breeding lines with six different disease resistance genes

 Gene combination  No. of breeding lines Resistance genes
6 genes39Xa4xa5Xa7xa13Xa21Pi-b
163Xa3Xa7Pi-bPi-taBph1Wbph
169Xa3xa5Pi-bPi-5Bph1Bph18(t)
170Xa3Xa4xa5Pi-bPi-taPi-5

5 genes61Xa4Pi-bPi-5Bph1Wbph
62Xa4Pi-bPi-5Bph1Bph18(t)
63Xa3Pi-bPi-taBph1Bph18(t)
69xa5Pi-bPi-taPi-5Bph18(t)
74xa5Pi-bPi-taPi-5Bph18(t)
80Xa4xa5Pi-bBph18(t)Wbph
85Xa3Xa4Pi-bBph18(t)Wbph
102Xa4Pi-bPi-taPi-5Bph18(t)
107Xa4Pi-bPi-5Bph1Bph18(t)
151Pi-bPi-taPi-5Bph1Wbph
153Xa7Pi-bPi-taPi-5Bph1
160Xa3Pi-bPi-taBph1Wbph
162Xa3Xa7Pi-bPi-taBph1

Cultivated crops are exposed to different stress factors. Climate change by increasing temperature affects the habitat range of pathogens and pests, which can facilitate spread of pathogens (Luck et al. 2011; Madgwick et al. 2011). Biotic stress adversely affect plant growth, yield losses, and nutritional values (Porter and Semenov 2005), as well as seed quality of crops. Although conventional breeding is an effective method to develop resistance rice variety, it takes a lot of time to develop a new rice variety. Cross-breeding via marker-assisted selection (MAS) is expected to play a role for the development and improvement of broad-spectrum stress-tolerant crops (Nogoy et al. 2016). Therefore, we investigated the amplification patterns of resistance genes against various biotic stresses such as bacterial blight, blast and brown planthopper pests to genotype 173 hybrid rice lines. It is important to screen candidate genetic resources in rice cross breeding lines through genotyping because it will help breeders to maintain several small base lines having resistance to biotic stress. Furthermore, the use of markers enables the usage and maintenance of diverse resistance genes, analysis of variation, and selection for pyramided genes (Lammerts van Bueren et al. 2010).

In this study, we report the distribution of biotic stress resistance genes such as dominant (Xa3, Xa4, Xa7, and Xa21) and recessive (xa5 and xa13) BB-resistance genes, rice blast (Pib, Pi-5, and Pi-ta), and BPH (Bph1, Bph18 and Wbph) resistance genes in hybrid breeding lines. A total of 173 hybrid rice breeding lines and germplasms were used for the screening of those lines with different resistance genes though MAS-based pyramiding approaches. We analyzed the distribution and genetic differences of BB resistant genes using six major resistant markers related to Xa3, Xa4, xa5, Xa7, xa13 and Xa21 genes. The results revealed that only 0.6% and 1.2% of the hybrid lines have xa13 and Xa21, respectively. However, we were also able to identify lines such as IR98161-2-1-1-k1-3 which contained five genes (Xa4, xa5, Xa7, Xa13 and Xa21), and IR98161-2-1-1-k1-2 and 7292s which possessed three respective gene combinations (Xa4, Xa7 and Xa21, and Xa3, Xa4 and xa5) (Table 4). Kinoshita (1995) reported that the combination of resistance genes into rice varieties is good way to develop durable resistance to BB disease. Recently, Pradhan et al. (2015) cited that a three-gene combination appeared to be the most effective with Xa21 contributing the largest component of resistance to BB. Therefore, in our future studies, we will use the identified hybrid lines with at least three gene combinations for phenotypic selection.

According to the report of Singh et al. (2015), several genes for blast resistance were found and effectively used to control rice blast disease in rice breeding and genetic studies (Chen et al. 2005; Ballini et al. 2008; Koide et al. 2009), and many blast resistant varieties have been developed. We used three major rice blast resistant genes, Pib, Pi-ta, and Pi-5 to confirm the distribution of resistance genes for improving rice blast resistance and multiple-resistance efficiency. Our results showed that Pi-ta on chromosome 12 was present in 33.5% of the hybrid rice lines while Pi-5 gene on chromosome 9 was amplified in 20.2% of 173 hybrid rice lines. Moreover, Pib gene in chromosome 2 was present in 68.8% of the total hybrid lines. Ramkumar et al. (2015) reported that Pib gene is a major resistance gene and offers resistance to wide range of isolates in India. Out of these hybrid lines, only 11 lines possessed three genes, Pib, Pi-5 and Pi-ta, which may be expected to provide high level of resistance against rice blast isolates, because Pib is one of significant rice blast resistant genes (Ramkumar et al. 2015; Wang et al. 1999).

Previous results have been reported that brown planthopper is a destructive phloem sap sucking pest of rice which has negative effect both qualitative and quantitative (Jena et al. 2006). In this study, we searched for hybrid lines having resistant genes for brown plant hopper, namely, Bph1, Bph18(t), and Wbph in 173 hybrid rice lines. Bph1 was present in 20.8%, Bph18 in 27.2%, and Wbph in 13.3% of the total hybrid lines. Among these lines, IR68, Laxmi, and Aromatic rice 6-3 have all three resistance genes (Table 4), which will be used in the future for phenotypic analysis.

In summary, we investigated hybrid rice lines with multiple- resistance to biotic disease and insect pests using DNA-based markers. Our results showed that most of the hybrid breeding lines contained one to four different resistant genes. We identified four lines harboring the maximum number of six resistance genes out of 12 resistance genes used and 13 lines with five resistant genes. The four lines were IR98161-2-1-1-k1-3, Damm- Noeub Khmau, 7290s, and 7292s lines (Table 5). These lines carrying a stack of six-genes are expected to provide higher level of multiple- resistance to biotic stresses. The information obtained and the selected lines with various resistance genes could be used to further facilitate rice disease resistance breeding especially in hybrid rice breeding programs. Available molecular analysis and transcriptome analysis of the various different genes present in each hybrid line may be able to propose a generalized stress response or points of cross-talk between signaling pathways (Atkinson and Urwin, 2012). Lastly, field performance and phenotypic reactions of the identified lines carrying multiple stack of genes in this study need to be verified to provide strong recommendations for use in hybrid rice breeding programs.

This work was supported by Golden Seed Project (No. 2130031-04-4-SB220) and by Agri-Bio industry Technology Development Program (313043-03-3-HD030), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea.

  1. Atkinson NJ, and Urwin PE. (2012) The interaction of plant biotic and abiotic stresses:from genes to the field. J. Exp. Bot 63, 3523-3543.
    Pubmed CrossRef
  2. Ballini E, Morel JB, Droc G, Price A, Courtois B, Notteghem JL, and Tharreau D. (2008) A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance. Mol. Plant Microbe Interact 21, 859-868.
    Pubmed CrossRef
  3. Bonman JM, Estrada BA, and Denton RJN., Blast management with rice cultivar mixture. Progress in Upland Rice Research (1986). Proceedings of the 2nd International Upland Rice Conference, 1985, Jakarta , pp.375-382. IRRI, Los Banos, Philippines.
  4. Chen S, Wang L, Que ZQ, Pan RQ, and Pan QH. (2005) Genetic and physical mapping of Pi37(t) a new gene conferring resistance to rice blast in the famous cultivar St. No. 1. Theor. Appl. Genet 111, 1563-1570.
    Pubmed CrossRef
  5. Couch BC, and Kohn LM. (2002) A multilocus gene genealogy concordant with host preference indicates segregation of a new species Magnaporthe oryzae from M. grisea. Mycologia 94, 683-693.
    CrossRef
  6. Chen X, Temnykh S, Xu Y, Cho YG, and McCouch SR. (1997) Development of a microsatellite framework map providing genomewide coverage in rice (Oryza sativa L.). Theor. Appl. Genet 95, 553-567.
    CrossRef
  7. Ghulam M, Muhammad MA, Sami UK, Muhammad N, and Abdul SM. (2013) Leaf rust resistance in semi dwarf wheat cultivars:a conspectus of post green revolution period in pakistan. Pakistan Journal of Botany 45, 415-422.
  8. Han SS, Ryu JD, Shim HS, Lee SW, Hong YK, and Cha KH. (2001) Breakdown of resistant cultivars by new race KI-1117a and race distribution of rice blast fungus during 1999-2000 in Korea. Res. Plant Dis 7, 86-92.
  9. Hibino H. (1983). Relations of rice tungro bacilliform and spherical viruses with their vector Nephotettix virescens Annals of the Phytopathological Society of Japan 49, 545-553.
    CrossRef
  10. Hittalmani S, Parco A, Mew T, Zeigler R, and Huang N. (2000) Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theor. Appl. Genet 100, 1121-1128.
    CrossRef
  11. Huang N, Angeles ER, Domingo J, Magpantay G, Singh S, Zhang G, Kumaravadivel N, Bennett J, and Khush GS. (1997) Pyramiding of bacterial blight resistance genes in rice:marker assisted selection using RFLP and PCR. Theor Appl Genet 95, 313-320.
    CrossRef
  12. Hur YJ, Jeung JU, Kim SY, Park HS, Cho JH, and Lee JY et al. (2013) Functional markers for bacterial blight resistance gene Xa3 in rice. Mol. Breed 31, 981-985.
    CrossRef
  13. CrossRef
  14. Jena KK, Jeung JU, Lee JH, Choi HC, and Brar DS. (2006) High resolution mapping of a new brown planthopper (BPH) resistance gene Bph18(t) and marker-assisted selection for BPH resistance in rice (Oryza sativa L.). Theor. Appl. Genet 112, 288-297.
    Pubmed CrossRef
  15. Jia Y, Wang Z, and Singh P. (2002) Development of dominant rice blast Pi-ta resistance gene markers. Crop Sci 42, 2145-2149.
    CrossRef
  16. Jia Y, Redus M, Wang Z, and Rutger JN. (2004) Development of a SNLP marker from the Pi-ta blast resistance gene by tri-primer PCR. Euphytica 138, 97-105.
    CrossRef
  17. Joseph M, Gopalakrishnan S, Sharma RK, Singh VP, Singh AK, Singh NK, and Mohapatra T. (2004) Combining bacterial blight resistance and basmati quality characteristics by phenotypic and molecular marker-assisted selection in rice. Mol Breed 13, 377-387.
    CrossRef
  18. Kim SM, and Sohn JK. (2005) Identification of a Rice Gene (Bph 1) Conferring Resistance to Brown Planthopper (Nilaparvata lugens Stal) Using STS Markers. Mol. Cells 20, 30-34.
    Pubmed
  19. Kinoshita T. (1995) Report of committee on gene symbolization, nomenclature and linkage groups. Rice Genet Newsl 12, 9-153.
  20. Koide Y, Kobayashi N, Xu D, and Fukuta Y. (2009) Resistance genes and selection DNA markers for blast disease in rice (Oryza sativa L.). JARQ 43, 255-280.
    CrossRef
  21. Koressaar T, and Remm M. (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23, 1289-91.
  22. Kump B, and Javornik B. (1996) Evaluation of genetic variability among common buckwheat (Fagopyrum esculentum Moench) populations by RAPD markers. Plant Science 114, 149-158.
    CrossRef
  23. Kwon SW, Cho YC, Kim YG, Suh JP, Jeung JU, and Roh JH et al. (2008) Development of Near-isogenic Japonica rice lines with enhanced resistance to Magnaporthe grisea. Mol. Cells 25, 407-416.
    Pubmed
  24. Lammerts van Bueren ET, Backes G, de Vriend H, and Ostergard H. (2010) The role of molecular markers and marker assisted selection in breeding for organic agriculture. Euphytica 175, 51-64.
    CrossRef
  25. Lee JH, Muhsin M, Atienza GA, Kwak DY, Kim SM, and Leon TB et al. (2010) Single nucleotide polymorphisms in a gene for translation initiation factor (eIF4G) of rice (Oryza sativa) associated with resistance to Rice tungro spherical virus. Mol. Plant Microbe Interact 23, 29-38.
    Pubmed CrossRef
  26. Luck J, Spackman M, Freeman A, Trebicki P, Griffiths W, Finlay K, and Chakraborty S. (2011) Climate change and diseases of food crops. Plant Pathology 60, 113-121.
    CrossRef
  27. Maclean JL, Dawe DC, Hardy B, and Hettel GP. (2002). Rice almanac (Third Edition) . IRRI, WARDA, CIAT and FAO, Philippines.
  28. Madgwick JW, West JS, White RP, Semenov MA, Townsend JA, Turner JA, and Fitt BDL. (2011) Impacts of climate change on wheat anthesis and fusarium ear blight in the UK. European Journal of Plant Pathology 130, 117-131.
    CrossRef
  29. Ni?o M, Lee HJ, Kim J, Abdula1 S, Jung YJ, Kang KK, Nou I, and Cho YG. (2015) Enhancement of Rice Resistance to Bacterial Blight by Overexpressing BrCP3 Gene of Brassica rapa Plant Breed. Biotech 3, 355-365.
    CrossRef
  30. McCouch SR, Teytelman L, Xu YB, Lobos KB, Clare K, and Walton M et al. (2002) Development and mapping of 2,240 new SSR markers for rice (Oryza sativa L.). DNA Res 9, 199-207.
    Pubmed CrossRef
  31. Mew TW. (1987) Current status and future prospects of research on bacterial blight of rice. Annu. Rev. Phytopathol 25, 359-382.
    CrossRef
  32. Nogoy FM, Song JY, Ouk S, Rahimi S, Kwon SW, Kang KK, and Cho YG. (2016) Current Applicable DNA Markers for Marker Assisted Breeding in Abiotic and Biotic Stress Tolerance in Rice (Oryza sativa L.) Plant Breed. Biotech 4, 271-284.
    CrossRef
  33. Porter BW, Chittoor JM, Yano M, and Sasaki T et al. (2003) Development and mapping of markers linked to the rice bacterial blight resistance gene Xa7. Crop Sci 43, 1484-1492.
    CrossRef
  34. Porter JR, and Semenov MA. (2005) Crop responses to climatic variation. Philosophical Transactions of the Royal Society B:Biological Sciences 360, 2021-2035.
    Pubmed KoreaMed CrossRef
  35. Pradhan SK, Nayak DK, Mohanty S, Behera L, Barik SR, and Pandit E et al. (2015) Pyramiding of three bacterial blight resistance genes for broad-spectrum resistance in deepwater rice variety, Jalmagna. Rice 8, 19.
    Pubmed KoreaMed CrossRef
  36. Rajpurohit D, Kumar R, Kumar M, Paul P, and Awasthi AA et al. (2011) Pyramiding of two bacterial blight resistance and a semi dwarfing gene in Type 3 Basmati using marker-assisted selection. Euphytica 178, 111-126.
    CrossRef
  37. Ramkumar G, Madhav MS, Rama Devi SJS, Prasad MS, and Ravindra Babu V. (2015) Nucleotide variation and identification of novel blast resistance alleles of Pib by allele mining strategy. Physiol Mol Biol Plants 21, 301-304.
    Pubmed KoreaMed CrossRef
  38. Sasaki T, and Burr B. (2000) International Rice Genome Sequencing Project:The effort to completely sequence the rice genome. Curr. Opin. Plant Biol 3, 138-141.
    CrossRef
  39. Singh AK, Singh PK, Arya M, Singh NK, and Singh US. (2015) Molecular screening of blast resistance genes in rice using SSR markers. Plant Pathol. J 31, 12-24.
    Pubmed KoreaMed CrossRef
  40. Singh AK, Gopala Krishnan S, Singh VP, Prabhu KV, Mohapatra T, and Singh NK et al. (2011) Marker assisted selection:a paradigm shift in Basmati breeding. Indian Journal of Genetics and Plant Breeding 71, 1-9.
  41. Song JY, Lee GA, Choi YM, Lee S, Lee KB, and Bae CH et al. (2014) Blast resistant genes distribution and resistance reaction to blast in Korean landraces of rice (Oryza sativa L.). Korean J. Plant Res 27, 687-700.
    CrossRef
  42. Sun L, Su C, Wang C, Zai H, and Wan J. (2005) Mapping of a major resistance gene to brown planthopper in the rice cultivar Rathu Heenati. Breed. Sci 55, 391-396.
    CrossRef
  43. Talbot NJ, and Foster AJ. (2001) Genetics and genomics of the rice blast fungus Magnaporthe grisea:developing an experimental model for understanding fungal diseases of cereals. Adv. Bot. Res 34, 263-287.
    CrossRef
  44. Talbot NJ. (2003) On the trail of a cereal killer:investigating the biology of Magnaporthe grisea. Annu. Rev. Microbiol 57, 177-202.
    Pubmed CrossRef
  45. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, and Rozen SG. (2012) Primer3 - new capabilities and interfaces. Nucleic Acids Research 40, e115.
    CrossRef
  46. Wang Q, Lu C, and Zhang Q. (2005) Midday photoinhibition of two newly developed super-rice hybrids. Photosynthetica 43, 277-281.
    CrossRef
  47. Wang ZX, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, and Sasaki T. (1999) The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine- rich repeat class of plant disease resistance genes. Plant J 19, 55-64.
    Pubmed CrossRef
  48. Yaegashi H. (1994) Use of resistant varieties and disease control for paddy rice. Agric. Hortic 69, 149-154.
  49. Yamasaki M, Yoshimura A, and Yasui H. (2003) Genetic basis of ovicidal response to whitebacked planthopper (Sogatella furcifera Horvath) in rice (Oryza sativa L.). Molecular Breeding 12, 133-143.
    CrossRef
  50. Zeigler RS, Thome J, Nelson J, Levy M, and Correa-Victoria FJ. (1994) Lineage exclusion:A proposal for linking blast population analysis to resistance breeding. In Rice Blast Disease, Zeigler R.S, Leong S.A, and Teng P (eds.) , pp.267-292. CAB International, Wallingford, UK.
  51. Zhang G, Angeles ER, Abenes MLP, and Khush GS et al. (1996) RAPD and RFLP mapping of the bacterial blight resistance gene Xa-13 in rice. Theor. Appl. Genet 93, 65-70.
    Pubmed CrossRef

Article

Research Article

J Plant Biotechnol 2016; 43(3): 317-331

Published online September 30, 2016 https://doi.org/10.5010/JPB.2016.43.3.317

Copyright © The Korean Society of Plant Biotechnology.

Application and utilization of marker assisted selection for biotic stress resistance in hybrid rice (Oryza sativa L.)

Jae-Young Song, Sothea Ouk, Franz Marielle Nogoy, Marjohn C. Ni?o, Soon Wook Kwon, Woongoo Ha, Kwon-Kyoo Kang, and Yong-Gu Cho*

Department of Crop Science, Chungbuk National University, Cheongju 28644, Korea,
Department of Plant Bioscience, Pusan National University, Busan 50463, Korea,
National Institute of Crop Science, Suwon 16429, Korea,
Department of Horticultural Life Science, Hankyong National University, Anseong 17579, Korea

Correspondence to: e-mail: ygcho@cbnu.ac.kr

Received: 13 September 2016; Revised: 13 September 2016; Accepted: 21 September 2016

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Development of disease resistant plant is one of the important objectives in rice breeding programs because biotic stresses can adversely affect rice growth and yield losses. This study was conducted to identify lines with multiple-resistance genes to biotic stress among 173 hybrid rice breeding lines and germplasms using DNA-based markers. Our results showed that one hybrid rice line [IR98161-2- 1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66)] possessed 5 bacterial blight resistance genes (Xa4, xa5, Xa7, Xa13 and Xa21) while two hybrid rice lines [IR98161-2-1-1-k1-2 (IR86409- 3-1-1-1-1-1/IRBB66) and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] possessed 3 bacterial blight resistance genes (Xa4, Xa7 and Xa21, and Xa3, Xa4 and xa5). Molecular survey on rice blast disease revealed that most of these lines had two different resistant genes. Only 11 lines possessed Pib, Pi-5, and Pi-ta. In addition, we further surveyed the distribution of insect resistant genes, such as Bph1, Bph18(t), and Wbph. Three hybrid breeding lines [IR98161-2-1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66), IR98161-2-1-1-k1-2 (IR86409- 3-1-1-1-1-1/IRBB66), and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] contained all three resistance genes. Finally, we obtained four hybrid rice breeding lines and germplasms [IR98161-2-1-1-k1-2 (IR86409-3-1-1-1-1-1/IRBB66), Damm- Noeub Khmau, 7290s, and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] possessing six-gene combination. They are expected to provide higher level of multiple resistance to biotic stress. This study is important for genotyping hybrid rice with resistance to diverse diseases and pests. Results obtained in this study suggest that identification of pyramided resistance genes is very important for screening hybrid rice breeding lines and germplasms accurately for disease and pest resistance. We will expand their cultivation safely through bioassays against diseases, pests, and disaster in its main export countries.

Keywords: Hybrid, Rice, MAS, MAB, biotic stress, resistance

Introduction

Rice (Oryza sativa L.) is one of the most important and essential source of food crops for many people in the world. Maintaining stable rice production is extremely important to feed the constantly growing human population (Maclean et al. 2002; Sasaki and Burr 2000) in this ever changing climate. Improvement of disease resistance is an important objective in rice breeding programs because rice is exposed to various pests and several diseases including bacterial leaf blight (BB), rice blast (BL), sheath blight (ShB), tungro, and brown planthopper (BPH). Among diseases, bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most destructive bacterial disease of rice that limits rice yield in Asia (Mew 1987) and most of the rice growing countries. Rice blast disease caused by the ascomycete fungus, Magnaporthe oryzae (Couch and Kohn 2002), is also one of the leading causes of great yield loss of rice worldwide and is the most devastating fungal disease on cultivated rice as well as other species of the Poaceae (Zeigler et al. 1994; Talbot and Foster 2001; Talbot 2003; Niño et al. 2015). Rice tungro disease (RTD) is caused by two viruses, Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV), and is one of the most serious diseases of rice in South and Southeast Asia (Lee et al. 2010). Rice damaged by RTD show symptoms, such as small growth and yellow-orange discoloration of leaves (Hibino 1983). Among the rice-feeding insects, Nilaparvata lugens, the rice brown planthopper (BPH) is the most damaging pest (Jiang et al. 2012). In addition, a rice phloem sap-sucking insect, whitebacked planthopper (WBPH) (Sogatella furcifera Horvath), can cause serious reductions in plant height, number of productive tillers, filled grains, and yield (Yamasaki et al. 2003).

Breeding for resistant varieties is the most effective control approach to prevent rice production losses as well as reduce pesticides usage. However, development of rice resistant cultivars by conventional breeding without checking the incorporation of resistant genes may lead to the breakdown of the disease resistance after new resistant cultivar is released, due to complexity of genetic control with complementary or additive effects as well as their environment interactions (Yaegashi 1994; Han et al. 2001). Therefore, it is necessary to develop durable resistance cultivars by incorporating several resistant genes against highly variable pathogen or pests (Bonman et al. 1986; Hittalmani et al. 2000) using marker- assisted selection (MAS). Molecular marker techniques such as MAS may provide new ways for identifying and pyramiding valuable genes to enhance the disease resistance and overcome the breakdown of resistance that frequently occurs in rice breeding programs (Song et al. 2014; Ghulam et al. 2013). Conventional breeding approaches are difficult due to dominance and epistatic effects of genes governing disease resistance by gene pyramiding (Joseph et al. 2004; Rajpurohit et al. 2011). However, DNA-based genetic markers identified in accordance with the phenotypic traits or closed linked to each of the resistance genes are recently expected to play an important role in marker-associated breeding (MAB) for assessment of stress-tolerance and disease-resistance in hybrid breeding plants (Nogoy et al. 2016). According to Wang et al. (2005), developing super hybrid rice depends largely on the genetic resources of the parental lines and the conventional breeding technology. However, improving rice varieties based on this breeding technology is complicated, cumbersome and time- consuming. Uncovering the molecular genetic control of rice heterosis would further improve hybrid rice technology.

To develop rice varieties that can withstand the current insect pest and disease problem in the rice growing regions, we initiate to establish rice breeding programs based on conventional breeding and advanced molecular breeding techniques.

Materials and methods

Plant materials

We used a rice panel comprised of 173 hybrid rice breeding lines and germplasms (Table 1) and control varieties (Table 2), which can be compared by size and presence/absence of amplicon between resistant and susceptible lines.

Table 1 . List of 173 hybrid rice breeding lines and germplasms used in disease resistance analysis in this study.

No Designation Cross/Origin Remark
1Damm-Noeub SarkCambodiaAromatic rice (Jasmine)
2Srau Damm-Noeub Banh-Chras KcharlCambodiaAromatic rice (Jasmine)
3SaigonVietnamRestorer
4Saigon DaratVietnamRestorer
5ReningSeoul University germplasmRestorer
6Jasmin 85ThailandAromatic rice (Jasmine)
7OM 341VietnamLocal variety
8OM 6312-1VietnamLocal variety
9OM 6312-2VietnamLocal variety
10OM 7347VietnamLocal variety
11OM 9538VietnamLocal variety
12HHZ 12-DT10-SAL1-DT1IRRIBreeding line
13VD 20VietnamRestorer
14Anhui collection 1Anhui province, ChinaRestorer
15Anhui collection 2 PTGMSJilin Province, ChinaRestorer
16Anhui collection 3 RAnhui province, ChinaRestorer
17Anhui collection 4 BulkAnhui province, ChinaP/TGMS
18Aromatic rice 10-1IndiaAromatic rice (Basmati)
19Aromatic rice 10-2IndiaAromatic rice (Basmati)
20IR101861-7-1-k1-2MingHui63/IR03A550Restorer
21IR101861-7-1-k1-3MingHui63/IR03A550Restorer
22IR101861-28-1-k1-2MingHui63/IR03A550Restorer
23IR101861-28-1-k1-3MingHui63/IR03A550Restorer
24IR101870-12-1-k1-2MingHui63/IR08N103Restorer
25IR101870-12-1-k1-3MingHui63/IR08N103Restorer
26IR101870-25-1-k1-2MingHui63/IR08N103Restorer
27IR101870-25-1-k1-3MingHui63/IR08N103Restorer
28IR101872-46-1-k1-2MingHui63/IR86590-22-2-2-1-3-1-1-1Restorer
29IR101907-27-2-k1-2IR85593-20-1-1-1-2-3-1-1-1-1/IR86522-29-4-2-1-1-1-1-1Restorer
30IR101907-27-2-k1-3IR85593-20-1-1-1-2-3-1-1-1-1/IR86522-29-4-2-1-1-1-1-1Restorer
31IR98073-3-1-1-k1-3IR72903-131-1-2-3R/IR85485-106-B-B-1-1-1-1Restorer
32IR98116-7-2-1-k1-2SACG7(GID: 2643634)/IRBB23Restorer, BB
33IR98116-7-2-1-k1-3SACG7(GID: 2643634)/IRBB23Restorer, BB
34IR98139-9-1-1-k1-2IR86505-6-15-2-1-1-1-1/IRBB60Restorer, BB
35IR98139-9-1-1-k1-3IR86505-6-15-2-1-1-1-1/IRBB60Restorer, BB
36IR98141-25-1-1-k1-2IR86526-8-8-1-1-1-1-1/IR86555-3-3-1-1-1-1-1-1-1-1Restorer
37IR98141-25-1-1-k1-3IR86526-8-8-1-1-1-1-1/IR86555-3-3-1-1-1-1-1-1-1-1Restorer
38IR98161-2-1-1-k1-2IR86409-3-1-1-1-1-1/IRBB66Restorer, BB
40IR98178-8-2-1-k1-2IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
41IR98178-18-1-1-k1-2IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
42IR98178-18-1-1-k1-3IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
43IR98187-30-1-1-k1-2IR85508-6-1-1-1-1-1-1-1/IR86409-3-1-1-1-1-1Restorer
44IR98187-30-1-1-k1-3IR85508-6-1-1-1-1-1-1-1/IR86409-3-1-1-1-1-1Restorer
45IR98194-9-2-1-k1-2IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
46IR98194-9-2-1-k1-3IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
47IR98200-25-1-1-k1-3IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
48IR98200-25-1-1-k1-4IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
49IR98212-18-2-1-k1-2IR06A169/IR86612-26-9-5-1-1-1-1-1Restorer
50IR98229-2-2-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
51IR98229-2-2-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
52IR98229-9-2-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
53IR98229-9-2-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
54IR98229-24-1-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
55IR98229-24-1-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
56IR98241-24-2-1-k1-2IR06N172/IR86612-38-2-2-1-1-1-1-1Restorer
57IR98241-24-2-1-k1-3IR06N172/IR86612-38-2-2-1-1-1-1-1Restorer
58IR98272-5-1-1-k1-39311/FL478(from Dr. Ismail)Restorer
59IR98272-5-1-1-k1-49311/FL478(from Dr. Ismail)Restorer
60IR98305-10-1-1-k1-3IR68897B/IRBB23maintainer, BB
61Com collection 2-1CambodiaHigh yielding ability
62IR96632-1-1-2-1-k1-2IR85592-7-1-1-1-5-1-1-1/WeedTolerantRice1Maintainer
63IR96632-1-1-2-1-k1-3IR85592-7-1-1-1-5-1-1-1/WeedTolerantRice1Maintainer
64IR96701-28-5-2-1-k1-2IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
65IR96701-28-5-2-1-k1-3IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
66IR96701-28-5-2-1-k1-2IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
67IR96701-28-5-2-1-k1-3IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
68IR 97727-11-1-2-2HANAREUM/NSIC RC 158//HANAREUM
69IR 97727-82-1-2-2HANAREUM/NSIC RC 158//HANAREUM
70IR 97730-27-2-3-1SAEGAEJINMI/IR 6 (PAKISTAN)//SAEGAEJINMI
71BK7 6089-74-40Seoul University germplasmRestorer
72IR2768-11-133-eIRRIRestorer
73RPW6-13Seoul University germplasmRestorer
74GIIB-si 213Seoul University germplasmRestorer
75IR20905-11-3-3IRRIRestorer
76J.P.5-IR946-2-2-2/IR1635-1FIRRIRestorer
77Oksusu byeoChinaRestorer
78IR 68IRRIRestorer
79IR5105-113-3IRRIRestorer
80IR2006IRRIRestorer
81IR2003-97-4-2IRRIRestorer
82IR2042-175-3-2-2IRRIRestorer
83IR1529-430-3IRRIRestorer
84IR1539-823-1-4IRRIRestorer
85Kianse-WuanChinaRestorer
86LaxmiIndiaRestorer
87Quella-IniaSeoul University germplasmRestorer
88IR9859-5-3-3IRRIRestorer
89CB435Seoul University germplasmRestorer
90IR23325-R-R-B-7-2-2IRRIRestorer
91IR4457-5-3-6IRRIRestorer
92VRH624Seoul University germplasmRestorer
939019ChinaRestorer
94Y4037ChinaRestorer
95K100520Seoul University germplasmRestorer
96CondeSeoul University germplasmRestorer
97N22IndiaWide compatibility
98GaruSeoul University germplasmRestorer
99Restorer 1ChinaRestorer
100Restorer 2ChinaRestorer
101Restorer 3ChinaRestorer
102Pare panjangSeoul University germplasmRestorer
103ESMET126Seoul University germplasmRestorer
104BD 43BangladeshRestorer
105BR 26BangladeshRestorer
106IR OM CS 2102IRRI- VietnamRestorer
107IR7760-4-8-2IRRIRestorer
108TGMSChinaTGMS
109Aromatic rice 1-1IndiaAromatic rice (Basmati)
110Aromatic rice 1-2IndiaAromatic rice (Basmati)
111Aromatic rice 1-3IndiaAromatic rice (Basmati)
112Aromatic rice 3-1IndiaAromatic rice (Basmati)
113Aromatic rice 3-2IndiaAromatic rice (Basmati)
114Aromatic rice 3-3IndiaAromatic rice (Basmati)
115Aromatic rice 5-1IndiaAromatic rice (Basmati)
116Aromatic rice 5-2IndiaAromatic rice (Basmati)
117Aromatic rice 5-3IndiaAromatic rice (Basmati)
118Aromatic rice 6-1IndiaAromatic rice (Basmati)
119Aromatic rice 6-2IndiaAromatic rice (Basmati)
120Aromatic rice 6-3IndiaAromatic rice (Basmati)
121Aromatic rice 9-1IndiaAromatic rice (Basmati)
122Aromatic rice 9-2IndiaAromatic rice (Basmati)
123Aromatic rice 9-3IndiaAromatic rice (Basmati)
124Aromatic rice 10-1IndiaAromatic rice (Basmati)
125Aromatic rice 10-2IndiaAromatic rice (Basmati)
127Aromatic rice 11-1IndiaAromatic rice (Basmati)
128Aromatic rice 11-2IndiaAromatic rice (Basmati)
129Aromatic rice 11-3IndiaAromatic rice (Basmati)
130Jasponica H-B-B-8-BSelected lineRestorer, Aromatic rice
131Jasponica H-B-B-7-BSelected lineRestorer, Aromatic rice
132Jasponica H-B-B-16-BSelected lineRestorer, Aromatic rice
133Jasponica H-B-B-21-BSelected lineRestorer, Aromatic rice
134Jasponica H-B-B-26-BSelected lineRestorer, Aromatic rice
135Jasponica H-B-B-7-1Selected lineRestorer, Aromatic rice
136Jasponica H-B-B-9-1Selected lineRestorer, Aromatic rice
137Jasponica H-B-B-12-1Selected lineRestorer, Aromatic rice
138Jasponica H-B-B-17-1Selected lineRestorer, Aromatic rice
139Jasponica H-B-B-18-1Selected lineRestorer, Aromatic rice
140Jasponica H-B-B-19-1Selected lineRestorer, Aromatic rice
141Jasponica H-B-B-21-1Selected lineRestorer, Aromatic rice
142Jasponica H-B-B-27-1Selected lineRestorer, Aromatic rice
143IRRI-A29 B12IRRI maintainerMaintainer, High natural crossing
144IRRI-A29 B13IRRI maintainerMaintainer, High natural crossing
145IR1487-327-1-1-3IRRIRestorer
146DF-1Seoul University germplasmRestorer
147Utri RajapanIRRI_IndonesiaRestorer
148IR4547-2-1-2IRRIRestorer
149IR 01W105IRRIRestorer
150IR 70IRRIRestorer
151Tjempo BrondolIndonesiaRestorer
152Burung PutarIndonesiaRestorer
153Phkar KhgneiCambodiaAromatic rice (Jasmine)
154Damm-Noeub Phka RoluohCambodiaAromatic rice (Jasmine)
155Karn Dal KbalCambodiaAromatic rice (Jasmine)
156Thmar Ror MealCambodiaAromatic rice (Jasmine)
157KaseKamCambodiaAromatic rice (Jasmine)
158Damm-Noeub Trar-sarkCambodiaAromatic rice (Jasmine)
159Damm-Noeub Krar MuonCambodiaAromatic rice (Jasmine)
160Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
161Damm-Noeub Trar-sarkCambodiaAromatic rice (Jasmine)
162Damm-Noeub Chherng MeannCambodiaAromatic rice (Jasmine)
163Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
164Damm-Noeub younCambodiaAromatic rice (Jasmine)
165Damm-Noeub younCambodiaAromatic rice (Jasmine)
166Krar HarmCambodiaAromatic rice (Jasmine)
167Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
1687289sIR75589-31-27-8-33S(S1)/IR102561BTGMS
1697290sIR75589-31-27-8-33S(S1)/IR102568BTGMS
1707292sIR75589-31-27-8-33S(S1)/IR102758BTGMS
1717293sIR75589-31-27-8-33S(S1)/IR58025BTGMS
1727306sIR75589-31-27-8-33S/IR105687BTGMS
173TGMS_bulksIRRITGMS

Table 2 . Information of control varieties used in several disease resistance analyses in this study.

GeneResistant Controls Susceptible Controls 
Xa3 IRBB3 IR24
Xa4 IRBB4 IR24
xa5 IRBB5 IR24
Xa7 IRBB7 IR24
xa13 IRBB13 IR24
Xa21 IRBB21 IR24
Pi-b IRBL-b LTH
Pi-ta IRBL-ta(K1), IRBL-ta(CT2), IRBL-ta2(Pi)  LTH
Pi-5 IRBL_3(CP4), IRBL_5(M), IRBL_i(F5) LTH
Bph1 Hangangchal1, IR26 Dongjin, IR24
Bph18(t) Anmi, Anda IR24, Ilpum
WBPH Utri merah, N22 Nipponbare, TN1

DNA extraction

Total genomic DNA was extracted from fresh leaves of two-week-old rice seedlings using modified Cetyl Trimethyl Ammonium Bromide (CTAB) method as previously described by Kump and Javornik (1996). DNA concentration was quantified using a spectrophotometer (NanoDrop One, Thermo Scientific). The DNA solution was then diluted to a working concentration with distilled water and stored at -20°C until use.

Genotyping

Polymerase chain reaction (PCR) was performed using resistant and susceptible gene-specific primers reported in previous studies and developed in this study (Table 3). Approximately 40 ng of genomic DNA was used in a 20 ul PCR reaction containing 2 ul of primer pairs (10 pmol/ul), 2.0 ul of 10 x PCR buffer, 1.6 ul of dNTP (2.5 mM), and 0.2 ul of Taq polymerase (5 unit/ul; Promega. USA). The reaction mixture was subjected to the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s and extension at 72°C for 45-60 s, and final extension at 72°C for 10 min. The PCR amplified products of bacterial blight (Xa3, Xa7, xa13 and Xa21), blast (Pi-b, Pi-ta and Pi-5), and brown planthopper (Bph1) resistant genes were separated on 1.5-2.0 % agarose gel and stained with ethidium bromide. The amplified products of Xa4, xa5, and Wbph were run on a fragment capillary gel electrophoresis system (Fragment analyzer, USA) and fragments were sized and scored using PROSize 2.0 software (Fragment analyzer, USA). The amplification of Bph18(t) is performed on an Eco™ Real-Time PCR System (Illumina, San Diego, California, USA) according to the user guide manual (Illumina). Allele calling of amplified fragments of hybrid lines and control varieties were based on their respective resistance and susceptible controls.

Table 3 . List of markers used for analysis of various diseases resistance.

Disease/InsectGeneMarkerTypePrimer sequence (5’ → 3’)Expected Size (bp)Reference
Bacterial blight (BB)Xa3BB3-SuFwCGGAGCGACACAGCTATCAT743Hur et al. 2013
RvCGTGAGGTTCCCTATGGCGATT
BB3-ReFwCCACAATGCCATGTCAGGTGGCATCCCTGCA255
RvAGGTGTTGGAGGATTGGCAT
Xa4RM 224FwATCGATCGATCTTCACGAGG150/120Chen et al. 1997;
RvTGCTATAAAAGGCATTCGGGMcCouch et al. 2002
xa5RM122FwGCACTGCAACCATCAATGAATC236/232Chen et al. 1997
RvCCTAGGAGAAACTAGCCGTCCA
Xa7M5FwCGATCTTACTGGCTCTGCAACTCTGT294/1170Porter et al. 2003
RvGCATGTCTGTGTCGATTCGTCCGTACGA
xa13xa13 promFwGGCCATGGCTCAGTGTTTAT1000/520Zhang et al. 1996;
RvGAGCTCCAGCTCTCCAAATGSingh et al. 2011
Xa21pTA248FwAGACGCGGAAGGGTGGTTCCCGGA1000/750Huang et al. 1997
RvAGACGCGGTAATCGAAAGATGAAA
BlastPibNSbFwATCAACTCTGCCACAAAATCC629Kwon et al. 2008
RvCCCATATCACCACTTGTTCCCC
Pi5JJ817FwGATATGGTTGAAAAGCTAATCTCA1450Kwon et al. 2008
RvATCATTGTCCTTCATATTCAGAGT
Pi-taYL155/87FwAGCAGGTTATAAGCTAGGCC1042Jia et al. 2002, 2004
RvCTACCAACAAGTTCATCAAA
BPHBph1BeP18-3FwCGCTGCGAGAGTGTGACACT523Kim and Sohn, 2005
RvTTGGGTTACACGGGTTTGAC
Bph18(t)SNP23FwCGATGGATTACCCTATCACCTCAA110Developed in this study
SNP24RvAACCCTCTGCACACCATCGG
WBPHWbphRM8213FwAGCCCAGTGATACAAAGATG177Sun et al. 2005
RvGCGAGGAGATACCAAGAAAG

Developing Bph18(t) primer for HRM

Bph18(t) (SNP23/SNP24) was designed based on a single nucleotide variation (G/C) found on the 18,379,251 region of the gene sequences in susceptible (HR 20654-39-3-5, IR 10 K153, IR 10K150, IR 10K152, Jinmi, Kanto 51 and Lemont) and resistant (IR65482, Anda, AG04208, SR14694-57-4- 2-1-3-2-2, SR21733-48-1-12-3-2, Backunchal, Backyang and Chupung) cultivars. The forward and reverse primer sequences that flank the SNP region were generated using Primer 3 (v.0.4.0) (Untergasser et al. 2012; Koressaar and Remm 2007) which were set to amplify 110 bp of product size. To test the primer, we used it to genotype a panel of 10 resistant and 31 susceptible varieties, as shown in Fig. 1.

Figure 1.

HRM curve profiles of 10 resistant and 31 susceptible varieties using developed specific SNP marker for Bph18(t) in this study


Results

Estimation of Genotypes for BB Resistance Genes

A total of 173 hybrid breeding lines genotyped using PCR- based markers related to Xa3, Xa4, xa5, Xa7, xa13, and Xa21 genes were analyzed for fragment size differences and presence/absence of genes (Fig. 2). The percentage of rice panel identified to contain the gene was 7.5% with Xa7, 22.5% with xa5, 0.6% with xa13, and 1.2% with Xa21 (Fig. 5A and Table 4). Among the hybrid breeding lines, IR98161- 2-1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66) possessed five bacterial blight resistance genes (Xa4, xa5, Xa7, Xa13 and Xa21) while two hybrid rice lines [IR98161-2-1-1-k1-2 (IR86409-3- 1-1-1-1-1/IRBB66) and 7292s (IR75589-31-27-8-33S(S1)/IR102758B)] possessed three bacterial blight resistance genes (Xa4, Xa7 and Xa21, and Xa3, Xa4 and xa5). Eighty-one lines did not contain any of the BB resistance genes (Fig. 2 and Table 4). The three multiple gene containing lines were selected for developing resistant varieties using MAS.

Figure 2.

PCR amplification patterns of markers that discriminate each of the six bacterial blight resistance genes in 173 hybrid rice breeding lines. A, Xa3; B, Xa7; C, xa13; D, Xa21 shows the band patterns for BB resistance specific primer on gel electrophoresis; E, Xa4 and F, xa5 indicates the size differentiation by fragment analyzer gel electrophoresis. R and S in first and second lanes indicate resistant and susceptible control variety


Figure 3.

PCR amplification patterns of markers that discriminate each of the three blast resistance genes in 173 hybrid breeding lines. A, Pib; B, Pi-ta; C, Pi-5 indicates the amplification patterns for each blast resistance genes in gel electrophoresis


Figure 4.

PCR amplification patterns for Bph1 and Wbph using specific primers on agarose and fragment analyzer gel electrophoresis. A and G indicates Bph1 and Wbph, respectively. HRM curve profiles of Bph18(t) specific primer in 173 rice hybrid breeding lines. B, control variety; C, 1-44; D, 45-88; E, 89-132; F, 133-173 hybrid breeding lines. R1 and R2 in G indicate resistant control variety such as Utri merah and N22, respectively


Figure 5.

A, frequency distribution of disease/insect resistant genes among 173 hybrid rice lines and B, the frequency of rice lines having different number of resistant genes


Table 4 . Distribution patterns of disease and insect resistant genes in 173 hybrid rice breeding lines and germplasms.

 NoEntryXa3 (Su)Xa3 (Re)Xa4xa5Xa7xa13Xa21No. of R genesPi-bPi-taPi-5No. of R genesBPH1BPH18WBPHNo. of R genesTotal no. of pyramiding R genes
1611504---+---10--01
2611506---+---10--01
3611507---+---10--01
4611509+--+--10--01
5611511---+--10--01
6611513+--+--10--01
7611517---+---10--01
8611531+--+---10--01
9611533+--+--10--01
10611535+--+---10--01
11611571+-----00++22
12611589+-----0+1--01
13611590-+----10-++23
14611611+-----00--00
15611626+--+---10--01
16611631+-+----10--+12
17611632+--+---10--01
18611639+-----0+1--01
19611650+-+---10--01
20611674+------0++2--02
21611675+-----0+1--01
22611677-------0+1--01
23611678-------0++2--02
24611680+---+--1++2--03
25611681+-----0+1++23
26611683+---+--1+1--02
27611684+-----00-+11
28611686-+----10--01
29611689+--+---10--01
30611690+--+---1+1--02
31611693------0++2--02
32611697+------0+1--01
33611698-------0+1--01
34611700+-----0++2--02
35611701+-----0++2--02
36611703+-----0+1--+12
37611704+-----0+1--01
38611707+-++-+3+1--04
39611708+-+++++5+1--06
40611710+-----0++2--02
41611712+-+---1+1--02
42611713+-+---1+1--02
43611715+--+--10--01
44611716+--+--10--01
45611719+-----00--00
46611720+-----00--00
47611722-+----10--01
48611723-+----1+1--02
49611725+-+---1+1++24
50611728+-----0++2-+13
51611729+-----0++2-+13
52611731++----1++2--03
53611732+-----0++2--02
54611734+-----0+++3--03
55611735+-----0+++3-+14
56611737+-----0+++3--03
57611738+-----0++2--02
58611740+-----0++2++24
59611741+--+--1+1--02
60611743-+----1+1--02
61611744+-+---1++2+-+25
62611747+-+---1++2++25
63611748++----1++2++25
64611750+-----0++2++24
65611751+-----0++2++24
66611525+-----00++22
67611527+--+--10--01
68611809+--+--1++2-+14
69611810+--+--1+++3-+15
70611811+-----0+1--01
71611815+-----0++2--02
72611529+--+--10--01
73611817+-----0+1-+12
74611818+--+--1+++3-+15
75611819+-++--20-+13
76611820+--+--10--01
77611821------0+++3-+14
78611822+-----0+1+++34
79611823+-+---1+1-+13
80611824+-++--2+1-++25
81611825+-----0+1-+12
82611826+-----0+1--01
83611827-+----1+1-+13
84611828+-----00--00
85611829-++---2+1-++25
86611830+-----0+1+++34
87611831+-----00++22
88611832+-----0+1--01
89611833---+--1++2-+14
90611834+-+---1++2-+14
91611835+-----00--00
92611836+-+---10--+12
93611837+-+---1+1--02
94611838------0++2--02
95611839+--+--1++2-+14
96611840+-----00--00
97611841+-----0+1-+12
98611842+--+--1+1--02
99611843-++---2+1--03
100611844-+----1+1--02
101611845+-----0+1-+12
102611846+-+---1+++3+-15
103611847+-----0++2--02
104611848+-----0+1++23
105611849+--+--1++2--+14
106611850+-+---1++2-+14
107611851+-+---1++2++25
108611852+-----0+1--+12
109611853+--++--2++2--04
110611854+--++--2++2--04
111611855+--++--2+1+-14
112611856+--+--1+1+-13
113611857+--+--1+1--02
114611858+--+--1+1--02
115611859------0+1--+12
116611860+-----0+1--01
117611861+-----0+1--01
118611862------0+1--+12
119611863------0+1+-+23
120611864+-----0+1+++34
121611865------0+1--+12
122611866------0+1--+12
123611867------0+1--+12
124611868+-----0++2--02
125611869+-+--1++2--03
126611870+-+---1++2+-14
127611871+-----0++2++24
128611872+-+---1+1--02
129611873+-----0++2--02
130611874-+----1++2--03
131611875-+----1+1--02
132611876++----1+1--02
133611877+-----0+1--01
134611878+-----0+1++23
135611879+-----0+1++23
136611880+-----0+1--01
137611881+-----0+1--01
138611882+-----0++2--02
139611883-+----1+1--02
140611884+-----0+1--01
141611885+-----0++2--02
142611886+-----0+1--01
143611887------0+1--01
144611888+---+--1+1--02
145611889+-----0+1--01
146611890+--+---1++2--03
147611891++-+---2+1--03
148611892+-+----1++2-+14
149611893+------0+1--01
150611894+--+--1++2-+14
151611895------0+++3+-+25
152611896+-----0++2--02
153611897----+-1+++3+-15
154611898-+--+-2+1--03
155611899++--+-2+1+-14
156611900-+---1+1--02
157611901-+----1+1--02
158611902-------0++2+-13
159611903-+----1++2+-14
160611904-+----1++2+-+25
161611905-----0++2--+13
162611906-+--+--2++2+-15
163611907-+--+-2++2+-+26
164611908+-----0+1+-12
165611909-+----1+1--02
166611910------0++2+-+24
167611911+------0+++3+-14
168611912+-+----1++2-+14
169611913++-+--2++2++26
170611914++++--3+++3--06
171611915+--+--1++2-+14
172611916+-++--2+1-+14
173611917+--+ --1+  1-+ 13

+: Positive response, -: negative response.


Estimation of Genotypes for Rice Blast Resistance Genes

We examined the amplification patterns of three major rice blast resistant genes (Pib, Pi-ta, and Pi-5) in 173 hybrid lines (Fig. 3). The tightly linked Pi-ta on chromosome 12 and Pi-5 genes on chromosome 9 were screened with respective primers, YL155/87 and JJ817 primers. Pi-ta gene was detected in 58 (33.5%) hybrid lines and Pi-5 gene was amplified in 35 (20.2%) hybrid lines, while most of the lines (68.8%) were found to carry Pib (Table 4 and Fig. 5A). According to Ramkumar et al. (2015), Pib gene is one of the major resistance genes located on chromosome 2 which confers resistance to wide range of isolates of rice blast pathogen. Of the total hybrid lines, only 11 were found to contain the three genes, Pib, Pi-5 and Pi-ta genes (Table 4) while none was found to possess at least two genes combination. The three-gene containing lines may provide stable resistance to rice blast isolates.

Estimation of Genotypes for BPH and WBPH Genes

We further surveyed the distribution of resistant genes to brown planthopper (BPH) and whitebacked planthopper (WBPH) in 173 hybrid rice breeding lines and germplasms (Fig. 4). Bph1 was present in 20.8%, Bph18 in 27.2%, and Wbph in 13.3% of the total hybrid rice breeding lines and germplasms (Fig. 5A and Table 4). Among these lines, 75 lines showed the amplification of more than one of BPH and WBPH resistance genes, ranged from 1 (27.2%) to 3 (1.7%) genes. Three hybrid breeding lines, IR68, Laxmi, and Aromatic rice 6-3 contained all three resistance genes, which later can be used to improve the insect resistance in rice.

Estimation of Genotypes for Gene Pyramiding

Distribution of different gene combination varied greatly among the lines. Number of lines with at least two genes was found highest (30.63%) followed by four gene containing lines (17.34%) and five gene containing lines (7.51%). Interestingly in this study, we were able to identify lines that contain up to six different resistance genes. These include IR98161- 2-1-1-k1-3, Damm-Noeub Khmau, 7290s, and 7292s (Table 5). These results prove that it is possible to develop hybrid lines with gene pyramiding through marker-assisted breeding approach.

Table 5 . List of hybrid breeding lines with six different disease resistance genes.

 Gene combination  No. of breeding lines Resistance genes
6 genes39Xa4xa5Xa7xa13Xa21Pi-b
163Xa3Xa7Pi-bPi-taBph1Wbph
169Xa3xa5Pi-bPi-5Bph1Bph18(t)
170Xa3Xa4xa5Pi-bPi-taPi-5

5 genes61Xa4Pi-bPi-5Bph1Wbph
62Xa4Pi-bPi-5Bph1Bph18(t)
63Xa3Pi-bPi-taBph1Bph18(t)
69xa5Pi-bPi-taPi-5Bph18(t)
74xa5Pi-bPi-taPi-5Bph18(t)
80Xa4xa5Pi-bBph18(t)Wbph
85Xa3Xa4Pi-bBph18(t)Wbph
102Xa4Pi-bPi-taPi-5Bph18(t)
107Xa4Pi-bPi-5Bph1Bph18(t)
151Pi-bPi-taPi-5Bph1Wbph
153Xa7Pi-bPi-taPi-5Bph1
160Xa3Pi-bPi-taBph1Wbph
162Xa3Xa7Pi-bPi-taBph1

Discussion

Cultivated crops are exposed to different stress factors. Climate change by increasing temperature affects the habitat range of pathogens and pests, which can facilitate spread of pathogens (Luck et al. 2011; Madgwick et al. 2011). Biotic stress adversely affect plant growth, yield losses, and nutritional values (Porter and Semenov 2005), as well as seed quality of crops. Although conventional breeding is an effective method to develop resistance rice variety, it takes a lot of time to develop a new rice variety. Cross-breeding via marker-assisted selection (MAS) is expected to play a role for the development and improvement of broad-spectrum stress-tolerant crops (Nogoy et al. 2016). Therefore, we investigated the amplification patterns of resistance genes against various biotic stresses such as bacterial blight, blast and brown planthopper pests to genotype 173 hybrid rice lines. It is important to screen candidate genetic resources in rice cross breeding lines through genotyping because it will help breeders to maintain several small base lines having resistance to biotic stress. Furthermore, the use of markers enables the usage and maintenance of diverse resistance genes, analysis of variation, and selection for pyramided genes (Lammerts van Bueren et al. 2010).

In this study, we report the distribution of biotic stress resistance genes such as dominant (Xa3, Xa4, Xa7, and Xa21) and recessive (xa5 and xa13) BB-resistance genes, rice blast (Pib, Pi-5, and Pi-ta), and BPH (Bph1, Bph18 and Wbph) resistance genes in hybrid breeding lines. A total of 173 hybrid rice breeding lines and germplasms were used for the screening of those lines with different resistance genes though MAS-based pyramiding approaches. We analyzed the distribution and genetic differences of BB resistant genes using six major resistant markers related to Xa3, Xa4, xa5, Xa7, xa13 and Xa21 genes. The results revealed that only 0.6% and 1.2% of the hybrid lines have xa13 and Xa21, respectively. However, we were also able to identify lines such as IR98161-2-1-1-k1-3 which contained five genes (Xa4, xa5, Xa7, Xa13 and Xa21), and IR98161-2-1-1-k1-2 and 7292s which possessed three respective gene combinations (Xa4, Xa7 and Xa21, and Xa3, Xa4 and xa5) (Table 4). Kinoshita (1995) reported that the combination of resistance genes into rice varieties is good way to develop durable resistance to BB disease. Recently, Pradhan et al. (2015) cited that a three-gene combination appeared to be the most effective with Xa21 contributing the largest component of resistance to BB. Therefore, in our future studies, we will use the identified hybrid lines with at least three gene combinations for phenotypic selection.

According to the report of Singh et al. (2015), several genes for blast resistance were found and effectively used to control rice blast disease in rice breeding and genetic studies (Chen et al. 2005; Ballini et al. 2008; Koide et al. 2009), and many blast resistant varieties have been developed. We used three major rice blast resistant genes, Pib, Pi-ta, and Pi-5 to confirm the distribution of resistance genes for improving rice blast resistance and multiple-resistance efficiency. Our results showed that Pi-ta on chromosome 12 was present in 33.5% of the hybrid rice lines while Pi-5 gene on chromosome 9 was amplified in 20.2% of 173 hybrid rice lines. Moreover, Pib gene in chromosome 2 was present in 68.8% of the total hybrid lines. Ramkumar et al. (2015) reported that Pib gene is a major resistance gene and offers resistance to wide range of isolates in India. Out of these hybrid lines, only 11 lines possessed three genes, Pib, Pi-5 and Pi-ta, which may be expected to provide high level of resistance against rice blast isolates, because Pib is one of significant rice blast resistant genes (Ramkumar et al. 2015; Wang et al. 1999).

Previous results have been reported that brown planthopper is a destructive phloem sap sucking pest of rice which has negative effect both qualitative and quantitative (Jena et al. 2006). In this study, we searched for hybrid lines having resistant genes for brown plant hopper, namely, Bph1, Bph18(t), and Wbph in 173 hybrid rice lines. Bph1 was present in 20.8%, Bph18 in 27.2%, and Wbph in 13.3% of the total hybrid lines. Among these lines, IR68, Laxmi, and Aromatic rice 6-3 have all three resistance genes (Table 4), which will be used in the future for phenotypic analysis.

In summary, we investigated hybrid rice lines with multiple- resistance to biotic disease and insect pests using DNA-based markers. Our results showed that most of the hybrid breeding lines contained one to four different resistant genes. We identified four lines harboring the maximum number of six resistance genes out of 12 resistance genes used and 13 lines with five resistant genes. The four lines were IR98161-2-1-1-k1-3, Damm- Noeub Khmau, 7290s, and 7292s lines (Table 5). These lines carrying a stack of six-genes are expected to provide higher level of multiple- resistance to biotic stresses. The information obtained and the selected lines with various resistance genes could be used to further facilitate rice disease resistance breeding especially in hybrid rice breeding programs. Available molecular analysis and transcriptome analysis of the various different genes present in each hybrid line may be able to propose a generalized stress response or points of cross-talk between signaling pathways (Atkinson and Urwin, 2012). Lastly, field performance and phenotypic reactions of the identified lines carrying multiple stack of genes in this study need to be verified to provide strong recommendations for use in hybrid rice breeding programs.

Acknowledgments

This work was supported by Golden Seed Project (No. 2130031-04-4-SB220) and by Agri-Bio industry Technology Development Program (313043-03-3-HD030), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea.

Fig 1.

Figure 1.

HRM curve profiles of 10 resistant and 31 susceptible varieties using developed specific SNP marker for Bph18(t) in this study

Journal of Plant Biotechnology 2016; 43: 317-331https://doi.org/10.5010/JPB.2016.43.3.317

Fig 2.

Figure 2.

PCR amplification patterns of markers that discriminate each of the six bacterial blight resistance genes in 173 hybrid rice breeding lines. A, Xa3; B, Xa7; C, xa13; D, Xa21 shows the band patterns for BB resistance specific primer on gel electrophoresis; E, Xa4 and F, xa5 indicates the size differentiation by fragment analyzer gel electrophoresis. R and S in first and second lanes indicate resistant and susceptible control variety

Journal of Plant Biotechnology 2016; 43: 317-331https://doi.org/10.5010/JPB.2016.43.3.317

Fig 3.

Figure 3.

PCR amplification patterns of markers that discriminate each of the three blast resistance genes in 173 hybrid breeding lines. A, Pib; B, Pi-ta; C, Pi-5 indicates the amplification patterns for each blast resistance genes in gel electrophoresis

Journal of Plant Biotechnology 2016; 43: 317-331https://doi.org/10.5010/JPB.2016.43.3.317

Fig 4.

Figure 4.

PCR amplification patterns for Bph1 and Wbph using specific primers on agarose and fragment analyzer gel electrophoresis. A and G indicates Bph1 and Wbph, respectively. HRM curve profiles of Bph18(t) specific primer in 173 rice hybrid breeding lines. B, control variety; C, 1-44; D, 45-88; E, 89-132; F, 133-173 hybrid breeding lines. R1 and R2 in G indicate resistant control variety such as Utri merah and N22, respectively

Journal of Plant Biotechnology 2016; 43: 317-331https://doi.org/10.5010/JPB.2016.43.3.317

Fig 5.

Figure 5.

A, frequency distribution of disease/insect resistant genes among 173 hybrid rice lines and B, the frequency of rice lines having different number of resistant genes

Journal of Plant Biotechnology 2016; 43: 317-331https://doi.org/10.5010/JPB.2016.43.3.317

Table 1 . List of 173 hybrid rice breeding lines and germplasms used in disease resistance analysis in this study.

No Designation Cross/Origin Remark
1Damm-Noeub SarkCambodiaAromatic rice (Jasmine)
2Srau Damm-Noeub Banh-Chras KcharlCambodiaAromatic rice (Jasmine)
3SaigonVietnamRestorer
4Saigon DaratVietnamRestorer
5ReningSeoul University germplasmRestorer
6Jasmin 85ThailandAromatic rice (Jasmine)
7OM 341VietnamLocal variety
8OM 6312-1VietnamLocal variety
9OM 6312-2VietnamLocal variety
10OM 7347VietnamLocal variety
11OM 9538VietnamLocal variety
12HHZ 12-DT10-SAL1-DT1IRRIBreeding line
13VD 20VietnamRestorer
14Anhui collection 1Anhui province, ChinaRestorer
15Anhui collection 2 PTGMSJilin Province, ChinaRestorer
16Anhui collection 3 RAnhui province, ChinaRestorer
17Anhui collection 4 BulkAnhui province, ChinaP/TGMS
18Aromatic rice 10-1IndiaAromatic rice (Basmati)
19Aromatic rice 10-2IndiaAromatic rice (Basmati)
20IR101861-7-1-k1-2MingHui63/IR03A550Restorer
21IR101861-7-1-k1-3MingHui63/IR03A550Restorer
22IR101861-28-1-k1-2MingHui63/IR03A550Restorer
23IR101861-28-1-k1-3MingHui63/IR03A550Restorer
24IR101870-12-1-k1-2MingHui63/IR08N103Restorer
25IR101870-12-1-k1-3MingHui63/IR08N103Restorer
26IR101870-25-1-k1-2MingHui63/IR08N103Restorer
27IR101870-25-1-k1-3MingHui63/IR08N103Restorer
28IR101872-46-1-k1-2MingHui63/IR86590-22-2-2-1-3-1-1-1Restorer
29IR101907-27-2-k1-2IR85593-20-1-1-1-2-3-1-1-1-1/IR86522-29-4-2-1-1-1-1-1Restorer
30IR101907-27-2-k1-3IR85593-20-1-1-1-2-3-1-1-1-1/IR86522-29-4-2-1-1-1-1-1Restorer
31IR98073-3-1-1-k1-3IR72903-131-1-2-3R/IR85485-106-B-B-1-1-1-1Restorer
32IR98116-7-2-1-k1-2SACG7(GID: 2643634)/IRBB23Restorer, BB
33IR98116-7-2-1-k1-3SACG7(GID: 2643634)/IRBB23Restorer, BB
34IR98139-9-1-1-k1-2IR86505-6-15-2-1-1-1-1/IRBB60Restorer, BB
35IR98139-9-1-1-k1-3IR86505-6-15-2-1-1-1-1/IRBB60Restorer, BB
36IR98141-25-1-1-k1-2IR86526-8-8-1-1-1-1-1/IR86555-3-3-1-1-1-1-1-1-1-1Restorer
37IR98141-25-1-1-k1-3IR86526-8-8-1-1-1-1-1/IR86555-3-3-1-1-1-1-1-1-1-1Restorer
38IR98161-2-1-1-k1-2IR86409-3-1-1-1-1-1/IRBB66Restorer, BB
40IR98178-8-2-1-k1-2IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
41IR98178-18-1-1-k1-2IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
42IR98178-18-1-1-k1-3IR86603-15-2-1-1-1-1-1-1/IR86515-4-7-2Restorer
43IR98187-30-1-1-k1-2IR85508-6-1-1-1-1-1-1-1/IR86409-3-1-1-1-1-1Restorer
44IR98187-30-1-1-k1-3IR85508-6-1-1-1-1-1-1-1/IR86409-3-1-1-1-1-1Restorer
45IR98194-9-2-1-k1-2IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
46IR98194-9-2-1-k1-3IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
47IR98200-25-1-1-k1-3IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
48IR98200-25-1-1-k1-4IR86590-22-2-2-1-3-1-1-1/IR86612-21-6-1-1-1-1-1Restorer
49IR98212-18-2-1-k1-2IR06A169/IR86612-26-9-5-1-1-1-1-1Restorer
50IR98229-2-2-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
51IR98229-2-2-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
52IR98229-9-2-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
53IR98229-9-2-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
54IR98229-24-1-1-k1-2IR08A138/IR72998-93-3-3-2RRestorer
55IR98229-24-1-1-k1-3IR08A138/IR72998-93-3-3-2RRestorer
56IR98241-24-2-1-k1-2IR06N172/IR86612-38-2-2-1-1-1-1-1Restorer
57IR98241-24-2-1-k1-3IR06N172/IR86612-38-2-2-1-1-1-1-1Restorer
58IR98272-5-1-1-k1-39311/FL478(from Dr. Ismail)Restorer
59IR98272-5-1-1-k1-49311/FL478(from Dr. Ismail)Restorer
60IR98305-10-1-1-k1-3IR68897B/IRBB23maintainer, BB
61Com collection 2-1CambodiaHigh yielding ability
62IR96632-1-1-2-1-k1-2IR85592-7-1-1-1-5-1-1-1/WeedTolerantRice1Maintainer
63IR96632-1-1-2-1-k1-3IR85592-7-1-1-1-5-1-1-1/WeedTolerantRice1Maintainer
64IR96701-28-5-2-1-k1-2IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
65IR96701-28-5-2-1-k1-3IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
66IR96701-28-5-2-1-k1-2IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
67IR96701-28-5-2-1-k1-3IR85552-37-1-1-1-2-1-1-1/IR80555Bmaintainer, BB
68IR 97727-11-1-2-2HANAREUM/NSIC RC 158//HANAREUM
69IR 97727-82-1-2-2HANAREUM/NSIC RC 158//HANAREUM
70IR 97730-27-2-3-1SAEGAEJINMI/IR 6 (PAKISTAN)//SAEGAEJINMI
71BK7 6089-74-40Seoul University germplasmRestorer
72IR2768-11-133-eIRRIRestorer
73RPW6-13Seoul University germplasmRestorer
74GIIB-si 213Seoul University germplasmRestorer
75IR20905-11-3-3IRRIRestorer
76J.P.5-IR946-2-2-2/IR1635-1FIRRIRestorer
77Oksusu byeoChinaRestorer
78IR 68IRRIRestorer
79IR5105-113-3IRRIRestorer
80IR2006IRRIRestorer
81IR2003-97-4-2IRRIRestorer
82IR2042-175-3-2-2IRRIRestorer
83IR1529-430-3IRRIRestorer
84IR1539-823-1-4IRRIRestorer
85Kianse-WuanChinaRestorer
86LaxmiIndiaRestorer
87Quella-IniaSeoul University germplasmRestorer
88IR9859-5-3-3IRRIRestorer
89CB435Seoul University germplasmRestorer
90IR23325-R-R-B-7-2-2IRRIRestorer
91IR4457-5-3-6IRRIRestorer
92VRH624Seoul University germplasmRestorer
939019ChinaRestorer
94Y4037ChinaRestorer
95K100520Seoul University germplasmRestorer
96CondeSeoul University germplasmRestorer
97N22IndiaWide compatibility
98GaruSeoul University germplasmRestorer
99Restorer 1ChinaRestorer
100Restorer 2ChinaRestorer
101Restorer 3ChinaRestorer
102Pare panjangSeoul University germplasmRestorer
103ESMET126Seoul University germplasmRestorer
104BD 43BangladeshRestorer
105BR 26BangladeshRestorer
106IR OM CS 2102IRRI- VietnamRestorer
107IR7760-4-8-2IRRIRestorer
108TGMSChinaTGMS
109Aromatic rice 1-1IndiaAromatic rice (Basmati)
110Aromatic rice 1-2IndiaAromatic rice (Basmati)
111Aromatic rice 1-3IndiaAromatic rice (Basmati)
112Aromatic rice 3-1IndiaAromatic rice (Basmati)
113Aromatic rice 3-2IndiaAromatic rice (Basmati)
114Aromatic rice 3-3IndiaAromatic rice (Basmati)
115Aromatic rice 5-1IndiaAromatic rice (Basmati)
116Aromatic rice 5-2IndiaAromatic rice (Basmati)
117Aromatic rice 5-3IndiaAromatic rice (Basmati)
118Aromatic rice 6-1IndiaAromatic rice (Basmati)
119Aromatic rice 6-2IndiaAromatic rice (Basmati)
120Aromatic rice 6-3IndiaAromatic rice (Basmati)
121Aromatic rice 9-1IndiaAromatic rice (Basmati)
122Aromatic rice 9-2IndiaAromatic rice (Basmati)
123Aromatic rice 9-3IndiaAromatic rice (Basmati)
124Aromatic rice 10-1IndiaAromatic rice (Basmati)
125Aromatic rice 10-2IndiaAromatic rice (Basmati)
127Aromatic rice 11-1IndiaAromatic rice (Basmati)
128Aromatic rice 11-2IndiaAromatic rice (Basmati)
129Aromatic rice 11-3IndiaAromatic rice (Basmati)
130Jasponica H-B-B-8-BSelected lineRestorer, Aromatic rice
131Jasponica H-B-B-7-BSelected lineRestorer, Aromatic rice
132Jasponica H-B-B-16-BSelected lineRestorer, Aromatic rice
133Jasponica H-B-B-21-BSelected lineRestorer, Aromatic rice
134Jasponica H-B-B-26-BSelected lineRestorer, Aromatic rice
135Jasponica H-B-B-7-1Selected lineRestorer, Aromatic rice
136Jasponica H-B-B-9-1Selected lineRestorer, Aromatic rice
137Jasponica H-B-B-12-1Selected lineRestorer, Aromatic rice
138Jasponica H-B-B-17-1Selected lineRestorer, Aromatic rice
139Jasponica H-B-B-18-1Selected lineRestorer, Aromatic rice
140Jasponica H-B-B-19-1Selected lineRestorer, Aromatic rice
141Jasponica H-B-B-21-1Selected lineRestorer, Aromatic rice
142Jasponica H-B-B-27-1Selected lineRestorer, Aromatic rice
143IRRI-A29 B12IRRI maintainerMaintainer, High natural crossing
144IRRI-A29 B13IRRI maintainerMaintainer, High natural crossing
145IR1487-327-1-1-3IRRIRestorer
146DF-1Seoul University germplasmRestorer
147Utri RajapanIRRI_IndonesiaRestorer
148IR4547-2-1-2IRRIRestorer
149IR 01W105IRRIRestorer
150IR 70IRRIRestorer
151Tjempo BrondolIndonesiaRestorer
152Burung PutarIndonesiaRestorer
153Phkar KhgneiCambodiaAromatic rice (Jasmine)
154Damm-Noeub Phka RoluohCambodiaAromatic rice (Jasmine)
155Karn Dal KbalCambodiaAromatic rice (Jasmine)
156Thmar Ror MealCambodiaAromatic rice (Jasmine)
157KaseKamCambodiaAromatic rice (Jasmine)
158Damm-Noeub Trar-sarkCambodiaAromatic rice (Jasmine)
159Damm-Noeub Krar MuonCambodiaAromatic rice (Jasmine)
160Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
161Damm-Noeub Trar-sarkCambodiaAromatic rice (Jasmine)
162Damm-Noeub Chherng MeannCambodiaAromatic rice (Jasmine)
163Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
164Damm-Noeub younCambodiaAromatic rice (Jasmine)
165Damm-Noeub younCambodiaAromatic rice (Jasmine)
166Krar HarmCambodiaAromatic rice (Jasmine)
167Damm-Noeub KhmauCambodiaAromatic rice (Jasmine)
1687289sIR75589-31-27-8-33S(S1)/IR102561BTGMS
1697290sIR75589-31-27-8-33S(S1)/IR102568BTGMS
1707292sIR75589-31-27-8-33S(S1)/IR102758BTGMS
1717293sIR75589-31-27-8-33S(S1)/IR58025BTGMS
1727306sIR75589-31-27-8-33S/IR105687BTGMS
173TGMS_bulksIRRITGMS

Table 2 . Information of control varieties used in several disease resistance analyses in this study.

GeneResistant Controls Susceptible Controls 
Xa3 IRBB3 IR24
Xa4 IRBB4 IR24
xa5 IRBB5 IR24
Xa7 IRBB7 IR24
xa13 IRBB13 IR24
Xa21 IRBB21 IR24
Pi-b IRBL-b LTH
Pi-ta IRBL-ta(K1), IRBL-ta(CT2), IRBL-ta2(Pi)  LTH
Pi-5 IRBL_3(CP4), IRBL_5(M), IRBL_i(F5) LTH
Bph1 Hangangchal1, IR26 Dongjin, IR24
Bph18(t) Anmi, Anda IR24, Ilpum
WBPH Utri merah, N22 Nipponbare, TN1

Table 3 . List of markers used for analysis of various diseases resistance.

Disease/InsectGeneMarkerTypePrimer sequence (5’ → 3’)Expected Size (bp)Reference
Bacterial blight (BB)Xa3BB3-SuFwCGGAGCGACACAGCTATCAT743Hur et al. 2013
RvCGTGAGGTTCCCTATGGCGATT
BB3-ReFwCCACAATGCCATGTCAGGTGGCATCCCTGCA255
RvAGGTGTTGGAGGATTGGCAT
Xa4RM 224FwATCGATCGATCTTCACGAGG150/120Chen et al. 1997;
RvTGCTATAAAAGGCATTCGGGMcCouch et al. 2002
xa5RM122FwGCACTGCAACCATCAATGAATC236/232Chen et al. 1997
RvCCTAGGAGAAACTAGCCGTCCA
Xa7M5FwCGATCTTACTGGCTCTGCAACTCTGT294/1170Porter et al. 2003
RvGCATGTCTGTGTCGATTCGTCCGTACGA
xa13xa13 promFwGGCCATGGCTCAGTGTTTAT1000/520Zhang et al. 1996;
RvGAGCTCCAGCTCTCCAAATGSingh et al. 2011
Xa21pTA248FwAGACGCGGAAGGGTGGTTCCCGGA1000/750Huang et al. 1997
RvAGACGCGGTAATCGAAAGATGAAA
BlastPibNSbFwATCAACTCTGCCACAAAATCC629Kwon et al. 2008
RvCCCATATCACCACTTGTTCCCC
Pi5JJ817FwGATATGGTTGAAAAGCTAATCTCA1450Kwon et al. 2008
RvATCATTGTCCTTCATATTCAGAGT
Pi-taYL155/87FwAGCAGGTTATAAGCTAGGCC1042Jia et al. 2002, 2004
RvCTACCAACAAGTTCATCAAA
BPHBph1BeP18-3FwCGCTGCGAGAGTGTGACACT523Kim and Sohn, 2005
RvTTGGGTTACACGGGTTTGAC
Bph18(t)SNP23FwCGATGGATTACCCTATCACCTCAA110Developed in this study
SNP24RvAACCCTCTGCACACCATCGG
WBPHWbphRM8213FwAGCCCAGTGATACAAAGATG177Sun et al. 2005
RvGCGAGGAGATACCAAGAAAG

Table 4 . Distribution patterns of disease and insect resistant genes in 173 hybrid rice breeding lines and germplasms.

 NoEntryXa3 (Su)Xa3 (Re)Xa4xa5Xa7xa13Xa21No. of R genesPi-bPi-taPi-5No. of R genesBPH1BPH18WBPHNo. of R genesTotal no. of pyramiding R genes
1611504---+---10--01
2611506---+---10--01
3611507---+---10--01
4611509+--+--10--01
5611511---+--10--01
6611513+--+--10--01
7611517---+---10--01
8611531+--+---10--01
9611533+--+--10--01
10611535+--+---10--01
11611571+-----00++22
12611589+-----0+1--01
13611590-+----10-++23
14611611+-----00--00
15611626+--+---10--01
16611631+-+----10--+12
17611632+--+---10--01
18611639+-----0+1--01
19611650+-+---10--01
20611674+------0++2--02
21611675+-----0+1--01
22611677-------0+1--01
23611678-------0++2--02
24611680+---+--1++2--03
25611681+-----0+1++23
26611683+---+--1+1--02
27611684+-----00-+11
28611686-+----10--01
29611689+--+---10--01
30611690+--+---1+1--02
31611693------0++2--02
32611697+------0+1--01
33611698-------0+1--01
34611700+-----0++2--02
35611701+-----0++2--02
36611703+-----0+1--+12
37611704+-----0+1--01
38611707+-++-+3+1--04
39611708+-+++++5+1--06
40611710+-----0++2--02
41611712+-+---1+1--02
42611713+-+---1+1--02
43611715+--+--10--01
44611716+--+--10--01
45611719+-----00--00
46611720+-----00--00
47611722-+----10--01
48611723-+----1+1--02
49611725+-+---1+1++24
50611728+-----0++2-+13
51611729+-----0++2-+13
52611731++----1++2--03
53611732+-----0++2--02
54611734+-----0+++3--03
55611735+-----0+++3-+14
56611737+-----0+++3--03
57611738+-----0++2--02
58611740+-----0++2++24
59611741+--+--1+1--02
60611743-+----1+1--02
61611744+-+---1++2+-+25
62611747+-+---1++2++25
63611748++----1++2++25
64611750+-----0++2++24
65611751+-----0++2++24
66611525+-----00++22
67611527+--+--10--01
68611809+--+--1++2-+14
69611810+--+--1+++3-+15
70611811+-----0+1--01
71611815+-----0++2--02
72611529+--+--10--01
73611817+-----0+1-+12
74611818+--+--1+++3-+15
75611819+-++--20-+13
76611820+--+--10--01
77611821------0+++3-+14
78611822+-----0+1+++34
79611823+-+---1+1-+13
80611824+-++--2+1-++25
81611825+-----0+1-+12
82611826+-----0+1--01
83611827-+----1+1-+13
84611828+-----00--00
85611829-++---2+1-++25
86611830+-----0+1+++34
87611831+-----00++22
88611832+-----0+1--01
89611833---+--1++2-+14
90611834+-+---1++2-+14
91611835+-----00--00
92611836+-+---10--+12
93611837+-+---1+1--02
94611838------0++2--02
95611839+--+--1++2-+14
96611840+-----00--00
97611841+-----0+1-+12
98611842+--+--1+1--02
99611843-++---2+1--03
100611844-+----1+1--02
101611845+-----0+1-+12
102611846+-+---1+++3+-15
103611847+-----0++2--02
104611848+-----0+1++23
105611849+--+--1++2--+14
106611850+-+---1++2-+14
107611851+-+---1++2++25
108611852+-----0+1--+12
109611853+--++--2++2--04
110611854+--++--2++2--04
111611855+--++--2+1+-14
112611856+--+--1+1+-13
113611857+--+--1+1--02
114611858+--+--1+1--02
115611859------0+1--+12
116611860+-----0+1--01
117611861+-----0+1--01
118611862------0+1--+12
119611863------0+1+-+23
120611864+-----0+1+++34
121611865------0+1--+12
122611866------0+1--+12
123611867------0+1--+12
124611868+-----0++2--02
125611869+-+--1++2--03
126611870+-+---1++2+-14
127611871+-----0++2++24
128611872+-+---1+1--02
129611873+-----0++2--02
130611874-+----1++2--03
131611875-+----1+1--02
132611876++----1+1--02
133611877+-----0+1--01
134611878+-----0+1++23
135611879+-----0+1++23
136611880+-----0+1--01
137611881+-----0+1--01
138611882+-----0++2--02
139611883-+----1+1--02
140611884+-----0+1--01
141611885+-----0++2--02
142611886+-----0+1--01
143611887------0+1--01
144611888+---+--1+1--02
145611889+-----0+1--01
146611890+--+---1++2--03
147611891++-+---2+1--03
148611892+-+----1++2-+14
149611893+------0+1--01
150611894+--+--1++2-+14
151611895------0+++3+-+25
152611896+-----0++2--02
153611897----+-1+++3+-15
154611898-+--+-2+1--03
155611899++--+-2+1+-14
156611900-+---1+1--02
157611901-+----1+1--02
158611902-------0++2+-13
159611903-+----1++2+-14
160611904-+----1++2+-+25
161611905-----0++2--+13
162611906-+--+--2++2+-15
163611907-+--+-2++2+-+26
164611908+-----0+1+-12
165611909-+----1+1--02
166611910------0++2+-+24
167611911+------0+++3+-14
168611912+-+----1++2-+14
169611913++-+--2++2++26
170611914++++--3+++3--06
171611915+--+--1++2-+14
172611916+-++--2+1-+14
173611917+--+ --1+  1-+ 13

+: Positive response, -: negative response.


Table 5 . List of hybrid breeding lines with six different disease resistance genes.

 Gene combination  No. of breeding lines Resistance genes
6 genes39Xa4xa5Xa7xa13Xa21Pi-b
163Xa3Xa7Pi-bPi-taBph1Wbph
169Xa3xa5Pi-bPi-5Bph1Bph18(t)
170Xa3Xa4xa5Pi-bPi-taPi-5

5 genes61Xa4Pi-bPi-5Bph1Wbph
62Xa4Pi-bPi-5Bph1Bph18(t)
63Xa3Pi-bPi-taBph1Bph18(t)
69xa5Pi-bPi-taPi-5Bph18(t)
74xa5Pi-bPi-taPi-5Bph18(t)
80Xa4xa5Pi-bBph18(t)Wbph
85Xa3Xa4Pi-bBph18(t)Wbph
102Xa4Pi-bPi-taPi-5Bph18(t)
107Xa4Pi-bPi-5Bph1Bph18(t)
151Pi-bPi-taPi-5Bph1Wbph
153Xa7Pi-bPi-taPi-5Bph1
160Xa3Pi-bPi-taBph1Wbph
162Xa3Xa7Pi-bPi-taBph1

References

  1. Atkinson NJ, and Urwin PE. (2012) The interaction of plant biotic and abiotic stresses:from genes to the field. J. Exp. Bot 63, 3523-3543.
    Pubmed CrossRef
  2. Ballini E, Morel JB, Droc G, Price A, Courtois B, Notteghem JL, and Tharreau D. (2008) A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance. Mol. Plant Microbe Interact 21, 859-868.
    Pubmed CrossRef
  3. Bonman JM, Estrada BA, and Denton RJN., Blast management with rice cultivar mixture. Progress in Upland Rice Research (1986). Proceedings of the 2nd International Upland Rice Conference, 1985, Jakarta , pp.375-382. IRRI, Los Banos, Philippines.
  4. Chen S, Wang L, Que ZQ, Pan RQ, and Pan QH. (2005) Genetic and physical mapping of Pi37(t) a new gene conferring resistance to rice blast in the famous cultivar St. No. 1. Theor. Appl. Genet 111, 1563-1570.
    Pubmed CrossRef
  5. Couch BC, and Kohn LM. (2002) A multilocus gene genealogy concordant with host preference indicates segregation of a new species Magnaporthe oryzae from M. grisea. Mycologia 94, 683-693.
    CrossRef
  6. Chen X, Temnykh S, Xu Y, Cho YG, and McCouch SR. (1997) Development of a microsatellite framework map providing genomewide coverage in rice (Oryza sativa L.). Theor. Appl. Genet 95, 553-567.
    CrossRef
  7. Ghulam M, Muhammad MA, Sami UK, Muhammad N, and Abdul SM. (2013) Leaf rust resistance in semi dwarf wheat cultivars:a conspectus of post green revolution period in pakistan. Pakistan Journal of Botany 45, 415-422.
  8. Han SS, Ryu JD, Shim HS, Lee SW, Hong YK, and Cha KH. (2001) Breakdown of resistant cultivars by new race KI-1117a and race distribution of rice blast fungus during 1999-2000 in Korea. Res. Plant Dis 7, 86-92.
  9. Hibino H. (1983). Relations of rice tungro bacilliform and spherical viruses with their vector Nephotettix virescens Annals of the Phytopathological Society of Japan 49, 545-553.
    CrossRef
  10. Hittalmani S, Parco A, Mew T, Zeigler R, and Huang N. (2000) Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theor. Appl. Genet 100, 1121-1128.
    CrossRef
  11. Huang N, Angeles ER, Domingo J, Magpantay G, Singh S, Zhang G, Kumaravadivel N, Bennett J, and Khush GS. (1997) Pyramiding of bacterial blight resistance genes in rice:marker assisted selection using RFLP and PCR. Theor Appl Genet 95, 313-320.
    CrossRef
  12. Hur YJ, Jeung JU, Kim SY, Park HS, Cho JH, and Lee JY et al. (2013) Functional markers for bacterial blight resistance gene Xa3 in rice. Mol. Breed 31, 981-985.
    CrossRef
  13. Biotechnology Advances. http://dx.doi.org/10.1016/j.biotechadv.2011.08.013
    CrossRef
  14. Jena KK, Jeung JU, Lee JH, Choi HC, and Brar DS. (2006) High resolution mapping of a new brown planthopper (BPH) resistance gene Bph18(t) and marker-assisted selection for BPH resistance in rice (Oryza sativa L.). Theor. Appl. Genet 112, 288-297.
    Pubmed CrossRef
  15. Jia Y, Wang Z, and Singh P. (2002) Development of dominant rice blast Pi-ta resistance gene markers. Crop Sci 42, 2145-2149.
    CrossRef
  16. Jia Y, Redus M, Wang Z, and Rutger JN. (2004) Development of a SNLP marker from the Pi-ta blast resistance gene by tri-primer PCR. Euphytica 138, 97-105.
    CrossRef
  17. Joseph M, Gopalakrishnan S, Sharma RK, Singh VP, Singh AK, Singh NK, and Mohapatra T. (2004) Combining bacterial blight resistance and basmati quality characteristics by phenotypic and molecular marker-assisted selection in rice. Mol Breed 13, 377-387.
    CrossRef
  18. Kim SM, and Sohn JK. (2005) Identification of a Rice Gene (Bph 1) Conferring Resistance to Brown Planthopper (Nilaparvata lugens Stal) Using STS Markers. Mol. Cells 20, 30-34.
    Pubmed
  19. Kinoshita T. (1995) Report of committee on gene symbolization, nomenclature and linkage groups. Rice Genet Newsl 12, 9-153.
  20. Koide Y, Kobayashi N, Xu D, and Fukuta Y. (2009) Resistance genes and selection DNA markers for blast disease in rice (Oryza sativa L.). JARQ 43, 255-280.
    CrossRef
  21. Koressaar T, and Remm M. (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23, 1289-91.
  22. Kump B, and Javornik B. (1996) Evaluation of genetic variability among common buckwheat (Fagopyrum esculentum Moench) populations by RAPD markers. Plant Science 114, 149-158.
    CrossRef
  23. Kwon SW, Cho YC, Kim YG, Suh JP, Jeung JU, and Roh JH et al. (2008) Development of Near-isogenic Japonica rice lines with enhanced resistance to Magnaporthe grisea. Mol. Cells 25, 407-416.
    Pubmed
  24. Lammerts van Bueren ET, Backes G, de Vriend H, and Ostergard H. (2010) The role of molecular markers and marker assisted selection in breeding for organic agriculture. Euphytica 175, 51-64.
    CrossRef
  25. Lee JH, Muhsin M, Atienza GA, Kwak DY, Kim SM, and Leon TB et al. (2010) Single nucleotide polymorphisms in a gene for translation initiation factor (eIF4G) of rice (Oryza sativa) associated with resistance to Rice tungro spherical virus. Mol. Plant Microbe Interact 23, 29-38.
    Pubmed CrossRef
  26. Luck J, Spackman M, Freeman A, Trebicki P, Griffiths W, Finlay K, and Chakraborty S. (2011) Climate change and diseases of food crops. Plant Pathology 60, 113-121.
    CrossRef
  27. Maclean JL, Dawe DC, Hardy B, and Hettel GP. (2002). Rice almanac (Third Edition) . IRRI, WARDA, CIAT and FAO, Philippines.
  28. Madgwick JW, West JS, White RP, Semenov MA, Townsend JA, Turner JA, and Fitt BDL. (2011) Impacts of climate change on wheat anthesis and fusarium ear blight in the UK. European Journal of Plant Pathology 130, 117-131.
    CrossRef
  29. Ni?o M, Lee HJ, Kim J, Abdula1 S, Jung YJ, Kang KK, Nou I, and Cho YG. (2015) Enhancement of Rice Resistance to Bacterial Blight by Overexpressing BrCP3 Gene of Brassica rapa Plant Breed. Biotech 3, 355-365.
    CrossRef
  30. McCouch SR, Teytelman L, Xu YB, Lobos KB, Clare K, and Walton M et al. (2002) Development and mapping of 2,240 new SSR markers for rice (Oryza sativa L.). DNA Res 9, 199-207.
    Pubmed CrossRef
  31. Mew TW. (1987) Current status and future prospects of research on bacterial blight of rice. Annu. Rev. Phytopathol 25, 359-382.
    CrossRef
  32. Nogoy FM, Song JY, Ouk S, Rahimi S, Kwon SW, Kang KK, and Cho YG. (2016) Current Applicable DNA Markers for Marker Assisted Breeding in Abiotic and Biotic Stress Tolerance in Rice (Oryza sativa L.) Plant Breed. Biotech 4, 271-284.
    CrossRef
  33. Porter BW, Chittoor JM, Yano M, and Sasaki T et al. (2003) Development and mapping of markers linked to the rice bacterial blight resistance gene Xa7. Crop Sci 43, 1484-1492.
    CrossRef
  34. Porter JR, and Semenov MA. (2005) Crop responses to climatic variation. Philosophical Transactions of the Royal Society B:Biological Sciences 360, 2021-2035.
    Pubmed KoreaMed CrossRef
  35. Pradhan SK, Nayak DK, Mohanty S, Behera L, Barik SR, and Pandit E et al. (2015) Pyramiding of three bacterial blight resistance genes for broad-spectrum resistance in deepwater rice variety, Jalmagna. Rice 8, 19.
    Pubmed KoreaMed CrossRef
  36. Rajpurohit D, Kumar R, Kumar M, Paul P, and Awasthi AA et al. (2011) Pyramiding of two bacterial blight resistance and a semi dwarfing gene in Type 3 Basmati using marker-assisted selection. Euphytica 178, 111-126.
    CrossRef
  37. Ramkumar G, Madhav MS, Rama Devi SJS, Prasad MS, and Ravindra Babu V. (2015) Nucleotide variation and identification of novel blast resistance alleles of Pib by allele mining strategy. Physiol Mol Biol Plants 21, 301-304.
    Pubmed KoreaMed CrossRef
  38. Sasaki T, and Burr B. (2000) International Rice Genome Sequencing Project:The effort to completely sequence the rice genome. Curr. Opin. Plant Biol 3, 138-141.
    CrossRef
  39. Singh AK, Singh PK, Arya M, Singh NK, and Singh US. (2015) Molecular screening of blast resistance genes in rice using SSR markers. Plant Pathol. J 31, 12-24.
    Pubmed KoreaMed CrossRef
  40. Singh AK, Gopala Krishnan S, Singh VP, Prabhu KV, Mohapatra T, and Singh NK et al. (2011) Marker assisted selection:a paradigm shift in Basmati breeding. Indian Journal of Genetics and Plant Breeding 71, 1-9.
  41. Song JY, Lee GA, Choi YM, Lee S, Lee KB, and Bae CH et al. (2014) Blast resistant genes distribution and resistance reaction to blast in Korean landraces of rice (Oryza sativa L.). Korean J. Plant Res 27, 687-700.
    CrossRef
  42. Sun L, Su C, Wang C, Zai H, and Wan J. (2005) Mapping of a major resistance gene to brown planthopper in the rice cultivar Rathu Heenati. Breed. Sci 55, 391-396.
    CrossRef
  43. Talbot NJ, and Foster AJ. (2001) Genetics and genomics of the rice blast fungus Magnaporthe grisea:developing an experimental model for understanding fungal diseases of cereals. Adv. Bot. Res 34, 263-287.
    CrossRef
  44. Talbot NJ. (2003) On the trail of a cereal killer:investigating the biology of Magnaporthe grisea. Annu. Rev. Microbiol 57, 177-202.
    Pubmed CrossRef
  45. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, and Rozen SG. (2012) Primer3 - new capabilities and interfaces. Nucleic Acids Research 40, e115.
    CrossRef
  46. Wang Q, Lu C, and Zhang Q. (2005) Midday photoinhibition of two newly developed super-rice hybrids. Photosynthetica 43, 277-281.
    CrossRef
  47. Wang ZX, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, and Sasaki T. (1999) The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine- rich repeat class of plant disease resistance genes. Plant J 19, 55-64.
    Pubmed CrossRef
  48. Yaegashi H. (1994) Use of resistant varieties and disease control for paddy rice. Agric. Hortic 69, 149-154.
  49. Yamasaki M, Yoshimura A, and Yasui H. (2003) Genetic basis of ovicidal response to whitebacked planthopper (Sogatella furcifera Horvath) in rice (Oryza sativa L.). Molecular Breeding 12, 133-143.
    CrossRef
  50. Zeigler RS, Thome J, Nelson J, Levy M, and Correa-Victoria FJ. (1994) Lineage exclusion:A proposal for linking blast population analysis to resistance breeding. In Rice Blast Disease, Zeigler R.S, Leong S.A, and Teng P (eds.) , pp.267-292. CAB International, Wallingford, UK.
  51. Zhang G, Angeles ER, Abenes MLP, and Khush GS et al. (1996) RAPD and RFLP mapping of the bacterial blight resistance gene Xa-13 in rice. Theor. Appl. Genet 93, 65-70.
    Pubmed CrossRef
JPB
Vol 51. 2024

Stats or Metrics

Share this article on

  • line

Related articles in JPB

Journal of

Plant Biotechnology

pISSN 1229-2818
eISSN 2384-1397
qr-code Download