J Plant Biotechnol (2023) 50:034-044
Published online April 26, 2023
https://doi.org/10.5010/JPB.2023.50.005.034
© The Korean Society of Plant Biotechnology
박태호
대구대학교 원예학과
Correspondence to : e-mail: thzoo@daegu.ac.kr
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.
The tetraploid Solanum hjertingii, a wild tuber-bearing species from Mexico is a relative of potato, S. tuberosum. The species has been identified as a potential source of resistance to blackening for potato breeding. It does not exhibit enzymatic browning nor blackspot which are physiological disorders. However, due to their sexual incompatibility, somatic hybridization between S. hjertingii and S. tuberosum must be used to introduce various traits from this wild species into potato. After somatic hybridization, molecular markers are essential for selecting fusion products. In this study, the chloroplast genome of S. hjertingii was sequenced by next-generation sequencing technology and compared with those of other Solanum species to develop specific markers for S. hjertingii. The chloroplast genome has a total sequence length of 155,545 bp, and its size, gene content, order and orientation are similar to those of the other Solanum species. Phylogenic analysis including 15 other Solanaceae species grouped S. hjertingii with S. demissum, S. hougasii, and S. stoloniferum. After detailed comparisons of the chloroplast genome sequence with eight other Solanum species, we identified one InDel and seven SNPs specific to S. hjertingii. Based on these, five PCR-based markers were developed for discriminating S. hjertingii from other Solanum species. The results obtained in this study will aid in exploring the evolutionary aspects of Solanum species and accelerating breeding using S. hjertingii.
Keywords cpDNA, InDels, PCR-based marker, Potato, SNPs, Solanum hjertingii
재배종 감자(
속씨식물의 엽록체 유전체는 일반적으로 원형의 이중가닥 분자구조를 가지고 있으며, 그 크기는 약 115-165 kb이고 large single copy (LSC), small single copy (SSC), 두 개의 inverted repeats (IRs) 영역을 가지는 전형적인 4분할 구조로 되어 있다(Yurina and Odintsova 1998). 재배종 감자와 감자의 근연야생종 또한 앞서 보고된 바와 같이 이와 매우 유사한 것으로 확인되었다(Palmer 1991; Raubeson and Jansen 2005; Saski et al. 2005; Sugiura et al. 1998) (Table 1). 그러나, 엽록체 유전체는 유전자의 재배열, 역위 등과 같은 구조적 변화에 의한 변이 발생으로 종간의 전체 엽록체 유전체를 비교해보면 종간의 염기서열 차이에 의해 발생하는 다수의 InDel과 SNP가 존재함을 알 수 있다(Calsa Junior et al. 2004; Cho et al. 2015; Jheng et al. 2012; Kim et al. 2005, 2018; Kim and Park 2019, 2020a, 2020b; Park 2021, 2022a, 2022b; Saski et al. 2005). 따라서, 본 연구에서는 Park (2022c)에 의해 알려진
Table 1 Chloroplast genome sequence characteristics of
Species | Accession no. | Total Length (bp) | GC content (%) | Total No. of genes | No. of tRNA | No. of rRNA | Reference |
---|---|---|---|---|---|---|---|
MK690623 | 155,545 | 37.88 | 134 | 36 | 4 | In this study | |
MK036506 | 155,570 | 37.84 | 135 | 36 | 4 | Park (2022b) | |
MK036507 | 155,531 | 37.87 | 135 | 36 | 4 | Park (2022a) | |
MK036508 | 155,558 | 37.87 | 135 | 36 | 4 | Park (2021) | |
MF471372 | 155,549 | 37.87 | 135 | 36 | 4 | Kim and Park (2020b) | |
MF471373 | 155,567 | 37.87 | 135 | 36 | 4 | Kim and Park (2020a) | |
MF471371 | 155,532 | 37.89 | 136 | 36 | 4 | Kim and Park (2019) | |
KY419708 | 155,533 | 37.88 | 137 | 39 | 4 | Kim et al. (2018) | |
KM489054 | 155,525 | 37.88 | 133 | 33 | 4 | Cho et al. (2016) | |
KM489055 | 155,432 | 37.90 | 139 | 39 | 4 | Cho and Park (2016) | |
KM489056 | 155,312 | 37.88 | 130 | 30 | 4 | Cho et al. (2016) | |
DQ347958 | 155,371 | 37.88 | 133 | 30 | 4 | Daniell et al. (2006) | |
NC008096 | 155,296 | 37.88 | 131 | 36 | 4 | Gargano et al. (2005) |
*The data have been partially adopted from Park (2022b).
DNA 분리에는 Genomic DNA Extraction kit (Plants) (RBC, New Taipei City, Taiwan)가 사용되었으며, 모든 식물재료의 기내식물체를 계대배양 후 약 100 mg의 유식물체를 채취하여 수행되었다.
Table 2 Primers for generating S. hjertingii specific and non-specific markers
InDel/SNP | System | Primer sequence (5’ → 3’) | Touchdown PCR | Annealing temperature (°C) | Amplicon size |
---|---|---|---|---|---|
SH1-16_InDel1 | Forward primer | AGTTTCTTATGTTTTAATTCC | No | 58 °C | 531 bp (insert sequence ‘tttattagg’ specific to |
Reverse primer | TCCTAATAAACCTAATAAAAC | ||||
SH1-16_SNP1 | Forward inner primer | GTATGAATAAAGGATCCATGGATGCAA | No | 54 °C | 313 bp (from two outer primers) 198 bp (A allele specific to 171 bp (G allele non- specific to |
Reverse inner primer | TAGTTACGATTAGAAATAAACTTTCTCTC | ||||
Forward outer primer | AGAACAAATACACATTATCGTGATTTGT | ||||
Reverse outer primer | TTTCTATTTAATAGGATCCTGAGGAAAA | ||||
SH1-16_SNP3 | Forward inner primer | GGACCTTTTAACCTTTTAAAAACCCTTGTG | Yes | 70 °C to 61 °C | 336 bp (from two outer primers) 246 bp (A allele specific to 181 bp (G allele non- specific to |
Reverse inner primer | AAGTCGTTTTTGGATCAATCCAAAGACT | ||||
Forward outer primer | CGCCAACAGTCAAATAATTAGTGAACCT | ||||
Reverse outer primer | TAGTAATGTCTAATGGCTACTCGCGTGA | ||||
SH1-16_SNP5 | Forward inner primer | AGAATCAGAACGTTCATTCAATTGAA | Yes | 70 °C to 61 °C | 397 bp (from two outer primers) 260 bp (C allele specific to 190 bp (A allele non- specific to |
Reverse inner primer | TTTATGGTTCTTTCATATACGTTTCCG | ||||
Forward outer primer | CGATTCCTTATAAAAGATATTCAGTCCG | ||||
Reverse outer primer | ATTGAACTAAAAAAAATTCTTGCTTTCG | ||||
SH1-16_SNP6 | Forward inner primer | CTATTAGTAATTTGTATATCTTGTTGGG | No | 54 °C | 214 bp (from two outer primers) 144 bp (A allele non- specific to |
Reverse inner primer | TTAGAAAACTACAAAGTTTAGTTATCTCTT | ||||
Forward outer primer | AGCTAGAATAGTCAATCTTAAGTTAAGAT | ||||
Reverse outer primer | AAATTTAGGACAATACAATAAAGGAC |
감자와 감자의 근연야생종의 엽록체 전장유전체의 전체 염기서열을 구명하고 이 정보를 이용하여 유전자형을 구분할 수 있는 감자 또는 감자의 근연야생종 특이적인 분자마커의 개발은 핵 내의 염색체 DNA를 대상으로 분자마커를 개발하는 것과 함께 감자의 신품종 육성과
멕시코 유래의 4배체 감자 근연야생종 중 하나인
이 성과는 정부(과학기술정보통신부)의 재원으로 한국연구재단의 지원을 받아 수행된 연구임(No. NRF-2021R1F1A1045981).
J Plant Biotechnol 2023; 50(1): 34-44
Published online April 26, 2023 https://doi.org/10.5010/JPB.2023.50.005.034
Copyright © The Korean Society of Plant Biotechnology.
박태호
대구대학교 원예학과
Tae-Ho Park
(Department of Horticulture, Daegu University, Gyeongsan 38453, South Korea)
Correspondence to:e-mail: thzoo@daegu.ac.kr
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.
The tetraploid Solanum hjertingii, a wild tuber-bearing species from Mexico is a relative of potato, S. tuberosum. The species has been identified as a potential source of resistance to blackening for potato breeding. It does not exhibit enzymatic browning nor blackspot which are physiological disorders. However, due to their sexual incompatibility, somatic hybridization between S. hjertingii and S. tuberosum must be used to introduce various traits from this wild species into potato. After somatic hybridization, molecular markers are essential for selecting fusion products. In this study, the chloroplast genome of S. hjertingii was sequenced by next-generation sequencing technology and compared with those of other Solanum species to develop specific markers for S. hjertingii. The chloroplast genome has a total sequence length of 155,545 bp, and its size, gene content, order and orientation are similar to those of the other Solanum species. Phylogenic analysis including 15 other Solanaceae species grouped S. hjertingii with S. demissum, S. hougasii, and S. stoloniferum. After detailed comparisons of the chloroplast genome sequence with eight other Solanum species, we identified one InDel and seven SNPs specific to S. hjertingii. Based on these, five PCR-based markers were developed for discriminating S. hjertingii from other Solanum species. The results obtained in this study will aid in exploring the evolutionary aspects of Solanum species and accelerating breeding using S. hjertingii.
Keywords: cpDNA, InDels, PCR-based marker, Potato, SNPs, Solanum hjertingii
재배종 감자(
속씨식물의 엽록체 유전체는 일반적으로 원형의 이중가닥 분자구조를 가지고 있으며, 그 크기는 약 115-165 kb이고 large single copy (LSC), small single copy (SSC), 두 개의 inverted repeats (IRs) 영역을 가지는 전형적인 4분할 구조로 되어 있다(Yurina and Odintsova 1998). 재배종 감자와 감자의 근연야생종 또한 앞서 보고된 바와 같이 이와 매우 유사한 것으로 확인되었다(Palmer 1991; Raubeson and Jansen 2005; Saski et al. 2005; Sugiura et al. 1998) (Table 1). 그러나, 엽록체 유전체는 유전자의 재배열, 역위 등과 같은 구조적 변화에 의한 변이 발생으로 종간의 전체 엽록체 유전체를 비교해보면 종간의 염기서열 차이에 의해 발생하는 다수의 InDel과 SNP가 존재함을 알 수 있다(Calsa Junior et al. 2004; Cho et al. 2015; Jheng et al. 2012; Kim et al. 2005, 2018; Kim and Park 2019, 2020a, 2020b; Park 2021, 2022a, 2022b; Saski et al. 2005). 따라서, 본 연구에서는 Park (2022c)에 의해 알려진
Table 1 . Chloroplast genome sequence characteristics of
Species | Accession no. | Total Length (bp) | GC content (%) | Total No. of genes | No. of tRNA | No. of rRNA | Reference |
---|---|---|---|---|---|---|---|
MK690623 | 155,545 | 37.88 | 134 | 36 | 4 | In this study | |
MK036506 | 155,570 | 37.84 | 135 | 36 | 4 | Park (2022b) | |
MK036507 | 155,531 | 37.87 | 135 | 36 | 4 | Park (2022a) | |
MK036508 | 155,558 | 37.87 | 135 | 36 | 4 | Park (2021) | |
MF471372 | 155,549 | 37.87 | 135 | 36 | 4 | Kim and Park (2020b) | |
MF471373 | 155,567 | 37.87 | 135 | 36 | 4 | Kim and Park (2020a) | |
MF471371 | 155,532 | 37.89 | 136 | 36 | 4 | Kim and Park (2019) | |
KY419708 | 155,533 | 37.88 | 137 | 39 | 4 | Kim et al. (2018) | |
KM489054 | 155,525 | 37.88 | 133 | 33 | 4 | Cho et al. (2016) | |
KM489055 | 155,432 | 37.90 | 139 | 39 | 4 | Cho and Park (2016) | |
KM489056 | 155,312 | 37.88 | 130 | 30 | 4 | Cho et al. (2016) | |
DQ347958 | 155,371 | 37.88 | 133 | 30 | 4 | Daniell et al. (2006) | |
NC008096 | 155,296 | 37.88 | 131 | 36 | 4 | Gargano et al. (2005) |
*The data have been partially adopted from Park (2022b)..
DNA 분리에는 Genomic DNA Extraction kit (Plants) (RBC, New Taipei City, Taiwan)가 사용되었으며, 모든 식물재료의 기내식물체를 계대배양 후 약 100 mg의 유식물체를 채취하여 수행되었다.
Table 2 . Primers for generating S. hjertingii specific and non-specific markers.
InDel/SNP | System | Primer sequence (5’ → 3’) | Touchdown PCR | Annealing temperature (°C) | Amplicon size |
---|---|---|---|---|---|
SH1-16_InDel1 | Forward primer | AGTTTCTTATGTTTTAATTCC | No | 58 °C | 531 bp (insert sequence ‘tttattagg’ specific to |
Reverse primer | TCCTAATAAACCTAATAAAAC | ||||
SH1-16_SNP1 | Forward inner primer | GTATGAATAAAGGATCCATGGATGCAA | No | 54 °C | 313 bp (from two outer primers) 198 bp (A allele specific to 171 bp (G allele non- specific to |
Reverse inner primer | TAGTTACGATTAGAAATAAACTTTCTCTC | ||||
Forward outer primer | AGAACAAATACACATTATCGTGATTTGT | ||||
Reverse outer primer | TTTCTATTTAATAGGATCCTGAGGAAAA | ||||
SH1-16_SNP3 | Forward inner primer | GGACCTTTTAACCTTTTAAAAACCCTTGTG | Yes | 70 °C to 61 °C | 336 bp (from two outer primers) 246 bp (A allele specific to 181 bp (G allele non- specific to |
Reverse inner primer | AAGTCGTTTTTGGATCAATCCAAAGACT | ||||
Forward outer primer | CGCCAACAGTCAAATAATTAGTGAACCT | ||||
Reverse outer primer | TAGTAATGTCTAATGGCTACTCGCGTGA | ||||
SH1-16_SNP5 | Forward inner primer | AGAATCAGAACGTTCATTCAATTGAA | Yes | 70 °C to 61 °C | 397 bp (from two outer primers) 260 bp (C allele specific to 190 bp (A allele non- specific to |
Reverse inner primer | TTTATGGTTCTTTCATATACGTTTCCG | ||||
Forward outer primer | CGATTCCTTATAAAAGATATTCAGTCCG | ||||
Reverse outer primer | ATTGAACTAAAAAAAATTCTTGCTTTCG | ||||
SH1-16_SNP6 | Forward inner primer | CTATTAGTAATTTGTATATCTTGTTGGG | No | 54 °C | 214 bp (from two outer primers) 144 bp (A allele non- specific to |
Reverse inner primer | TTAGAAAACTACAAAGTTTAGTTATCTCTT | ||||
Forward outer primer | AGCTAGAATAGTCAATCTTAAGTTAAGAT | ||||
Reverse outer primer | AAATTTAGGACAATACAATAAAGGAC |
감자와 감자의 근연야생종의 엽록체 전장유전체의 전체 염기서열을 구명하고 이 정보를 이용하여 유전자형을 구분할 수 있는 감자 또는 감자의 근연야생종 특이적인 분자마커의 개발은 핵 내의 염색체 DNA를 대상으로 분자마커를 개발하는 것과 함께 감자의 신품종 육성과
멕시코 유래의 4배체 감자 근연야생종 중 하나인
이 성과는 정부(과학기술정보통신부)의 재원으로 한국연구재단의 지원을 받아 수행된 연구임(No. NRF-2021R1F1A1045981).
Table 1 . Chloroplast genome sequence characteristics of
Species | Accession no. | Total Length (bp) | GC content (%) | Total No. of genes | No. of tRNA | No. of rRNA | Reference |
---|---|---|---|---|---|---|---|
MK690623 | 155,545 | 37.88 | 134 | 36 | 4 | In this study | |
MK036506 | 155,570 | 37.84 | 135 | 36 | 4 | Park (2022b) | |
MK036507 | 155,531 | 37.87 | 135 | 36 | 4 | Park (2022a) | |
MK036508 | 155,558 | 37.87 | 135 | 36 | 4 | Park (2021) | |
MF471372 | 155,549 | 37.87 | 135 | 36 | 4 | Kim and Park (2020b) | |
MF471373 | 155,567 | 37.87 | 135 | 36 | 4 | Kim and Park (2020a) | |
MF471371 | 155,532 | 37.89 | 136 | 36 | 4 | Kim and Park (2019) | |
KY419708 | 155,533 | 37.88 | 137 | 39 | 4 | Kim et al. (2018) | |
KM489054 | 155,525 | 37.88 | 133 | 33 | 4 | Cho et al. (2016) | |
KM489055 | 155,432 | 37.90 | 139 | 39 | 4 | Cho and Park (2016) | |
KM489056 | 155,312 | 37.88 | 130 | 30 | 4 | Cho et al. (2016) | |
DQ347958 | 155,371 | 37.88 | 133 | 30 | 4 | Daniell et al. (2006) | |
NC008096 | 155,296 | 37.88 | 131 | 36 | 4 | Gargano et al. (2005) |
*The data have been partially adopted from Park (2022b)..
Table 2 . Primers for generating S. hjertingii specific and non-specific markers.
InDel/SNP | System | Primer sequence (5’ → 3’) | Touchdown PCR | Annealing temperature (°C) | Amplicon size |
---|---|---|---|---|---|
SH1-16_InDel1 | Forward primer | AGTTTCTTATGTTTTAATTCC | No | 58 °C | 531 bp (insert sequence ‘tttattagg’ specific to |
Reverse primer | TCCTAATAAACCTAATAAAAC | ||||
SH1-16_SNP1 | Forward inner primer | GTATGAATAAAGGATCCATGGATGCAA | No | 54 °C | 313 bp (from two outer primers) 198 bp (A allele specific to 171 bp (G allele non- specific to |
Reverse inner primer | TAGTTACGATTAGAAATAAACTTTCTCTC | ||||
Forward outer primer | AGAACAAATACACATTATCGTGATTTGT | ||||
Reverse outer primer | TTTCTATTTAATAGGATCCTGAGGAAAA | ||||
SH1-16_SNP3 | Forward inner primer | GGACCTTTTAACCTTTTAAAAACCCTTGTG | Yes | 70 °C to 61 °C | 336 bp (from two outer primers) 246 bp (A allele specific to 181 bp (G allele non- specific to |
Reverse inner primer | AAGTCGTTTTTGGATCAATCCAAAGACT | ||||
Forward outer primer | CGCCAACAGTCAAATAATTAGTGAACCT | ||||
Reverse outer primer | TAGTAATGTCTAATGGCTACTCGCGTGA | ||||
SH1-16_SNP5 | Forward inner primer | AGAATCAGAACGTTCATTCAATTGAA | Yes | 70 °C to 61 °C | 397 bp (from two outer primers) 260 bp (C allele specific to 190 bp (A allele non- specific to |
Reverse inner primer | TTTATGGTTCTTTCATATACGTTTCCG | ||||
Forward outer primer | CGATTCCTTATAAAAGATATTCAGTCCG | ||||
Reverse outer primer | ATTGAACTAAAAAAAATTCTTGCTTTCG | ||||
SH1-16_SNP6 | Forward inner primer | CTATTAGTAATTTGTATATCTTGTTGGG | No | 54 °C | 214 bp (from two outer primers) 144 bp (A allele non- specific to |
Reverse inner primer | TTAGAAAACTACAAAGTTTAGTTATCTCTT | ||||
Forward outer primer | AGCTAGAATAGTCAATCTTAAGTTAAGAT | ||||
Reverse outer primer | AAATTTAGGACAATACAATAAAGGAC |
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