J Plant Biotechnol (2023) 50:045-055
Published online April 26, 2023
https://doi.org/10.5010/JPB.2023.50.006.045
© 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 diploid Solanum cardiophyllum, a wild tuberbearing species from Mexico is one of the relatives to potato, S. tuberosum. It has been identified as a source of resistance to crucial pathogens and insects such as Phytophthora infestans, Potato virus Y, Colorado potato beetle, etc. and is widely used for potato breeding. However, the sexual hybridization between S. cardiophyllum and S. tuberosum is limited due to their incompatibility. Therefore, somatic hybridization can introduce beneficial traits from this wild species into the potato. After somatic hybridization, selecting fusion products using molecular markers is essential. In the current study, the chloroplast genome of S. cardiophyllum was sequenced by next-generation sequencing technology and compared with those of other Solanum species to develop S. cardiophyllum-specific markers. The total length of the S. cardiophyllum chloroplast genome was 155,570 bp and its size, gene content, order and orientation were similar to those of the other Solanum species. Phylogenic analysis with 32 other Solanaceae species revealed that S. cardiophyllum was expectedly grouped with other Solanum species and most closely located with S. bulbocastanum. Through detailed comparisons of the chloroplast genome sequences of eight Solanum species, we identified 13 SNPs specific to S. cardiophyllum. Further, four SNP-specific PCR markers were developed for discriminating S. cardiophyllum from other Solanum species. The results obtained in this study would help to explore the evolutionary aspects of Solanum species and accelerate breeding using S. cardiophyllum.
Keywords cpDNA, PCR-based marker, Potato, SNPs, Solanum cardiophyllum
멕시코에 자생하는
일반적으로 속씨식물의 엽록체 유전체는 large single copy (LSC) 영역과 small single copy (SSC) 영역이 각각 하나씩 그리고 두 개의 inverted repeats (IRs) 영역으로 구성된 4분할의 원형 이중가닥의 분자구조를 가지고 있다(Yurina and Odintosova 1998). 재배종 감자 및 감자의 근연야생종을 대상으로 한 엽록체 유전체 연구의 결과가 보고된 바 있으며(Table 1), 이들
Table 1 Comparative analysis of the chloroplast genome sequence of
Species | Accession no. | Total Length (bp) | GC content (%) | Total No. of genes | No. of tRNA | No. of rRNA | Reference |
---|---|---|---|---|---|---|---|
MK690622 | 155,570 | 37.90 | 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 (2021b) | |
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 분리는 Genomic DNA Extraction kit (Plants) (RBC, New Taipei City, Taiwan)를 이용하여 모든 식물재료의 기내식물체를 계대배양 후 약 3주 이내의 유식물체 약 100 mg를 채취하여 수행되었다.
Table 2 Primer information used to generate
SNP | System | Primer sequence (5’ → 3’) | Touchdown PCR | Annealing temperature (°C) | Amplicon size |
---|---|---|---|---|---|
SC1-9_SNP2 | Forward inner primer | AAAAGCAATCTATATTGTCAGAGAATACAG | No | 58°C | 256 bp (from two outer primers) 136 bp (T allele non-specific to |
Reverse inner primer | GTTCGAATCCTTCCGTCCCAGAACGTA | ||||
Forward outer primer | AAACGATTCATCGAAGAAAAAAATCAGA | ||||
Reverse outer primer | ATTTGGGCTAGAGTTGACAAACAAACAA | ||||
SC1-9_SNP6 | Forward inner primer | TTTTATTGTCGTTTTATGTTCTATTCGAGG | Yes | 72°C to 63°C | 336 bp (from two outer primers) 210 bp (T allele specific to |
Reverse inner primer | GTTTTCGGAGAGCACAACCTGTGTCGA | ||||
Forward outer primer | TCATTGTTCAAAAAATGATTCGCAGAGA | ||||
Reverse outer primer | CCCCCCAAGATAAATTGTTAGACGGATA | ||||
SC1-9_SNP9 | Forward inner primer | TTATTAGAGTTGAGATAACTTTGCTCGC | No | 62°C | 306 bp (from two outer primers) 169 bp (C allele specific to |
Reverse inner primer | TTTTTTCTTTGTAAAGAGATCCTAATCGT | ||||
Forward outer primer | TCTGTTTCATCTTTTTAGGTTTATGCTC | ||||
Reverse outer primer | TCAATTAGAATAATTTATGGTTGGCTTG | ||||
SC1-9_SNP10 | Forward inner primer | ATGTTAATAAATTCCAAAATCCATTTAGC | Yes | 72°C to 63°C | 404 bp (from two outer primers) 252 bp (T allele specific to |
Reverse inner primer | AAAAGACAGTCGTCGCTACTGGAAGA | ||||
Forward outer primer | GTAATGTAGGCTTAGCCGTTTTAGAACC | ||||
Reverse outer primer | TTCGCTTTGAAGAAACTATTCCCTAGAT |
감자의 신품종 육성을 위하여 다양한 유용 형질을 보유한 감자의 근연야생종을 활용한 연구가 지속적으로 이루어지고 있다. 하지만, 많은 근연야생종이 재배종 감자와 배수성 및 EBN의 차이로 인하여 교배육종의 방법으로는 유용 형질의 도입이 쉽지 않아 체세포융합에 의한 체세포잡종을 만들어 신품종 육성에 활용하고 있으며, 그 과정에서 체세포잡종의 유전자형 확인이 필요하다. 유전자형의 확인은 서로 다른 종의 염색체의 조합 뿐만 아니라 미토콘드리아 및 엽록체 유전체의 전달 확인도 필요한데, 다양한 식물종의 체세포융합 과정에서 미토콘드리아 유전체는 높은 빈도의 재조합 발생이 이루어지는 것으로 알려져 있으나 엽록체 유전체의 경우 한 쪽의 엽록체 유전체만 무작위로 전달이 되는 경우 뿐 만 아니라 양친의 유전체 모두가 전달되는 경우도 있었다(Chen et al. 2013; Cho et al. 2016; Lössl et al. 2000; Mohapatra et al. 1998; Smyda-Dajmund et al. 2016; Wang et al. 2011; Xiang et al. 2004).따라서, 본 연구의 결과인 SNP 기반의 분자마커는 감자의 신품종을 육성 과정에서
멕시코 유래의 2배체 감자 근연야생종
이 성과는 정부(과학기술정보통신부)의 재원으로 한국연구재단의 지원을 받아 수행된 연구임(No. NRF-2021R1F1A1045981).
J Plant Biotechnol 2023; 50(1): 45-55
Published online April 26, 2023 https://doi.org/10.5010/JPB.2023.50.006.045
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 diploid Solanum cardiophyllum, a wild tuberbearing species from Mexico is one of the relatives to potato, S. tuberosum. It has been identified as a source of resistance to crucial pathogens and insects such as Phytophthora infestans, Potato virus Y, Colorado potato beetle, etc. and is widely used for potato breeding. However, the sexual hybridization between S. cardiophyllum and S. tuberosum is limited due to their incompatibility. Therefore, somatic hybridization can introduce beneficial traits from this wild species into the potato. After somatic hybridization, selecting fusion products using molecular markers is essential. In the current study, the chloroplast genome of S. cardiophyllum was sequenced by next-generation sequencing technology and compared with those of other Solanum species to develop S. cardiophyllum-specific markers. The total length of the S. cardiophyllum chloroplast genome was 155,570 bp and its size, gene content, order and orientation were similar to those of the other Solanum species. Phylogenic analysis with 32 other Solanaceae species revealed that S. cardiophyllum was expectedly grouped with other Solanum species and most closely located with S. bulbocastanum. Through detailed comparisons of the chloroplast genome sequences of eight Solanum species, we identified 13 SNPs specific to S. cardiophyllum. Further, four SNP-specific PCR markers were developed for discriminating S. cardiophyllum from other Solanum species. The results obtained in this study would help to explore the evolutionary aspects of Solanum species and accelerate breeding using S. cardiophyllum.
Keywords: cpDNA, PCR-based marker, Potato, SNPs, Solanum cardiophyllum
멕시코에 자생하는
일반적으로 속씨식물의 엽록체 유전체는 large single copy (LSC) 영역과 small single copy (SSC) 영역이 각각 하나씩 그리고 두 개의 inverted repeats (IRs) 영역으로 구성된 4분할의 원형 이중가닥의 분자구조를 가지고 있다(Yurina and Odintosova 1998). 재배종 감자 및 감자의 근연야생종을 대상으로 한 엽록체 유전체 연구의 결과가 보고된 바 있으며(Table 1), 이들
Table 1 . Comparative analysis of the chloroplast genome sequence of
Species | Accession no. | Total Length (bp) | GC content (%) | Total No. of genes | No. of tRNA | No. of rRNA | Reference |
---|---|---|---|---|---|---|---|
MK690622 | 155,570 | 37.90 | 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 (2021b) | |
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 분리는 Genomic DNA Extraction kit (Plants) (RBC, New Taipei City, Taiwan)를 이용하여 모든 식물재료의 기내식물체를 계대배양 후 약 3주 이내의 유식물체 약 100 mg를 채취하여 수행되었다.
Table 2 . Primer information used to generate
SNP | System | Primer sequence (5’ → 3’) | Touchdown PCR | Annealing temperature (°C) | Amplicon size |
---|---|---|---|---|---|
SC1-9_SNP2 | Forward inner primer | AAAAGCAATCTATATTGTCAGAGAATACAG | No | 58°C | 256 bp (from two outer primers) 136 bp (T allele non-specific to |
Reverse inner primer | GTTCGAATCCTTCCGTCCCAGAACGTA | ||||
Forward outer primer | AAACGATTCATCGAAGAAAAAAATCAGA | ||||
Reverse outer primer | ATTTGGGCTAGAGTTGACAAACAAACAA | ||||
SC1-9_SNP6 | Forward inner primer | TTTTATTGTCGTTTTATGTTCTATTCGAGG | Yes | 72°C to 63°C | 336 bp (from two outer primers) 210 bp (T allele specific to |
Reverse inner primer | GTTTTCGGAGAGCACAACCTGTGTCGA | ||||
Forward outer primer | TCATTGTTCAAAAAATGATTCGCAGAGA | ||||
Reverse outer primer | CCCCCCAAGATAAATTGTTAGACGGATA | ||||
SC1-9_SNP9 | Forward inner primer | TTATTAGAGTTGAGATAACTTTGCTCGC | No | 62°C | 306 bp (from two outer primers) 169 bp (C allele specific to |
Reverse inner primer | TTTTTTCTTTGTAAAGAGATCCTAATCGT | ||||
Forward outer primer | TCTGTTTCATCTTTTTAGGTTTATGCTC | ||||
Reverse outer primer | TCAATTAGAATAATTTATGGTTGGCTTG | ||||
SC1-9_SNP10 | Forward inner primer | ATGTTAATAAATTCCAAAATCCATTTAGC | Yes | 72°C to 63°C | 404 bp (from two outer primers) 252 bp (T allele specific to |
Reverse inner primer | AAAAGACAGTCGTCGCTACTGGAAGA | ||||
Forward outer primer | GTAATGTAGGCTTAGCCGTTTTAGAACC | ||||
Reverse outer primer | TTCGCTTTGAAGAAACTATTCCCTAGAT |
감자의 신품종 육성을 위하여 다양한 유용 형질을 보유한 감자의 근연야생종을 활용한 연구가 지속적으로 이루어지고 있다. 하지만, 많은 근연야생종이 재배종 감자와 배수성 및 EBN의 차이로 인하여 교배육종의 방법으로는 유용 형질의 도입이 쉽지 않아 체세포융합에 의한 체세포잡종을 만들어 신품종 육성에 활용하고 있으며, 그 과정에서 체세포잡종의 유전자형 확인이 필요하다. 유전자형의 확인은 서로 다른 종의 염색체의 조합 뿐만 아니라 미토콘드리아 및 엽록체 유전체의 전달 확인도 필요한데, 다양한 식물종의 체세포융합 과정에서 미토콘드리아 유전체는 높은 빈도의 재조합 발생이 이루어지는 것으로 알려져 있으나 엽록체 유전체의 경우 한 쪽의 엽록체 유전체만 무작위로 전달이 되는 경우 뿐 만 아니라 양친의 유전체 모두가 전달되는 경우도 있었다(Chen et al. 2013; Cho et al. 2016; Lössl et al. 2000; Mohapatra et al. 1998; Smyda-Dajmund et al. 2016; Wang et al. 2011; Xiang et al. 2004).따라서, 본 연구의 결과인 SNP 기반의 분자마커는 감자의 신품종을 육성 과정에서
멕시코 유래의 2배체 감자 근연야생종
이 성과는 정부(과학기술정보통신부)의 재원으로 한국연구재단의 지원을 받아 수행된 연구임(No. NRF-2021R1F1A1045981).
Table 1 . Comparative analysis of the chloroplast genome sequence of
Species | Accession no. | Total Length (bp) | GC content (%) | Total No. of genes | No. of tRNA | No. of rRNA | Reference |
---|---|---|---|---|---|---|---|
MK690622 | 155,570 | 37.90 | 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 (2021b) | |
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 . Primer information used to generate
SNP | System | Primer sequence (5’ → 3’) | Touchdown PCR | Annealing temperature (°C) | Amplicon size |
---|---|---|---|---|---|
SC1-9_SNP2 | Forward inner primer | AAAAGCAATCTATATTGTCAGAGAATACAG | No | 58°C | 256 bp (from two outer primers) 136 bp (T allele non-specific to |
Reverse inner primer | GTTCGAATCCTTCCGTCCCAGAACGTA | ||||
Forward outer primer | AAACGATTCATCGAAGAAAAAAATCAGA | ||||
Reverse outer primer | ATTTGGGCTAGAGTTGACAAACAAACAA | ||||
SC1-9_SNP6 | Forward inner primer | TTTTATTGTCGTTTTATGTTCTATTCGAGG | Yes | 72°C to 63°C | 336 bp (from two outer primers) 210 bp (T allele specific to |
Reverse inner primer | GTTTTCGGAGAGCACAACCTGTGTCGA | ||||
Forward outer primer | TCATTGTTCAAAAAATGATTCGCAGAGA | ||||
Reverse outer primer | CCCCCCAAGATAAATTGTTAGACGGATA | ||||
SC1-9_SNP9 | Forward inner primer | TTATTAGAGTTGAGATAACTTTGCTCGC | No | 62°C | 306 bp (from two outer primers) 169 bp (C allele specific to |
Reverse inner primer | TTTTTTCTTTGTAAAGAGATCCTAATCGT | ||||
Forward outer primer | TCTGTTTCATCTTTTTAGGTTTATGCTC | ||||
Reverse outer primer | TCAATTAGAATAATTTATGGTTGGCTTG | ||||
SC1-9_SNP10 | Forward inner primer | ATGTTAATAAATTCCAAAATCCATTTAGC | Yes | 72°C to 63°C | 404 bp (from two outer primers) 252 bp (T allele specific to |
Reverse inner primer | AAAAGACAGTCGTCGCTACTGGAAGA | ||||
Forward outer primer | GTAATGTAGGCTTAGCCGTTTTAGAACC | ||||
Reverse outer primer | TTCGCTTTGAAGAAACTATTCCCTAGAT |
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