Development of PCR-based markers for discriminating Solanum berthaultii using its complete chloroplast genome sequence

Abstract

Solanum berthaultii is one of the wild diploid Solanum species, which is an excellent resource in potato breeding owing to its resistance to several important pathogens. On the other hand, sexual hybridization between S. berthaultii and S. tuberosum (potato) is limited because of their sexual incompatibility. Therefore, cell fusion can be used to introgress various novel traits from this wild species into the cultivated potatoes. After cell fusion, it is crucial to identify fusion products with the aid of molecular markers. In this study, the chloroplast genome sequence of S. berthaultii obtained by next-generation sequencing technology was described and compared with those of five other Solanum species to develop S. berthaultii specific markers. A total sequence length of the chloroplast genome is 155,533 bp. The structural organization of the chloroplast genome is similar to those of the five other Solanum species. Phylogenic analysis with 25 other Solanaceae species revealed that S. berthaultii is most closely located with S. tuberosum. Additional comparison of the chloroplast genome sequence with those of the five Solanum species revealed 25 SNPs specific to S. berthaultii. Based on these SNPs, six PCR-based markers for differentiating S. berthaultii from other Solanum species were developed. These markers will facilitate the selection of fusion products and accelerate potato breeding using S. berthaultii.

PCR-based markers developed by comparison of complete chloroplast genome sequences discriminateSolanum chacoensefrom otherSolanumspecies

“…To develop molecular markers which could discriminate S. chacoense cytoplasm, the complete S. chacoense cpDNA sequence was aligned with those of the five other species in the Solanum genus. A number of InDels or SNPs was previously reported by the alignment of the cpDNA sequences in the Solanum species Chung et al, 2006;Kim et al, 2018). Overall 280 and 1,490 InDels and SNPs through the whole cpDNA sequences were detected by the multiple alignment with six species generated using ClustalW2 in EMBL (https://www.ebi.ac.uk/Tools/msa/ clustalw2), respectively.…”

“…For the cpDNA sequencing of S. chacoense, the total genomic DNA of the line SC3-12 of S. chacoense was extracted and sequencing was conducted using the Illumina Daniell et al (2006) *The data have been partially adopted from Kim et al (2018).…”

“…Molecular markers can effectively be developed by the application of PCR on the regions covering SNPs followed by the digestion of PCR product with the appropriate restriction enzymes that called the cleaved amplified polymorphism sequence (CAPS) marker (Konieczny and Ausubel 1993). The CAPS markers were widely used to compare with polymorphism in different genotypes of Solanum species Kim et al 2018;Park et al 2005;Smilde et al 2005;Uribe et al 2014) and other plant species (Komori and Nitta 2005;Uncu et al 2015;Wang et al 2017). Therefore, 17 primer pairs which can amplify regions including each SNPs were designed and the proper restriction enzymes had been identified.…”

“…Most cpDNA contain approximately 110~130 genes encoding diverse unique proteins, rRNAs, and tRNAs. Several cpDNA sequences of Solanum species such as S. berthaultii (KY419708, Kim et al 2018), S. tuberosum (KM489056, and (NC008096, Gargano et al 2005), S. commersonii (KM489055, , S. bulbocastanum (NC007943, Daniell et al 2006), and S. nigrum (KM489054, Cho and Park 2016) have previously been completed (Table 1). Their size and gene organization are very similar and their structures are exactly same with two copies of IRs, LSC, and SSC regions (Palmer 1991;Raubeson and Jansen 2005;Saski et al 2005;Sugiura et al 1998).…”

Development ofSolanum hougasii-specific markers using the complete chloroplast genome sequences ofSolanumspecies

“…GC의 함량은 37.87%이며, 유전자, tRNA, rRNA 등의 개수, 순 서 등이 모두 다른 Solanum 속의 종들과 유사한 것으로 나타 났을 뿐 만 아니라, S. stoloniferum과는 거의 일치하였다(Table 1)Kim et al 2018;Kim and Park, 2019). 체계적인 분류의 결과를 보여주었을 뿐 만 아니라 높은 부트스트랩(bootstrap) 값은 계통수의 결과를 뒷받침 해 주었다.…”