[논문]엽록체 전장유전체 비교를 통한 PCR 기반의 Solanum brevicaule 특이적 분자마커 개발

박태호

doi:10.5010/jpb.2022.49.1.030

[국내논문] 엽록체 전장유전체 비교를 통한 PCR 기반의 Solanum brevicaule 특이적 분자마커 개발
Development of PCR-based markers specific to Solanum brevicaule by using the complete chloroplast genome sequences of Solanum species 원문보기

Journal of plant biotechnology = 식물생명공학회지, v.49 no.1, 2022년, pp.30 - 38

초록
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Solanum brevicaule는 괴경을 형성하는 감자 야생종 중의 하나로 감자재배에서 문제가 되는 중요한 몇 가지 병에 대해 저항성 보여 감자의 신품종 육성을 위한 재료로 이용될 수 있다. 하지만, 본 연구에서 이용된 S. brevicaule의 EBN이 2인 사실로 인하여 재배종 감자와의 생식에 의한 종자생산에 장벽이 되고 있다. 본 연구에서는 차세대 유전체 기술에 의해 완성된 S. brevicaule의 엽록체 전장 유전체와 다른 7개 Solanum 종의 엽록체 전장 유전체를 비교하여 S. brevicaule를 다른 Solanum 종과 구별할 수 있는 Solanum 종 특이적인 분자마커를 개발하였다. S. brevicaule의 엽록체 전장 유전체의 총길이는 155,531 bp였으며, Blastn을 통해 S. spegazzinii 및 S. kurtzianum과 각각 99.99% 및 99.89%의 유사도를 확인할 수 있었다. 또한, 그 구조와 유전자의 구성이 다른 Solanum 종과 매우 유사하였으며, 계통수 분석에서도 다른 Solanum 종들과 매우 가까운 유연관계를 가지는 것으로 확인되었다. 엽록체 전장 유전체 다중 정렬에서는 총 27개의 S. brevicaule 특이적인 SNP 영역이 확인되었으며, 이들 중 세 개의 SNP 영역을 대상으로 최종적으로 S. brevicaule 특이적인 PCR 기반의 CAPS 분자마커를 개발하였다. 본 연구를 통해 얻은 S. brevicaule의 엽록체 전장 유전체와 S. brevicaule 특이적인 분자마커의 결과는 향후 Solanum 종을 대상으로 한 진화와 S. brevicaule를 이용한 감자품종 육성 연구에 기여를 할 수 있을 것이다.

Abstract ▼ AI-Helper

Solanum brevicaule is one of the tuber-bearing wild Solanum species. Because of its resistance to several important pathogens infecting potatoes during cultivation, it can be used for potato breeding. However, the fact that S. brevicaule used in this study has an EBN value of two causes the sexual reproduction barriers between the species and cultivated potatoes. In this study, specific markers for discriminating S. brevicaule from other Solanum species were developed on the basis of the results of sequence alignments with the whole chloroplast genomes of S. brevicaule and seven other Solanum species. The chloroplast genome of S. brevicaule was completed by next-generation sequencing technology described in other recent studies. The total sequence length of the chloroplast genome of S. brevicaule is 155,531 bp. Its structure and gene composition are similar to those of other Solanum species. Phylogenetic analysis revealed that S. brevicaule was closely grouped with other Solanum species. BLASTN search showed that its genome sequence had 99.99% and 99.89% identity with those of S. spegazzinii (MH021562) and S. kurtzianum (MH021495), respectively. Sequence alignment identified 27 SNPs that were specific to S. brevicaule. Thus, three PCR-based CAPS markers specific to S. brevicaule were developed on the basis of these SNPs. This study will facilitate in further studies on evolutionary and breeding aspects in Solanum species.

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표/그림 (7)

그림 Fig. 1 Assembly result of the complete chloroplast genome sequence of S. brevicaule (MK036507). Four representative contigs of the chloroplast genome of S. brevicaule and comparison with the corresponding regions of the chloroplast genome of S. berthaultii (KY419708; Kim et al. 2018) are combined. Blue and yellow bars indicate contig matching with the reference sequence in forward and reverse orientations, respectively
그림 Fig. 2 Gene map of the chloroplast genome of S. brevicaule. Genes on the outside and inside of the map are transcribed in the clockwise and counterclockwise directions, respectively
그림 Fig. 3 Maximum-likelihood phylogenetic tree of S. brevicaule with 11 species belonging to the Solanaceae family based on the chloroplast protein-coding sequences of each chloroplast genome. The numbers on each node indicate the bootstrap values from 1,000 replicates. The figure has been partially adopted from Park (2019)
그림 Fig. 4 Multiple alignment of the sequences on the intergenic or intragenic regions containing SNPs used to develop the PCRbased markers. SA11, SB2-1, SD9, Stub, Scmr, Sblb, Scha, and Ssto indicate the chloroplast genome sequences of S. acaule (MK036506; Park 2021a), S. brevicaule (MK036507; In this study), S. demissum (MK036508; Cho et al. 2019), S. tuberosum (KM489056; Cho et al. 2016), S. commersonii (KM489054; Cho et al. 2016), S. bulbocastanum (DQ347958; Daniell et al. 2006), S. chacoense (MF471371; Kim and Park 2019), and S. stoloniferum (MF471373; Kim and Park 2020a), respectively. They are listed from top to bottom in each region of the SNPs. The regions of the SNPs detected on that of S. brevicaule are highlighted and underlined. Restriction sites are indicated in red and bold font
표 Table 1 Comparative analysis of the chloroplast genome sequence of S. brevicaule with those of nine Solanaceae species
그림 Fig. 5 PCR-based markers for the discrimination of S. brevicaule from other Solanum species. A: SB2_SNP_3. B: SB2_SNP_5. C: SB2_SNP_8. The three markers all generated bands positively and/or negatively specific to S. brevicaule. Red and yellow allows indicate positive and negative bands on S. brevicaule, respectively. SB2-1 and SB2-17 indicate two different lines of S. brevicaule (PI205394). M, SH, HR, DJ, PT56, SA, SB1, SD, SC4, SC1, SC2, SI, SK, SJ, SM2, SM1, SP, SS, SV2, and SV1 indicate a size marker ladder, the potato varieties 'Seohong', 'Haryeong', 'Daeji', a potato breeding line 'PT56', S. acaule (PI310970), S. berthaultii (PI310981), S. demissum (PI218047), S. candolleanum (PI210035), S. cardiophyllum (PI341233), S. commersonii (PI558050), S. iopetalum (PI230459), S. kurtzianum (PI578236), S. jamesii (PI578326), S. microdontum (PI310979), S. mochiquense (PI338616), S. pinnatisectum (PI190115), S. stoloniferum (PI160224), S. vernei (PI230468) and S. verrucosum (PI160228), respectively
표 Table 2 Primers and restriction enzymes for generating S. brevicaule-specific markers

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