Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq
Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq
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DOI：
10.1007/s00122-015-2584-z
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发表时间：
2015-11-01
影响因子：
5.4
通讯作者：
Choi, Doil
中科院分区：
文献类型：
--
作者：
Kim, Sunggil;Kim, Cheol-Woo;Choi, Doil
结合BSA和RNA-seq鉴定洋葱恢复育性基因的候选基因。参与DNA错配修复的AcPMS1被确定为最佳候选。为了确定洋葱细胞质雄性不育育性恢复基因（Ms）的候选基因，采用了大量分离分析和RNA-seq相结合的方法。从32,674个从头组装的contig中，在141个contig中鉴定出430个在雄性可育（MF）和雄性不育（MS）块之间完全纯合的snp。经PCR扩增和测序验证snp的纯合子性，其中139个contigs上的snp为两个重组体的基因型，其中Ms位点与两个紧密连锁的分子标记之间存在交叉事件。结果在大分离群体中鉴定出30个与Ms位点完全连锁的contigs。对251份国产选育材料进行基因分型，发现其中14份与Ms位点存在完全连锁不平衡（LD）。此外，标记14个contigs的分子标记在21个国家引进的124个外来种质中也表现出几乎完全的LD，除了一个种质存在交叉事件，14个标记被分成两组。对具有完全LD的14个等位基因进行全长cDNA测序后，比较了推断的MF和MS等位基因的氨基酸序列。四个基因被证明在已知结构域中存在假定的关键氨基酸变化。其中编码PMS1的基因参与DNA错配修复途径，被认为是洋葱雄性不育育性恢复的最佳候选基因。
A combination of BSA and RNA-seq was performed to identify candidates for the restorer-of-fertility gene in onion. The AcPMS1 involved in DNA mismatch repair was identified as the best candidate.To identify candidate genes of the restorer-of-fertility gene (Ms) responsible for fertility restoration of onion cytoplasmic male-sterility, a combined approach of bulked segregant analysis and RNA-seq was employed. From 32,674 de novo assembled contigs, 430 perfectly homozygous SNPs between male-fertile (MF) and male-sterile (MS) bulks were identified in 141 contigs. After verifying the homozygosity of the SNPs by PCR amplification and sequencing, the SNPs on 139 of the contigs were genotypes for the two recombinants which contained crossover events between the Ms locus and two tightly linked molecular markers. As a result, 30 contigs showing perfect linkage with the Ms locus in the large-sized segregating population were identified. Among them, 14 showed perfect linkage disequilibrium (LD) with the Ms locus, as determined by genotyping 251 domestic breeding lines. Furthermore, molecular markers tagging the 14 contigs also showed almost perfect LD with each other in 124 exotic accessions introduced from 21 countries, except for one accession which contained a crossover event by which the 14 markers were divided into two groups. After sequencing of the full-length cDNA of the 14 contigs showing perfect LD, the deduced amino acids sequences of the MF and MS alleles were compared. Four genes were shown to harbor putative critical amino acid changes in the known domains. Among them, the gene encoding PMS1, involved in the DNA mismatch repair pathway, was assumed to be the best candidate gene responsible for fertility restoration of male-sterility in onion.