Quantitative Genotyping for the Astringency Locus in Hexaploid Persimmon Cultivars using Quantitative Real-time PCR
Quantitative Genotyping for the Astringency Locus in Hexaploid Persimmon Cultivars using Quantitative Real-time PCR
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DOI：
10.21273/jashs.135.1.59
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发表时间：
2010-01-01
影响因子：
1.9
通讯作者：
Kanzaki, Shinya
中科院分区：
文献类型：
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作者：
Akagi, Takashi;Takeda, Yumi;Kanzaki, Shinya
柿子 (Diospyros kaki Thunb.) 通常是六倍体，单个 AST 基因座控制着六个相应染色体中每一个的授粉恒定非涩性性状。授粉恒定非涩性 (PCNA) 基因型是零复合体，并且需要 AST 基因座上的纯合隐性等位基因 (ast)。有几个非 PCNA 品种/选择可以成为交叉亲本；然而，产生零复合体后代的概率取决于隐性等位基因（ast）的数量。在六倍体柿子的AST位点基因分型中，与二倍体植物的情况相反，我们需要检测AST/ast等位基因的剂量；这不能通过常见的共显性标记检测到。在本研究中，我们利用柿品种基因组中保守的肌动蛋白（DkAct）、花青素还原酶（DkANR）和L5R这三个参考位点，通过定量实时聚合酶链反应（qPCR）检测了M-ast的等位基因剂量，M-ast是品种间与ast等位基因强连锁的标记等位基因。根据 M-ast 等位基因剂量，估计了 63 个非涩性品种/选择的 AST/ast 基因型，其中只有 5 个品种/选择估计为单链或双链。使用 qPCR 的定量基因分型方法可能对多倍体植物普遍有效。
Persimmon (Diospyros kaki Thunb.) is generally hexaploid, and a single AST locus controls the pollination-constant non-astringency trait on each of six corresponding chromosomes. The pollination-constant non-astringent (PCNA) genotype is nulliplex and requires homozygous recessive alleles (ast) at the AST locus. There are several non-PCNA cultivars/selections that could be cross parents; however, the probability of yielding nulliplex offspring depends on the number of recessive alleles (ast). In genotyping for the AST locus in hexaploid persimmon, in contrast to the situation in diploid plants, we need to detect the AST/ast allele dosage; this cannot be detected by common codominant markers. In this study, we detected the allele dosage of M-ast, which is a marker allele strongly linked to the ast allele among cultivars, by quantitative real-time polymerase chain reaction (qPCR) using three reference sites, actin (DkAct), anthocyanin reductase (DkANR), and L5R, whose sequences are conserved in the genome of persimmon cultivars. Based on the allele dosage of the M-ast, AST/ast genotypes were estimated for 63 non-astringent cultivars/selections, of which only five cultivars/selections were estimated to be simplex or duplex. The quantitative genotyping method using qPCR may be generally effective for polyploid plants.