Quantitative real-time PCR to determine allele number for the astringency locus by analysis of a linked marker in Diospyros kaki Thunb
Quantitative real-time PCR to determine allele number for the astringency locus by analysis of a linked marker in Diospyros kaki Thunb
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DOI：
10.1007/s11295-009-0202-4
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发表时间：
2009-07-01
影响因子：
2.4
通讯作者：
Yonemori, Keizo
中科院分区：
文献类型：
--
作者：
Akagi, Takashi;Kanzaki, Shinya;Yonemori, Keizo
柿子（Diospyros kaki Thunb.）是东亚一种具有重要经济价值的多倍体果树。天然涩味损失是柿子育种中的一个重要性状。采用实时荧光定量PCR技术对柿子果实涩味的AST/ AST等位基因数量进行了测定。为此，用一个编码nadp依赖性山梨醇-6-磷酸脱氢酶（S6PDH）基因的1或2个拷贝转化了品种Jiro，该基因被用作测量与隐性基因座紧密相连的序列标记的等位基因数量的标准。然后使用与AST或AST等位基因相关的标记引物直接测量全同胞杂交后代的AST与AST比率。通过AST/ AST比值的测定和S6PDH拷贝数的测定，估计AST/ AST位点上AST和AST等位基因的数量。本研究支持了kaki是具有6个AST和/或AST等位基因的六倍体的假设。除了确定AST位点的等位基因状态外，还证明了real-time PCR在自多倍体或异源多倍体中用于确认靶基因的倍性水平和等位基因组成的应用。
Persimmon (Diospyros kaki Thunb.) is a polyploidy fruit tree species of economic importance to East Asia. Natural astringency loss is an important trait in persimmon breeding programs. Quantitative real-time PCR was used to determine the number of AST/ast alleles for fruit astringency in persimmon (D. kaki Thunb.). To this end, the cultivar Jiro was transformed with one or two copies of a gene encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), which was used as a standard for measuring the allele number of a sequenced marker tightly linked to the recessive ast locus for nonastringency. Primers for markers linked to the AST or ast allele were then used to measure the AST to ast ratio directly in the progeny of a full-sib cross. From determination of the AST to ast ratio and the results of the S6PDH copy number, the number of AST and ast alleles at the AST/ast locus was estimated. This research supported the hypothesis that D. kaki is a hexaploid with six AST and/or ast alleles. In addition to the determination of the allelic status of the AST locus, the application of real-time PCR for confirmation of the ploidy level and allelic composition of target genes in autopolyploids or allopolyploids was demonstrated.