Identification of molecular markers linked to natural astringency loss of persimmon

授粉恒定和非涩（PCNA）型果实是最理想的鲜食柿，并具有良好的果实品质的新品种的PCNA型一直在世界各地等待。然而，PCNA型的遗传是定性的，并且PCNA型对非PCNA（PVNA、PVA和PCA）型是隐性的。与天然失涩性状紧密连锁的分子标记是育种的有力工具。因此，我们试图通过以下实验来建立它，使育种过程更有效.用128对AFLP引物组合对6个PCNA型和10个非PCNA型的杂交后代进行群体分离分析。结果表明，共检测到4个潜在的AFLP标记，可用于区分PCNA和非PCNA类型.从4个潜在的AFLP标记中筛选出最可靠的标记并测序。用此标记作探针，经HindIII酶切后进行Southern杂交分析，可根据6.5kb或8.0kb条带的存在与否，区分PCNA型和非PCNA型。这些带的分离率表明柿属多体遗传。Perceptide可能是一种同源或同源异源六倍体。用AFLP标记作探针对现有的品种进行Southern分析，PCNA品种与非PCNA品种的鉴别准确率为100%。这一结果表明，该分析方法对于在更广泛的育种群体中选择PCNA类型具有较高的可靠性.两个RFLP标记（6.5 kb和8.0 kb）的测序分析，以表征他们和引物的PCR分析，更容易检测这些标记是不完全成功的。8.0kb条带测序成功，6.5kb条带测序失败。因此，需要进一步研究设计PCR引物，使其比Southern分析更容易区分PCNA型和非PCNA型。

The pollination-constant and non-astringent (PCNA)-type fruit is the most desirable persimmon for fresh consumption, and new PCNA-type cultivars with good fruit quality have been awaited worldwide. However, the inheritance of PCNA-type is qualitative and the PCNA-type is recessive to non-PCNA (PVNA, PVA, and PCA) types. Molecular marker closely linked to the trait of natural astringency-loss is a powerful tool for the breeding project. So, we tried to establish it by the following experiments to make the breeding process more efficient.1. Bulked segregant analysis (BSA) of 6 PCNA-type and 10 non-PCNA-type offspring in breeding population was performed by AFLP method using 128 primer combinations. As a result, 4 potential AFLP markers were detected for discriminating PCNA- and non-PCNA types.2. The most reliable marker among 4 potential AFLP markers was selected and sequenced. When this marker was used as a probe for Southern analysis after genomic DNA was digested HindlII, all progenies examined could be distinguished between PCNA-type and non-PCNA type by the existence or absence of 6.5kb or 8.0 kb band. Furthermore, segregation rate of these bands indicated that persimmon shows polysomic inheritance. Persimmon seemed to be an auto- or autoallo-hexaploid.3. When Southern analysis using AFLP maker as a probe was examined to existing cultivars, PCNA cultivars could be distinguished from non-PCNA culitvars with 100% accuracy. This result indicates a high reliability of this analysis for selecting PCNA-type among wider breeding populations.4. The sequencing analysis of two RFLP markers (6.5 kb and 8.0 kb) for characterizing them and making primers for PCR analysis for easier detection of these makers was not completely successful. We could sequence 8.0 kb band but not 6.5 kb band. So, further study was needed to design PCR primers for more easier method than Southern analysis to distinguish PCNA-type from non-PCNA-type.