完全甜柿及部分雄性种质间的亲缘关系研究
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摘要
中国柿(Diospyros kaki Thunb．)在长期栽培过程中，由于遗传变异和自然、人工选择的作用，分化出了许多宝贵的甜柿资源。本研究对部分柿属雄性种质及甜柿种质采用RAPD、ISSR等分子标记分析，结合对新发现原产中国大别山区雄性种质花粉基础生物学研究，建立和优化柿属植物ITS-PCR及AFLP技术体系，对部分柿属雄性种质间亲缘关系、系统演化及遗传多样性进行了分析比较，探讨柿属雄性种质在中国原产甜柿起源演化中可能的作用。主要结果如下：
1．12份供试雄性种质花粉萌芽率在20.1％—38.5％之间；除台湾正柿及油柿外，其余试材的花粉均能在罗田甜柿的柱头上萌发；雄株2号、雄株3号、雄株8号不含巨大花粉，其余试材有0.18％—4.44％不同比例的巨大花粉存在。
2．对29份柿属雄性种质RAPD分析结果如下：从118条随机引物中筛选得到32条引物，共扩增得到499条谱带，平均多态性比率87.17％，26个种质特异性标记，平均多态性信息含量为0.6097。3种方法(UPGMA法，ME法，Wagner简约法)聚类分析表明：中国柿种质与日本柿种质明显聚为2个类群，甜柿与涩柿不能截然分开；完全雄性资源同罗田甜柿亲缘关系密切；湘西甜柿和日本甜柿关系密切，宝盖甜柿和磨盘柿(罗田)亲缘关系密切；3种聚类方法结果基本聚类分组结果类似，同时也存在极少数差异；主成分分析和主坐标分析支持聚类分析结果。
3．建立了适合柿属植物的ISSR分析技术体系：25μL反应体系中，含1×Buffer，Mg~(2+)2.0或2.5mmol／L，dNTPs 0.2mmol／L，引物0.6μmol／L，Taq DNA聚合酶1U，甲酰胺2％，模板DNA 2.4ng／μL；PCR扩增程序：94℃变性3min；94℃ 30s，(Tm+2)℃(根据引物不同设定)45s，72℃ 1.5min，35个循环；72℃延伸7min；2％琼脂糖电泳分离PCR产物。对52份柿属雄性种质ISSR分析结果如下：从58条ISSR引物中筛选得到20条随机引物，共扩增得到274条谱带，平均多态性比率92.33％，30个种质特异性标记，平均多态性信息含量为0.7119。3种方法聚类分析结果表明中国柿种质、日本柿种质可以分成不同的3-5组，甜柿与涩柿虽不能截然分开，但甜、涩柿种质基本可以分为不同的组，仅有2个完全甜柿种质分在涩柿1组中；3个韩国涩柿品种和中国涩柿品种亲缘关系密切，分组结果和地理起源较为一致；完全雄性种质和罗田甜柿亲缘关系密切，宝盖甜柿
There are abundant valuable persimmon (Diospyros kaki Thunb.) genetic resources existing in China because of the genetic variation, natural and artificial selection occurring during the long history of cultivation. In the present study, we investigated the phylogenetic relationships among some staminate Diospyros spp. and its relatives using RAPD and ISSR, as while we examined the pollen germinability and the occurrence of giant pollen in the new discovering staminate persimmon germplasm distributed in Dabie mountains. The technique of ITS-PCR and AFLP in Diospyros were also established and optimized. The study on the phylogentics of Diospyros may provide useful information for the analysis of the genetic diversity and molecular systematics in Diospyros as while as the evaluation of the staminate germplasm effects in the evolution of Diospyros kaki Thunb.. The main results were as follows:
1. 12 Diospyros spp. staminate germplasm materials were used to test themselves' pollen germinability in vitro and which on the stigma of D.kaki Thunb. cv. Luotiantianshi and the occurrence of giant pollen. The results showed that the pollen germinability in vitro of those materials varied from 20.1% to 38.5%. 10 materials except 'Taiwanzhengshi' (D.kaki Thunb.) and 'Oily persimmon' (D. oleifera Cheng.) had germinability on the stigma of'Luotiantianshi'. There are different percentage of giant pollen existing in 9 germplasm genotypes with variation 0.18% to 4.44% but Male strain No.2、Male strain No.3 and Male strain No.8.
2. A total of 499 bands generated by 32 informative RAPD primers screened from 118 RAPD primer, 26 of which were unique to specific accessions in 29 Diospyros spp. The polymorphic rate of RAPD markers was 87.17% and the polymorphic information content was 0.6097. The results of clustering analysis using UPGMA, ME and Wanger Parsimony showed that the 29 genotypes could be divided into 3 groups. The first group included the Chinese persimmon germplasm, while the second consisted of the Japanese persimmon cultivars. Diospyros lotus L. and Diospyros oleifera Cheng. formed the third group. PCNA couldn't be separated from
1．12份供试雄性种质花粉萌芽率在20.1％—38.5％之间；除台湾正柿及油柿外，其余试材的花粉均能在罗田甜柿的柱头上萌发；雄株2号、雄株3号、雄株8号不含巨大花粉，其余试材有0.18％—4.44％不同比例的巨大花粉存在。
2．对29份柿属雄性种质RAPD分析结果如下：从118条随机引物中筛选得到32条引物，共扩增得到499条谱带，平均多态性比率87.17％，26个种质特异性标记，平均多态性信息含量为0.6097。3种方法(UPGMA法，ME法，Wagner简约法)聚类分析表明：中国柿种质与日本柿种质明显聚为2个类群，甜柿与涩柿不能截然分开；完全雄性资源同罗田甜柿亲缘关系密切；湘西甜柿和日本甜柿关系密切，宝盖甜柿和磨盘柿(罗田)亲缘关系密切；3种聚类方法结果基本聚类分组结果类似，同时也存在极少数差异；主成分分析和主坐标分析支持聚类分析结果。
3．建立了适合柿属植物的ISSR分析技术体系：25μL反应体系中，含1×Buffer，Mg~(2+)2.0或2.5mmol／L，dNTPs 0.2mmol／L，引物0.6μmol／L，Taq DNA聚合酶1U，甲酰胺2％，模板DNA 2.4ng／μL；PCR扩增程序：94℃变性3min；94℃ 30s，(Tm+2)℃(根据引物不同设定)45s，72℃ 1.5min，35个循环；72℃延伸7min；2％琼脂糖电泳分离PCR产物。对52份柿属雄性种质ISSR分析结果如下：从58条ISSR引物中筛选得到20条随机引物，共扩增得到274条谱带，平均多态性比率92.33％，30个种质特异性标记，平均多态性信息含量为0.7119。3种方法聚类分析结果表明中国柿种质、日本柿种质可以分成不同的3-5组，甜柿与涩柿虽不能截然分开，但甜、涩柿种质基本可以分为不同的组，仅有2个完全甜柿种质分在涩柿1组中；3个韩国涩柿品种和中国涩柿品种亲缘关系密切，分组结果和地理起源较为一致；完全雄性种质和罗田甜柿亲缘关系密切，宝盖甜柿
There are abundant valuable persimmon (Diospyros kaki Thunb.) genetic resources existing in China because of the genetic variation, natural and artificial selection occurring during the long history of cultivation. In the present study, we investigated the phylogenetic relationships among some staminate Diospyros spp. and its relatives using RAPD and ISSR, as while we examined the pollen germinability and the occurrence of giant pollen in the new discovering staminate persimmon germplasm distributed in Dabie mountains. The technique of ITS-PCR and AFLP in Diospyros were also established and optimized. The study on the phylogentics of Diospyros may provide useful information for the analysis of the genetic diversity and molecular systematics in Diospyros as while as the evaluation of the staminate germplasm effects in the evolution of Diospyros kaki Thunb.. The main results were as follows:
1. 12 Diospyros spp. staminate germplasm materials were used to test themselves' pollen germinability in vitro and which on the stigma of D.kaki Thunb. cv. Luotiantianshi and the occurrence of giant pollen. The results showed that the pollen germinability in vitro of those materials varied from 20.1% to 38.5%. 10 materials except 'Taiwanzhengshi' (D.kaki Thunb.) and 'Oily persimmon' (D. oleifera Cheng.) had germinability on the stigma of'Luotiantianshi'. There are different percentage of giant pollen existing in 9 germplasm genotypes with variation 0.18% to 4.44% but Male strain No.2、Male strain No.3 and Male strain No.8.
2. A total of 499 bands generated by 32 informative RAPD primers screened from 118 RAPD primer, 26 of which were unique to specific accessions in 29 Diospyros spp. The polymorphic rate of RAPD markers was 87.17% and the polymorphic information content was 0.6097. The results of clustering analysis using UPGMA, ME and Wanger Parsimony showed that the 29 genotypes could be divided into 3 groups. The first group included the Chinese persimmon germplasm, while the second consisted of the Japanese persimmon cultivars. Diospyros lotus L. and Diospyros oleifera Cheng. formed the third group. PCNA couldn't be separated from
引文
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