梨品种亲缘关系、S基因型鉴定及其克隆研究
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摘要
本文以梨属植物材料为试材，应用现代分子生物学等研究方法，研究了梨品种间的亲缘关系、梨品种自交不亲和性基因型(S基因型)及克隆了部分新S-RNase基因，主要的结果如下：
1．利用SSR标记技术对56个梨栽培品种的遗传多样性和亲缘关系进行了分析。6对SSR引物共产生38个等位位点，平均每对引物产生6.3个等位位点。聚类分析结果显示，梨属内56份材料在相似系数0.71处可分为4个组。第三组又可以分为5个亚组；2．首次开展了SRAP标记技术在梨属植物上应用的探讨，以3个不同梨品种为试验材料，对影响SRAP-PCR反应体系的Mg~(2+)、dNTP、引物浓度、Taq酶等因子设置梯度试验，筛选和建立可扩增多态性高、重复性好、带型清晰的最佳反应条件，并且利用72对引物进行高效引物的筛选。结果表明：在20μL反应体系中，模板DNA 30 ng，MgCl_2 2.0mmol·L~(-1)，dNTP 200μmol·L~(-1)，正、反向引物各0.2μmol·L~(-1)，DNA聚类酶1U；
3．利用SRAP标记技术对56个梨栽培品种的遗传多样性和亲缘关系分析，11对引物组合共扩增到188个谱带，其中样品间共同扩增的条带有4条，呈现多态性的条带有184条，多态百分率达到97.9％。NTSYS软件进行相似系数计算，UPGMA法聚类分析发现在相似系数0.662处，将56个梨品种分为4组，第二组又可分3亚组。结果表明SRAP技术可以反映各梨品种的亲缘关系，我国梨品种间具有高度的遗传多样性，梨品种演化中起主导作用的是有性杂交重组，为明确这些梨品种间的遗传背景提供理论依据：
4．利用PCR技术，根据梨S基因高度保守区C1和C3区，设计引物P1和P2，且结合根据梨S_2-RNase序列设计特异引物PS2，对梨品种的基因组DNA进行S基因特异扩增、克隆、测序，并在GenBank中BLAST比较，确定S基因特异性片段，确定了京白梨等90个供试自交不亲和性品种的S基因型，并且通过田间杂交试验及应用荧光显微镜观察异花授粉后花柱内的花粉管生长情况来验证鉴定出的梨品种S基因型的可靠性。首次鉴定了我国主栽的一些梨品种的S基因，所获得的梨品种S基因型列表对于生产上合理配置授粉树提供了理论依据；
5．利用分子生物学技术，根据日本梨S-RNase基因的C1和C3区的保守序列，对7个中国梨品种的基因组DNA进行序列测定和生物信息学分析，鉴定了8个新的梨自交不亲和S-RNase基因，分别命名为S_(28)-RNase，S_(30)-RNase，S_(31)-RNase，S_(32)-RNase，S_(33)-RNase，S_(35)-RNase，S_(36)-RNase，S_(37)-RNase，其登录号依次为AY562394，AY876945，DQ124366，DQ124367，DQ138081，DQ323732，DQ417607，DQ448239，这些新S-RNase基因的发现和梨品种S基因型的确定为中国梨品种的优化配置、丰产栽培和遗传改良提供了科学依据。
Pear(Pyrus) is a commercially important fruit crop worldwide and most come from China, which resoure is abundant. It's necessary to classify and analyze the relationship between the different cultivars in heredity. The phylogenetic relationships of 56 Pyrus were analyzed by using SSR and SRAP markers.
Self-incompatibility(SI) is a genetic mechanism employed by flowering plants to prevent inbreeding and promoting out-crossing, which involved a complex set of cell-cell interactions between the pistil and the pollen, thence, a model system for studying of intercellular information transmission, cell-cell recognition and gene spatial-temporal expression. Pear exhibits S-RNase-based gametophytic self-incompatiility, as other Rosaceae species do. Therefore, they will not set fruit unless pollinated with cultivars with different S-genotypes. Determination of correct pollen incompatibility groups and assignment of cultivars to the groups are essential for good crop production. Pollen incompatibility groups in pear have been identified by controlled pollination tests and pollen tube growth tests, but these tests is time-consuming and prone to be affected by environmental factors. The allele-specific PCR system was established to identify the S-genotype of 90 pear cultivars in this study.
All results were summarized as follows:
1. Simple sequence repeat(SSR) markers were used to assess relationship of 56 Pyrus cultivars. Atotal of 38 putative alleles were generated from six primer-pairs. All the SSR markers showed a high level of genetic polymorphism with a mean of 6.3 putative alleles per locus. Four groups were generated from all the accessions by UPGMA clusters analysis at Dice's similarity coefficient of 0.71. The third group was classified into three subgroup;
2. Factors influencing SRAP-PCR analysis were studied using three pear cultivars. A reliable, effective and reproductive PCR reaction system for detecting SRAP was developed and 72 primer-pairs were filtered. Each 20μL PCR reaction mixture consisted of template DNA 30 ng, 2.0 mmol·L~(-1) of MgCl_2, 200μmol·L~(-1) of dNTPs, 0.2μmol·L~(-1) of primer and 1 unit of Taq polymerase;
3. Further study on relationship of Pyrus cultivars native mainly to Eastern Asia had been carried out with sequence-related amplified polymorphism markers. The eleven primer could successfully differentiate all the materials and generated 188 fragments of which 184 fragments were polymorphic to 97.9%. Dendrogram analysis clusted 56 Pyrus cultivars into four group at Dice's similarity coefficient of 0.662. The research supported the close relationship between Japanese pears and Chinese sand pears. All these results showed that SRAP markers were economic, effective, and reliable. Pear has widely genetic diversity;
4. Classifying pear S-genotypes are useful for breeding programs and may help selection. Using primers(P1 and P2) based on the conserved regions C1 and C3 of pear S-RNase structure and specific primers(PS2) based on sequence of the pear S_3-RNase, DNA of ninety Chinese-bred pear cultivars were carried on PCR and PAGE. S-genotypes of these cultivars were identified by analysis of DNA sequencing. The results were confirmed by fruit set of some cultivars and microscopic observation of pollen tube. The S-genotypes of ninety cultivars were determined;
5. Partial S-RNase genes of pear were amplified and cloned in seven cultivars of unknown S-geontypes. The PCR products were sequenced and blasted in GenBank. S_(28)-RNase, S_(30)-RNase, S_(31)-RNase, S_(32)-RNase, S_(33)-RNase, S_(35)-RNase, S_(36)-RNase and S_(37)-RNase were deposited in NCBI under the accession numbers of AY562394, AY876945, DQ124366, DQ124367, DQ138081, DQ323732, DQ417607, DQ448239.
1．利用SSR标记技术对56个梨栽培品种的遗传多样性和亲缘关系进行了分析。6对SSR引物共产生38个等位位点，平均每对引物产生6.3个等位位点。聚类分析结果显示，梨属内56份材料在相似系数0.71处可分为4个组。第三组又可以分为5个亚组；2．首次开展了SRAP标记技术在梨属植物上应用的探讨，以3个不同梨品种为试验材料，对影响SRAP-PCR反应体系的Mg~(2+)、dNTP、引物浓度、Taq酶等因子设置梯度试验，筛选和建立可扩增多态性高、重复性好、带型清晰的最佳反应条件，并且利用72对引物进行高效引物的筛选。结果表明：在20μL反应体系中，模板DNA 30 ng，MgCl_2 2.0mmol·L~(-1)，dNTP 200μmol·L~(-1)，正、反向引物各0.2μmol·L~(-1)，DNA聚类酶1U；
3．利用SRAP标记技术对56个梨栽培品种的遗传多样性和亲缘关系分析，11对引物组合共扩增到188个谱带，其中样品间共同扩增的条带有4条，呈现多态性的条带有184条，多态百分率达到97.9％。NTSYS软件进行相似系数计算，UPGMA法聚类分析发现在相似系数0.662处，将56个梨品种分为4组，第二组又可分3亚组。结果表明SRAP技术可以反映各梨品种的亲缘关系，我国梨品种间具有高度的遗传多样性，梨品种演化中起主导作用的是有性杂交重组，为明确这些梨品种间的遗传背景提供理论依据：
4．利用PCR技术，根据梨S基因高度保守区C1和C3区，设计引物P1和P2，且结合根据梨S_2-RNase序列设计特异引物PS2，对梨品种的基因组DNA进行S基因特异扩增、克隆、测序，并在GenBank中BLAST比较，确定S基因特异性片段，确定了京白梨等90个供试自交不亲和性品种的S基因型，并且通过田间杂交试验及应用荧光显微镜观察异花授粉后花柱内的花粉管生长情况来验证鉴定出的梨品种S基因型的可靠性。首次鉴定了我国主栽的一些梨品种的S基因，所获得的梨品种S基因型列表对于生产上合理配置授粉树提供了理论依据；
5．利用分子生物学技术，根据日本梨S-RNase基因的C1和C3区的保守序列，对7个中国梨品种的基因组DNA进行序列测定和生物信息学分析，鉴定了8个新的梨自交不亲和S-RNase基因，分别命名为S_(28)-RNase，S_(30)-RNase，S_(31)-RNase，S_(32)-RNase，S_(33)-RNase，S_(35)-RNase，S_(36)-RNase，S_(37)-RNase，其登录号依次为AY562394，AY876945，DQ124366，DQ124367，DQ138081，DQ323732，DQ417607，DQ448239，这些新S-RNase基因的发现和梨品种S基因型的确定为中国梨品种的优化配置、丰产栽培和遗传改良提供了科学依据。
Pear(Pyrus) is a commercially important fruit crop worldwide and most come from China, which resoure is abundant. It's necessary to classify and analyze the relationship between the different cultivars in heredity. The phylogenetic relationships of 56 Pyrus were analyzed by using SSR and SRAP markers.
Self-incompatibility(SI) is a genetic mechanism employed by flowering plants to prevent inbreeding and promoting out-crossing, which involved a complex set of cell-cell interactions between the pistil and the pollen, thence, a model system for studying of intercellular information transmission, cell-cell recognition and gene spatial-temporal expression. Pear exhibits S-RNase-based gametophytic self-incompatiility, as other Rosaceae species do. Therefore, they will not set fruit unless pollinated with cultivars with different S-genotypes. Determination of correct pollen incompatibility groups and assignment of cultivars to the groups are essential for good crop production. Pollen incompatibility groups in pear have been identified by controlled pollination tests and pollen tube growth tests, but these tests is time-consuming and prone to be affected by environmental factors. The allele-specific PCR system was established to identify the S-genotype of 90 pear cultivars in this study.
All results were summarized as follows:
1. Simple sequence repeat(SSR) markers were used to assess relationship of 56 Pyrus cultivars. Atotal of 38 putative alleles were generated from six primer-pairs. All the SSR markers showed a high level of genetic polymorphism with a mean of 6.3 putative alleles per locus. Four groups were generated from all the accessions by UPGMA clusters analysis at Dice's similarity coefficient of 0.71. The third group was classified into three subgroup;
2. Factors influencing SRAP-PCR analysis were studied using three pear cultivars. A reliable, effective and reproductive PCR reaction system for detecting SRAP was developed and 72 primer-pairs were filtered. Each 20μL PCR reaction mixture consisted of template DNA 30 ng, 2.0 mmol·L~(-1) of MgCl_2, 200μmol·L~(-1) of dNTPs, 0.2μmol·L~(-1) of primer and 1 unit of Taq polymerase;
3. Further study on relationship of Pyrus cultivars native mainly to Eastern Asia had been carried out with sequence-related amplified polymorphism markers. The eleven primer could successfully differentiate all the materials and generated 188 fragments of which 184 fragments were polymorphic to 97.9%. Dendrogram analysis clusted 56 Pyrus cultivars into four group at Dice's similarity coefficient of 0.662. The research supported the close relationship between Japanese pears and Chinese sand pears. All these results showed that SRAP markers were economic, effective, and reliable. Pear has widely genetic diversity;
4. Classifying pear S-genotypes are useful for breeding programs and may help selection. Using primers(P1 and P2) based on the conserved regions C1 and C3 of pear S-RNase structure and specific primers(PS2) based on sequence of the pear S_3-RNase, DNA of ninety Chinese-bred pear cultivars were carried on PCR and PAGE. S-genotypes of these cultivars were identified by analysis of DNA sequencing. The results were confirmed by fruit set of some cultivars and microscopic observation of pollen tube. The S-genotypes of ninety cultivars were determined;
5. Partial S-RNase genes of pear were amplified and cloned in seven cultivars of unknown S-geontypes. The PCR products were sequenced and blasted in GenBank. S_(28)-RNase, S_(30)-RNase, S_(31)-RNase, S_(32)-RNase, S_(33)-RNase, S_(35)-RNase, S_(36)-RNase and S_(37)-RNase were deposited in NCBI under the accession numbers of AY562394, AY876945, DQ124366, DQ124367, DQ138081, DQ323732, DQ417607, DQ448239.
引文
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