萝卜自交不亲和性测定与相关基因的鉴定
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摘要
萝卜杂种优势明显，自交不亲和是萝卜杂交优势利用的一种重要途径。本研究结合亲和指数法与荧光显微观察法对高代自交材料进行自交不亲和测定。利用SLG和SRK的特异引物，通过限制性片段长度多态性分析(PCR-RFLP)研究萝卜的SLG和SRK基因，并对材料的SLG和SRK基因进行克隆验证。为萝卜自交不亲和性杂优利用、了解SLG和SRK基因在十字花科中的进化、萝卜自交不亲和性机理的研究奠定基础。
1以萝卜高代自交系为试材，利用荧光显微镜观察其花期和蕾期自花授粉不同时期柱头与花柱，结果发现自交不亲和与自交亲和材料花粉萌发和花粉管伸长状况存在明显差异。自交不亲和材料花期授粉后柱头上出现严重的胼胝质反应，花粉很少萌发，即使萌发也不能正常生长，出现弯曲或背向生长，花粉管顶端膨大不能穿越柱头乳突细胞；自交亲和系萝卜单株花期、蕾期以及自交不亲和系单株蕾期授粉后花粉多数能够正常萌发并穿越柱头进入子房。对供试材料进行了田间自交亲和指数测定，与荧光显微镜测定法鉴定结果高度一致。因此利用荧光显微镜观察法可以准确快速鉴定萝卜的自交不亲和性，从而提高优良自交不亲和系选育效率。
2萝卜S位点糖蛋白基因(SLG)和S位点受体激酶(SRK)基因参与了花粉和柱头之间的自交不亲和反应。设计SLG和SRK特异引物，利用PCR-RFLP技术对SLG和SRK基因的多态性进行分析。根据classⅠSLG特异引物扩增产物限制性内切酶酶切后DNA多态性，19个自交系被分为12个S单元型；SRK特异引物扩增酶切后，20个自交系也分为13个S单元型。classⅡSLG特异引物扩增产物限制性内切酶酶切后，根据DNA多态性17个自交系被分为6个S单元型。因此PCR-RFLP技术在区分萝卜S单元型中有着重要作用。对5个SLG等位基因和6个SRK等位基因的DNA序列进行了验证，研究发现氨基酸序列与甘蓝SLG6基因的相似性达80.72％～84.26％，SRK基因与甘蓝中SRK3基因也具有很高的相似性。分析萝卜和甘蓝的SLG序列发现，萝卜的SLG基因并不自己独立成一组，而是分散在树形图中，说明SLG单元型分化在萝卜属和芸薹属分类之前。
The ascendent of crossbreed is very obvious in Raphanus sativus. self-incompatibility of Raphanus sativus is one of the important routes in ascendent of crossbreed using. The self-incompatibility (SI) of advanced inbred lines were tested with the traditional index method and the fluoroscope observation method. Polymorphism of the S-locus glycoprotein gene, SLG and the S locus receptor kinase gene,SRK which participate in the pollen-stigma interaction of self-incompatibility in Raphanus sativus were analyzed by PCR-RFLP using SLG-and SRK-specific primers. The DNA fragments of R.sativus SLG alleles and R.sativus SRK alleles were cloned and determinded. These establish base for self-incompat--ibility ascendent of crossbreed using in Raphanus sativus, understanding the evolution of SLG alleles and SRK alleles in Cruciferae, and researching of self-incompatibility mechanism in Raphanus sativus.
1. Using the advanced inbred lines as the materials, the interaction of pollen-stigmas at different periods was investigated in radish of self-pollination done both in the bud and at anthesis with the fluoroscope. The results indicated that there were obvious differences in the germination of pollen grains in stigmas and growth of pollen tubes in styles between the self-incompatible and self-compatible lines. Few pollen grains of open flower self-pollina--tion attached and germinated in the stigma of self-incompatible individuals, and intensive callose reaction occurred on the surface of stigma. The germinated pollen grains could not grown naturally and fail to penetrate into the stigma with the abnormality of distorting of pollen tubes, swelling of the tops and failure to further growth. Most pollen grains of self-pollination both in the bud and at anthesis germinated naturally and penetrated into the stigmas and finally succeeded in reaching to the ovary in self-compatible individuals, and theses also occurred in the bud self-pollination in self-incompatible dividuals. The self-incompatibility (SI) indexes of all the lines were tested with the traditional index method, and it is high accordant with the results from the fluoroscope observation method, indicating that self-incompatibility of radish lines can be identified with the fluoroscope observation method. Therefore fluoroscope observation method is a good efficiently tool for development of elite self-incompatibility in radish breeding program.
2. Polymorphism of the S-locus glycoprotein gene, SLG and the S locus receptor kinase gene, SRK which participate in the pollen-stigma interaction of self-incompatibility in Raphanus sativus were analyzed by PCR-RFLP using SLG- and SRK- specific primers. 19 inbred lines of R.sativus could be grouped into 12 S haplotypes by PCR with class I SLG specific primers showed different profiles up polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. 20 inbred lines of R.sativus could be grouped into 13 S haplotypes by PCR with SRK specific primers. 17 inbred lines of R.sativus could be grouped into 6 S haplotypes by PCR with class II SLG specific primers. Therefore the PCR-RFLP technology is very important in identification and classification of S haplotypes in Raphanus sativus. The nucleotide sequences of the DNA fragments of 5 R.sativus SLG alleles and 6 R.sativus SRK alleles were determinded. Degrees of similarity of the R.sativus SLG deduced amino-acid sequences to a Brassica SLG6 were 80.72%~84.26%. Phylogenetic analysis of the SLG sequence from Raphanus and Brassica revealed that the Raphanus SLGs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs. These results suggested that diversification of the SLG alleles of Raphanus and Brassica before differentiation of the genera.
1以萝卜高代自交系为试材，利用荧光显微镜观察其花期和蕾期自花授粉不同时期柱头与花柱，结果发现自交不亲和与自交亲和材料花粉萌发和花粉管伸长状况存在明显差异。自交不亲和材料花期授粉后柱头上出现严重的胼胝质反应，花粉很少萌发，即使萌发也不能正常生长，出现弯曲或背向生长，花粉管顶端膨大不能穿越柱头乳突细胞；自交亲和系萝卜单株花期、蕾期以及自交不亲和系单株蕾期授粉后花粉多数能够正常萌发并穿越柱头进入子房。对供试材料进行了田间自交亲和指数测定，与荧光显微镜测定法鉴定结果高度一致。因此利用荧光显微镜观察法可以准确快速鉴定萝卜的自交不亲和性，从而提高优良自交不亲和系选育效率。
2萝卜S位点糖蛋白基因(SLG)和S位点受体激酶(SRK)基因参与了花粉和柱头之间的自交不亲和反应。设计SLG和SRK特异引物，利用PCR-RFLP技术对SLG和SRK基因的多态性进行分析。根据classⅠSLG特异引物扩增产物限制性内切酶酶切后DNA多态性，19个自交系被分为12个S单元型；SRK特异引物扩增酶切后，20个自交系也分为13个S单元型。classⅡSLG特异引物扩增产物限制性内切酶酶切后，根据DNA多态性17个自交系被分为6个S单元型。因此PCR-RFLP技术在区分萝卜S单元型中有着重要作用。对5个SLG等位基因和6个SRK等位基因的DNA序列进行了验证，研究发现氨基酸序列与甘蓝SLG6基因的相似性达80.72％～84.26％，SRK基因与甘蓝中SRK3基因也具有很高的相似性。分析萝卜和甘蓝的SLG序列发现，萝卜的SLG基因并不自己独立成一组，而是分散在树形图中，说明SLG单元型分化在萝卜属和芸薹属分类之前。
The ascendent of crossbreed is very obvious in Raphanus sativus. self-incompatibility of Raphanus sativus is one of the important routes in ascendent of crossbreed using. The self-incompatibility (SI) of advanced inbred lines were tested with the traditional index method and the fluoroscope observation method. Polymorphism of the S-locus glycoprotein gene, SLG and the S locus receptor kinase gene,SRK which participate in the pollen-stigma interaction of self-incompatibility in Raphanus sativus were analyzed by PCR-RFLP using SLG-and SRK-specific primers. The DNA fragments of R.sativus SLG alleles and R.sativus SRK alleles were cloned and determinded. These establish base for self-incompat--ibility ascendent of crossbreed using in Raphanus sativus, understanding the evolution of SLG alleles and SRK alleles in Cruciferae, and researching of self-incompatibility mechanism in Raphanus sativus.
1. Using the advanced inbred lines as the materials, the interaction of pollen-stigmas at different periods was investigated in radish of self-pollination done both in the bud and at anthesis with the fluoroscope. The results indicated that there were obvious differences in the germination of pollen grains in stigmas and growth of pollen tubes in styles between the self-incompatible and self-compatible lines. Few pollen grains of open flower self-pollina--tion attached and germinated in the stigma of self-incompatible individuals, and intensive callose reaction occurred on the surface of stigma. The germinated pollen grains could not grown naturally and fail to penetrate into the stigma with the abnormality of distorting of pollen tubes, swelling of the tops and failure to further growth. Most pollen grains of self-pollination both in the bud and at anthesis germinated naturally and penetrated into the stigmas and finally succeeded in reaching to the ovary in self-compatible individuals, and theses also occurred in the bud self-pollination in self-incompatible dividuals. The self-incompatibility (SI) indexes of all the lines were tested with the traditional index method, and it is high accordant with the results from the fluoroscope observation method, indicating that self-incompatibility of radish lines can be identified with the fluoroscope observation method. Therefore fluoroscope observation method is a good efficiently tool for development of elite self-incompatibility in radish breeding program.
2. Polymorphism of the S-locus glycoprotein gene, SLG and the S locus receptor kinase gene, SRK which participate in the pollen-stigma interaction of self-incompatibility in Raphanus sativus were analyzed by PCR-RFLP using SLG- and SRK- specific primers. 19 inbred lines of R.sativus could be grouped into 12 S haplotypes by PCR with class I SLG specific primers showed different profiles up polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. 20 inbred lines of R.sativus could be grouped into 13 S haplotypes by PCR with SRK specific primers. 17 inbred lines of R.sativus could be grouped into 6 S haplotypes by PCR with class II SLG specific primers. Therefore the PCR-RFLP technology is very important in identification and classification of S haplotypes in Raphanus sativus. The nucleotide sequences of the DNA fragments of 5 R.sativus SLG alleles and 6 R.sativus SRK alleles were determinded. Degrees of similarity of the R.sativus SLG deduced amino-acid sequences to a Brassica SLG6 were 80.72%~84.26%. Phylogenetic analysis of the SLG sequence from Raphanus and Brassica revealed that the Raphanus SLGs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs. These results suggested that diversification of the SLG alleles of Raphanus and Brassica before differentiation of the genera.
引文
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