| Sugar beet(Beta vulgaris L.) is one of the most important sugar-production crop in the world, in order to find new sugar beet germline with advantaged traits and breeding more popular lines,more and more scientists have realized the necessity of molecular assisted breeding in cross-breeding. Cytoplasmic male sterility(CMS),which is caused by mitochondrial genes with coupled nuclear genes, is significant to breeding maintainer line plants.In this study,we used 200 strains of one-year-breeding sugar beet and 54 strains of two-year-breeding sugar beet as experimental materials,and identified their fertility genotype by the cytoplasm and nucleus types test.With this method,we can save a lot of breeding time and labor costs to select maintaining line and sterile line of sugar beet.We also observed the flower morphology of the experimental materials while flowering stage and test their pollen viability. Also,we designed primers to verify the difference between maintainer line and sterile line,the sugar beet DY5-O(maintainer line) and DY5-CMS(CMS line) were used as experimental materials,our conclusions are as following :(1) According to the mitochondrial VNTR polymorphism of different cytoplasmic genotypes of sugar beet material, 200 strains of one-year-breeding sugar beet cytoplasmic genotype were identified, except 4 strains are not identified, 196 strains were N type cytoplasm, which is the same as maintainer line; according to the polymorphism of beet nuclear genes bv ORF17- upstream region, we identified nucleus gene type,and found the ’xxzz’ type, which is the same as maintainer line,combined with cytoplasmic we selected 45 strains of maintainer line of sugar beet. The flower morphology showed these sugar beets’ anther is yellow, and the pollen can be stained,indicating that they are fertile.(2) Using the same method, the fertility genotypes of 54 strains of two-year-breeding sugar beet were identified, and 19 strains of them were maintainer line, while 12 strains were CMS line.The flower fertility type observation and pollen staining showed that the maintainer line of sugar beet were full pollen and can be stained, while CMS sugar beet’s anther is white, almost no pollen and cannot be stained.The fertility idenfication of their F1 strains were consistent with the expected, showed that our method in this study is correct.(3) We designed 29 pairs of specific primers according to the differences betweenmaintainer line and sterile line’s leaf genome and buds transcriptome. Using these primers we amplified the leaf DNA of the maintainer line(DY5-O) and sterile line(DY5-CMS), and successfully found that 7 pairs can identify their gene differences(some genes in CMS lines are absent in maintainer lines), the PCR reaction conditions were also been optimized, thus we found a new approach to identificate maintainer line and CMS line by molecular biological method.In conclusion, in this paper we found a new way to breeding maintainer line and sterile line of sugar beet, which laid a good foundation for molecular assisted breeding,and significantly shorten the breeding time of sugar beet. |
