| Tartary buckwheat, a diploid (2n=16) annual crop plant, is widely cultivated in Asia and Europe. Because tartary buckwheat can be grown at high altitudes, has a short growing season, and its grains contain high-quality proteins, it is an important alimentary crop in mountainous regions of China and in other countries in the northern hemisphere. Tartary buckwheat proteins contain a good balance of such amino acids and flavonoids as rutin, quercetin and kaempferol-3-rutinoside. Most tartary buckwheat grows in barren hill field thus the yield is not satisfactory. It is a challenging research for tartary buckwheat industrial development to increase resistance, quality and yield, moving up the integrated value of tartary buckwheat by molecular marker assisted breeding.Construction of genetic linkage map is the basis for molecular marker assisted breeding and also an important step of plant genome research. The accomplishment of genetic linkage map in tartary buckwheat will be invaluable for both targeting excellent gene and important agronomic traits and achieving marker assisted selection breeding.In this study,136 F2 obtained from controlled cross Dianningyihao and Wild tartary buckwheat is recognized as mapping population. SRAP and SSR reaction system of tartary buckwheat and the separation of markers were studied. SRAP and SSR markers were used to construct the first genetic linkage map of tartary buckwheat. Specific contents are as following:SRAP-PCR,SSR-PCR amplification system on tartary buckwheat was optimized by interactive orthogonal design L27(313) in 5 elements (Mg2+, dNTP, Taq DNA polymerase, template DNA and primer) at 3 levels, and were validated. An optimal reaction system of SRAP-PCR was established containing 1.5 mmol/L Mg2+,0.2 mmol/L dNTP,1.5 U Taq DNA polymerase,40 ng DNA,0.25μmol/L primer,10×buffer 2μl. An optimal reaction system of SSR-PCR was established containing 1 mmol/L Mg2+,0.3 mmol/L dNTP,1.5 U Taq DNA polymerase,30 ng DNA,0.25μmol/L primer,10×buffer 2μl.The PCR products were tested by denaturing and non-denaturing PAGE, and the results showed that the non-denaturing PAGE, DYCZ-24F operating system was more suitable for SRAP analysis.165 SRAP primers were used to amplify DNA of parental and Fl and 43(26.1%) primers gave polymorphic bands in the parents. The 43 polymorphic primers revealed 50 polymorphic SRAP loci in F2 population of these parents. Primers(MlE2,M3E3,M6E19,M10E4,MllE17)gave two polymorphic bands respectively and M1E6 gave three polymorphic bands. After Chi-square Test, among 50 polylmorphic loci obtained before,35 showed the expected Mendelian segregation ratio, consisting of a proportion of 70%. Among 148 SSR tested primers,26(17.6%) primers gave polymorphic bands in the parents. Among which,1 of 26 polymorphic loci with 3.8% proportion showed the expected Mendelian segregation ratio responding to Chi-square Test.Combining the all polymorphic SRAP and SSR markers in the population, a primary molecular genetic linkage map in tartary buckwheat was set up. The map consist 38 markers (37 SRAP and 1 SSR markers) in 10 linkage groups with a total length of 725.1 cM and an average interval of 25.9 cM. The size of the linkage groups ranged from 2.2 cM to 46.6 cM. The number of marker located in linkage group is from 2 to 6.The greatness of Genetic linkage map of tartary buckwheat constructed in this paper is significant markers in the map, specific amplification and good repeatability. Weakness are less markers and low density. We will prefect it by using SRAP markers to establish a more complete molecular genetic linkage map of tartary buckwheat. |
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