Analysis Of Multi-Ovary Wheat's Cytoplasm And Unclear Gene By SSR Molecular Marker

Wheat is one of the most widely grown crop in the world and it is the most important food crop of northern China. Heterosis exists in crops and horticultural crops generally. Wheat is conferred with observable heterosis as well as other crops. Therefore, research and application of hybrid wheat has great economic and social benefits. The research on wheat heterosis has made great advance after several decades, but it is still difficult to extend hybrid wheat in a larger area because of low propagation coefficient of wheat seed breeding. Multi-ovary character as a new properties of wheat was discovered in our country, has 2-3 pistils and three stamens in its flower. It has the advantage of stable seed-setting and multiple grain leading to the improvement of propagation coefficient. Multi-ovary wheat has a great future in improving the benefit of hybrid wheat seed production. In this study, part of multi-ovary wheat's nuclear gene and cytoplasmic gene was used to SSR molecular marker and genetic diversity analysis. The following achievements were obtained.1. A suitable SSR-PCR amplification system for multi-ovary wheat's gene was established by orthogonal experimental design and gradient PCR. The optimized SSR-PCR reaction system was: 10×Buffer2.0μl, Mg²⁺(25mM)1.5μl, dNTP(2.5mM) 1.6μl, SSR primers (2μmol/L) 2.0μl, DNA templates(50ng/μl) 2.0μl, ddH2O10.9μl.The appropriate primer annealing temperature should be lower than its Tm value of 4-6℃.2. In this study, dwarfing sterile wheat was used as instrumental material, duo II was used as donor parent, and 77(2)was used as recurrent male parent. The multi-ovary near-isogenic lines (NIL) was preliminary cultivated after successive backcrossing(5 times) and crossing. Genetic similarity and genetic distance of 23 NIL materials and their recurrent parents were compared and analysised by using SSR. The results showed:(1)All the selected 42 pairs of SSR primers had amplified 92 bands, 53 bands(57.61%) were polymorphic and 30 pairs(71.43%) of primers were polymorphic. (2)The genetic similarity coefficient of 23 materials and their recurrent parents was between 0.77 and 0.95, with an average of 0.88. (3)Cluster analysis revealed 23 NIL materials and their recurrent parents into 7 classes with the similarity coefficient of 0.84. 70.83% of them were clustered into the first class; the first class included 3 subclasses. The multi-ovary material 2008H69 was the least different from the recurrent parent, it was clustered into a small class with the similarity coefficient of 0.93. 3. Multi-ovary wheat duo II was reciprocal crossed with Zangye. All plants F1 were multi-ovary, the ratio of multi-ovary: mono-ovary in F2 was suitable 3:1. The result indicates that on the premise of cytoplasm of common wheat, the multi-ovary character of wheat was controlled by one dominant nuclei-gene. A multi-ovary and a mono-ovary gene pools were established from F2 segregation population by BSA method. 80 primers were used to SSR and the result showed that there wasn't any amplification products of two pairs of primers. Other 78 pairs of primers had amplified 202 bands, 31(15.35%) were polymorphic and 10 pairs of primers were polymorphic. All of 10 pairs of primers can be used as the molecular marker between the two parents.4. Multi-ovary wheat duo II was reciprocal crossed with 11 kinds of wheat with different types of cytoplasm, the result showes: 7 kinds of alloplasmic wheat and a material with common wheat cytoplasm have cytoplasm effects, make the multi-ovary gene expression obstruct in F1 heterozygous dominant gene type.5. mtDNA of multi-ovary wheat and alloplasmic wheat was extracted and used to SSR and polymorphism analysis. The result shows: (1) mtDNA extracted by our method has the character of high purity, good quality, but low concentration. So we can use wheat leaves of the field in stead of etiolated tissues. (2) All the selected 21 pairs of SSR primers had amplified 57 bands, 43 bands (78.95%) were polymorphic and 13 pairs of primers (61.90%) were polymorphic. Two primers WMt1 and WMt8 which can marker the multi-ovary character were fround. (3) Cluster analysis revealed 12 kinds of wheat into 2 classes with the similarity coefficient of 0.71. Two alloplasmic wheat with Ae.gilops cytoplasm which had no cytoplasm effects 02-11-29,20-11-30 clustered together with multi-ovary wheat duo II, the other nine kinds of wheat existing cytoplasm effects in the second category. (4) F1 population have high genetic similarity with their donor parents, both at 0.89 level or above.