Identification And Screening On Allelic Variations For Hardness In Wheat

In this research, the hardness types of 85 varieties of Chinese wheat were screened and classified by such methods as SKCS,PCR amplification using specific primers and modified SDS-PAGE while the screening was eliminated one kind by one kind. The feasibility and improvement were further discussed at the same time as the theory bases were supplied for the variety resources screening, identification in early generations, breeding materials selecting, quality and character analyzing of the wheat hardness. Then we got the results as follows: 1. Measured by SKCS, there are 37 hard wheat varieties whose hardness indexes were more than 50, 17 mixed varieties whose hardness indexes were between 25-65 and 31 soft varieties whose hardness indexes were less that 41. They occupy 43.5%,20% and 36.5% respectively in whole. Both the hard and mixed varieties have mutations in the major genes controlling the grain hardness. The mixed types may be caused by people's behavior or environment reasons to make the hardness index of some kernels lower. 2. 54 varieties were amplified and identified by STS specific primers which were designed on the mutated-type site of Pinb-D1b and the corresponding wild-type site of Pinb-D1a. The results show that at 56℃ of the annealing temperature, there are polymorphism between the two amplified clear bands which is good on stability. So the primers designed on the mutated-type site of Pinb-D1b are feasible for screening. 42 varieties of wheat are determined to be the mutated-type of Pinb-D1b and occupy 77.8% in the whole 54 hard and mixed varieties. 3. The amplified whole Pinb can be cleaved to two bands by restriction enzyme PvuⅡwhen the site is wild, or can't be cleaved. Except the wild and Pinb-D1b two alle types, all the other 12 varieties can be cleaved and determined to be not the mutated-type of Pinb-D1c. The 12 varieties were amplified and identified by STS primers which were designed on the mutated-type site of Pinb-D1d,Pinb-D1e,Pinb-D1f and Pinb-D1g and the corresponding wild-type site of the four. The amplified products are not good on stability. We can't use these designed primers as primers designed on the mutated-type site of Pinb-D1b. 4. The subunits Pruoindoline-a and Pruoindoline-b can be discerned easily in the gel of SDS-PAGE when the N'-N' acrylamide was displaced of PDA. And the expressed protein's absence or presence of pinA can be discerned easily too. Detected by SDS-PAGE, 6 varieties which occupy 11.1% were pinA-null in the whole 54 hard and mixed varieties. 5. Though there are a lot of mutated types, the designed screening methods are limited. Measured to the gene sequences of remained 6 varieties unidentified, we can find all of them are Pinb-D1p which is abundant in Chinese varieties and they occupy 11.1% in the whole 54 hard and mixed varieties too. 6. The grain hardness are affected by different mutation types: the hardness mean of pinA-null is 69.000 and the largest in all the three types which means that pinA-null affect the hardness most. The hardness means of Pina-D1a/Pinb-D1b and Pina-D1a/Pinb-D1p are 58.810 and 55.500. The smaller means mean pinb mutation types affect the hardness comparatively less.