Research On Ssr Molecular Markers In Genetic Diversity Of Wheat Hardness

Grain hardness, influencing the particle size of flour, flour yield, the number ofdamaged starch grains, wheat processing quality of the final production, is one of theimportant quality traits of wheat, and also an important parameter for grading andpricing in the wheat market at home and abroad. In this study, a region, containingmajor Pina and Pinb gene affecting hardness trait, had been searched for findingmicrosatellite repeat loci, and primers were designed in some microsatellite repeatloci. Through agarose gel electrophoresis, polyacrylamide electrophoresis, capillaryelectrophoresis and nucleotide sequencing, the genetic diversity of Pina and Pinb genein Qinghai bred, Tibet Wheat cultivars and Aegilops tauschii was studied. The resultswere followed:1. By analyzing187kb nucleotide sequence containing Pina and Pinb gene fromNCBI data bank,20microsatellite loci were found. Primers were designed in four lociwith high repeat motif. An effective amplification was found about primer of SSR7.After sequencing, the nucleotide sequence of amplification segment was consistentwith target sequence containing AG repeat region. The site locates in the promoterregion of Pina, and at495bp before the initiation codon of Pina, which means that itcan be used as a molecular marker for Pina gene.2.18types of segment were found in amplification products of SSR7primer inQinghai and Tibet wheat cultivars with capillary electrophoresis. The shortest typewas154bp, and the longest type was182bp. All varieties with fragment length greaterthan176bp were from Tibet cultivars. The Type with most varieties was171bp,containing22cultivars. The154bp and166bp type only contain one cultivar. Theywere y1904and Xiangnong3respectively.3. Pina-promoter primers containing SSR7microsatellite regions, was designedand used to amplify genome DNA of different type of Qinghai and Tibet wheatcultivars with different segments in capillary electrophoresis experiment, andAegilops tauschii. After sequencing amplification products, it was found that AGrepeats existed in all sequenced cultivars. Six kinds of repeat types were found, andthey were6,10,16,18,19,20times respectively. Ae.tauschii had only two kinds ofrepeat types,6and10, and the rest was common wheat varieties. So pina and pinb in common wheat should not come from Ae.tauschii material in this study.4. Compared with Ae.tauschii, common wheat contained one or two AC at the5’of SSR7region. Except AS61and AS82, Ae.tauschii had13bases deletion at660bpupstream of the start region of Pina. The differences of Nucleotide sequence betweenAe.tauschii and common wheat in SSR7sites and Pina gene promoter regions shouldgive a possible way to identifying the origin of Pina and Pinb gene in common wheat,Ae.tauschii and synthetic wheat.5. The same fragment length of the amplification production of SSR7Primer incapillary electrophoresis contained different grain hardness and hardness of allelicvariation type gene. This was most likely due to the change frequency of SSR lociwas much higher than SNP loci, and the region except SSR loci may also mutate.Further research should be employed for verifying this reason.These findings should be benefited for detecting allelic variation of Pina andPinb gene, and providing experimental materials and theoretical basis for molecularmarker-assisted selection breeding.