Research On Construction Of Xiangzamian SSR Fingerprint And Detection Of Seed Purity

This paper conducted research on Xiangzamian series and parts of their parents-line materials planted on the Yangtze River area,using the method of SSR molecular markers,and then a set of highly effective,accurate and reliable molecular technique identification system was set up.Primers with polymorphism on Xiangzamian and its parent lines were screened and the DNA fingerprints of Xiangzamian were constructed.It can be applied on the detection of Xiangzamian seed purity and the unknown status recognition.The main results were summarized as follows:1.The establishment of SSR technology system fitted for cotton seed DNA:the total reaction volume was 10μl,containing 0.4μl of Mg²⁺(25mM),0.2μl of dNTPs (10mM).0.6μl of SSR forward and reverse primers(10μM) respectively,0.5μl of TaqDNA polymerase(1U/μl),1.0μl of 10×Reaction buffer,0.5μl of template DNA (50ng/μl).The outputs of PCR was shown through electrophoresis on 8% poly-crylamide at constant voltage(180V) for 70 minute,and then revealed the bands through modified silver stained method.2.The construction of fingerprinting for Xiangzamian by SSR markers:331 pairs of SSR primers were selected from 26 pairs of chromosomes of cotton.Among these, there were 31 pairs of primers performed polymorphisms,accounting for 9.37%.19 pairs of primers out of 31 were used to establish the Xiangzamian DNA fingerprints.3.The detection of Xiangzamian seed purity:detected the seed purity of Xiangzamian 2 and 6 respectively by using single multiple PCR and double multiple PCR.The purity obtained were 96%and 98%,this is quite close to the field purity about 96.2%and 97.8%.It proved that it was feasible to use the method of SSR molecular markers to detect hybrid cotton.At the same time,it testified the important principle concerning multiple combination of PCR should follows.4.The recognition of unknown status:using the constructed fingerprint bands, combining with calculation about the genetic similarity coefficient and clustering analytic comparison,we can recognize the unknown status.