Construction and Application of SSR Marker Identification System for Bitter Gourd Varieties
[Objective]In order to provide a practical support for authenticity identification,purity identification and protection of new variety rights of bitter gourd,a set of SSR core primers were screened,and then,the molecular identification system and SSR fingerprint database were established.[Method]Firstly,by using 8 representative varieties with large phenotypic differences,the preliminary 138 pairs SSR primer screening were carried out through 6％polyacrylamide-gel electrophoresis,and the preliminarily screened primers were synthesized into fluorescent primers.Secondly,the primer re-screening was carried out by fluorescence capillary electrophoresis against 95 bitter gourd varieties with diverse geographical sources.Then,the discrimination rate,PIC value,number of alleles,and other parameters of each re-screened primer were calculated respectively,and a set of SSR primer combinations with high discrimination rate and good polymorphism were selected.Finally,the total of 208 bitter gourd varieties were tested by the re-screened primers and a set of SSR core primers were further selected and used to construct a DNA fingerprint database of bitter gourd varieties.[Result]45 pairs of SSR primers with high specificity,high polymorphism and clear bands were screened by polyacrylamide gel electrophoresis.20 pairs of SSR core primers were finally screened from small populations to large populations by fluorescence capillary electrophoresis.The core primers were divided into 4 groups for multiple electrophoresis,and DNA fingerprint data of 208 bitter gourd varieties were collected.The total of 65 alleles and 102 genotypes were detected by 20 pairs of SSR core primers.12 reference varieties were selected,which could cover all alleles.Through phylogenetic tree construction,208 bitter gourd varieties were divided into 2 categories,indicating that 20 pairs of SSR core primers were suitable for genetic diversity analysis of bitter gourd population.202 of 208 bitter gourd varieties could be identified by core primers,and the discrimination rate was 97.11％.11 groups of parents and hybrid materials were used to analyze genetic relationship and then conformed to Mendel's genetic law.There were only 1 locus of fragment loss in each 2 groups,which provided a reference for the decision threshold value of identification of bitter gourd hybrids.[Conclusion]In this study,the identification system of bitter gourd varieties constructed based on 20 pairs of SSR core primers had superior applicability and excellent identification effect,and could also be used for authenticity identification,purity identification,hybrids identification of bitter gourd varieties,similar varieties selection in DUS test and protection of new variety rights of bitter gourd.
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