基于荧光检测技术的青稞品种鉴定方法的建立

Establishment of a Method Identifying the Varieties of Hulless Barley Based on Fluorescence Detection Technology

本研究通过筛选出一套SSR核心引物,建立青稞DNA指纹鉴定体系,为青稞品种选育、品种鉴定提供高通量检测技术方法。首先以形态差异大的24份种质为材料,对198对SSR引物进行初筛,筛选出42对多态性好、扩增稳定、条带清晰的SSR引物,并对之进行荧光标记。采用筛选出的42对引物对226份材料进行PCR扩增,扩增产物经毛细管电泳检测,最终筛选出28对SSR荧光引物建立基于高通量荧光SSR标记的青稞品种鉴定体系。采用28对SSR引物构建的DNA指纹图谱进行226个青稞材料的鉴定。28对SSR引物共检测到252个等位基因,每对引物可检测到有效等位基因数为5-16个,平均等位基因数为9;基因多样性指数变异范围为0.48-0.86,平均为0.68;多态信息量(PIC)变幅为0.44-0.84,平均为0.65。226份材料的遗传距离为0-0.99,该套引物可以将大部分参试材料区分开。从28对SSR引物中选择了7对分布于不同染色体上、扩增和检测效果较好的引物(Bmag0211、Scssr07759、HVM62、EBmac0679、Scssr03907、Bmag0870、EBmac0827),构建青稞品种(系)DNA的指纹图谱库。为消除不同检测平台、不同试验批次等引起的误差,为7对SSR核心引物各主要等位变异选择相应的参照品种,作为普通聚丙烯酰胺凝胶电泳的读带依据。最终构建了在2个检测平台(毛细管电泳平台及聚丙烯酰胺凝胶电泳平台)通用的SSR分子标记青稞品种鉴定体系。

A series of SSR core primers were screened to provide a DNA fingerprinting of hulless barley varieties,which may provide high-throughput detection method for the breeding and variety identification of hulless barley.First of all,24 hulless barley germplasms with large morphological differences were used,42 primers were screened from the original 198 SSR primers,which had clear amplification bands,rich polymorphism and stable amplification.These screened primers were labeled with fluorescent,and used for the PCR amplification of the total 226 cultivars.The amplified products were detected by capillary electrophoresis.Finally,28 pairs of SSR fluorescent primers were screened to establish an identification system for hulless barley variety based on the high-throughput fluorescent SSR marker.And the selected 28 primers were used to construct DNA fingerprinting for identifying the 226 cultivars of hulless barley.A total of 252 alleles were detected by 28 pairs of SSR primers,the effective number of alleles ranged from 5 to 16,and the average number of alleles was 9.The genetic diversity ranged from 0.48 to 0.86 and the average value was 0.68.The polymorphism information content(PIC)changed from 0.44 to 0.84 and the average value of PIC was 0.65.The genetic distance of 226 materials was 0-0.99.This set of primers distinguished the most of the test materials.Seven SSR primers(Bmag0211,Scssr07759,HVM62,EBmac0679,Scssr03907,Bmag0870,and EBmac0827)with good amplification and detection effect as well as distributed in different chromosomes were chosen from the 28 SSR primers,and they were used for constructing DNA fingerprinting spectrum.The corresponding reference cultivars to major allelic variations of 7 pairs of primers were selected to eliminate the errors caused by different platforms and experimental batches.These reference cultivars can be used as the basis for reading bands of ordinary polyacrylamide gel electrophoresis.In this study,a SSR high-throughput identification system was constructed in two detection platforms(fluorescent capillary electrophoresis platform and polyacrylamide gel electrophoresis platform)based on 28 pairs of primers.