基于向日葵锈菌转录组测序的SSR标记及遗传多态性分析
发布时间：2018-08-08 19:54
【摘要】：向日葵锈病(Pucciniahelianthi Schw.)是油料作物向日葵上的重要病害之一。本研究利用新一代的高通量Illumina测序技术获得的向日葵锈菌转录组数据,通过软件进行大规模转录组简单重复序列(simple sequence repeat,SSR)标记的发掘,同时对其组成、分布及特征进行分析。使用primer5.0软件,对挖掘的包含SSR序列的转录本设计引物探究向日葵锈菌SSR-PCR反应的最佳条件。使用UPGMA法聚类40个向日葵锈菌菌样SSR分析结果,并进行了遗传多态性分析和组群划分。1.根据向日葵锈菌转录组数据,使用Perl操作平台M1SA软件对向日葵锈菌转录组序列进行高通量微卫星SSR位点发掘。包含SSR的序列有1728条,共1815个SSR。有192种重复基元(motif)模式出现在1815条完美型SSR中共,其中各个重复基元出现频率最多类型和百分百如下,二碱基重复(AT/AT)n 56.22%、三碱基重复(AAT/ATT)n 13.06%、四碱基重复(TTTG/CAAA)n 15.07%、五碱基重复(TTTTC/GAAAA)n 13.33%和六碱基重复(ATCCCT/AGGGAT)n 50.00%。微卫星长度从18bp到84bp不等,平均长21.34bp。长度在18到25之间的序列在向日葵锈菌微卫星中占极大比例,长度大于25的微卫星仅占微卫星共有42条。研究结果显示,微卫星与基因平均表达水平存在关联,含微卫星基因的平均表达水平低于不含微卫星基因的平均表达水平。1728条Unigene中只有649个(37.56%)得到了相应的GO分类号,62.44%不能成功注释,其中587条序列被注释为分子功能,264条被注释到生物过程,423条被注释到细胞组分。2.设计试验探索适宜向日葵锈菌SSR-PCR反应的最佳条件。根据普通PCR反应条件,改变影响PCR反应的4个因素(模板DNA、引物浓度、dNTPs和Taq酶催化活性浓度)之一,进行反应体系优化,并通过温度梯度试验优化引物退火温度。PCR最佳优化体系:25μL体系中,模板DNA质量浓度为20ng,引物用量为0.6μL(10mm),dNTPs用量为1μL(2.5mmol/L),Taq酶催化活性浓度为2.OμL(3U/L)。最佳扩增程序:预变性94℃ 5min,变性94℃ 1min,退火Tm℃(退火温度随引物不同而定)1min20s,延伸72℃ 1 min,35个循环后72 ℃延伸10min;25μL反应体系。3.设计了 170对向日葵锈菌SSR引物,30对引物可以在向日葵锈菌中有效扩增。选取10对引物作为核心引物分析40份向日葵锈菌样品遗传多态性。10条引物共扩增出35条带,32条条带具有多态性,多态性条带比率为91.42%,每个引物可扩增2～4条多态性条带。以遗传相似性系数GS=0.81456为阈值,40份向日葵锈菌样品按照地理位置大致聚类到三个组群,分别是群1:内蒙古东部、吉林、黑龙江;群2:内蒙古中部、山西、河北;群3:内蒙古西部、新疆、宁夏。按照地理纬度分布,向日葵锈菌的三个组群分别对应湿润、半湿润大陆性季风气候、干旱或半干旱内陆气候和半湿润半干旱内陆高原气候。
[Abstract]:Sunflower rust (Pucciniahelianthi Schw.) It is one of the important diseases of sunflower. In this study, we used the new generation of high-throughput Illumina sequencing technology to obtain the sunflower rust transcriptional data, and used software to excavate the simple repeat sequence (simple sequence repeat SSR markers of large-scale transcriptional group, and to analyze its composition, distribution and characteristics. Primer5.0 software was used to design primers to explore the optimal conditions for SSR-PCR reaction of sunflower rust. UPGMA method was used to cluster 40 samples of sunflower rust by SSR analysis, and genetic polymorphism analysis and cluster division. 1. Based on the transcriptional data of sunflower rust, the high-throughput microsatellite SSR sites were detected by Perl operating platform M1SA software. There are 1728 sequences containing SSR, 1815 SSRs. There are 192 types of repetitive primitive (motif) patterns appearing in 1815 perfect SSR, of which each repeats the most frequent types and 100% of them are as follows. Two base repeats (AT/AT) n 56.22, three base repeats (AAT/ATT) n 13.06, four base repeats (TTTG/CAAA) n 15.07, five base repeats (TTTTC/GAAAA) n 13.33% and six base repeats (ATCCCT/AGGGAT) n 50.00g. The length of microsatellite ranged from 18bp to 84bp with an average length of 21.34bp. Sequences between 18 and 25 account for a large proportion of sunflower rust microsatellites, and only 42 microsatellites are larger than 25 in length. The results showed that microsatellites were associated with the average gene expression level. The average expression level of microsatellite gene was lower than that without microsatellite gene. Only 649 (37.56%) of the 1728 Unigene had the corresponding go classification number and 62.44% could not be successfully annotated. Of these, 587 sequences were annotated as molecular functional 264, and 423 in biological processes were annotated as cell components. The optimum conditions for SSR-PCR reaction of sunflower rust were investigated. According to the general PCR reaction conditions, the reaction system was optimized by changing one of the four factors (template DNA, primer concentration and Taq enzyme catalytic activity concentration) affecting the PCR reaction. Temperature gradient test was used to optimize the optimal system of primer annealing. The optimal system was: the concentration of template DNA was 20ng, the amount of primer was 0.6 渭 L (10mm), the dosage of primer was 1 渭 L (2.5mmol/L) Taq enzyme activity concentration was 2.0 渭 L (3U/L). The optimal amplification procedure was as follows: predenaturation at 94 鈩，
本文编号：2172879
[Abstract]:Sunflower rust (Pucciniahelianthi Schw.) It is one of the important diseases of sunflower. In this study, we used the new generation of high-throughput Illumina sequencing technology to obtain the sunflower rust transcriptional data, and used software to excavate the simple repeat sequence (simple sequence repeat SSR markers of large-scale transcriptional group, and to analyze its composition, distribution and characteristics. Primer5.0 software was used to design primers to explore the optimal conditions for SSR-PCR reaction of sunflower rust. UPGMA method was used to cluster 40 samples of sunflower rust by SSR analysis, and genetic polymorphism analysis and cluster division. 1. Based on the transcriptional data of sunflower rust, the high-throughput microsatellite SSR sites were detected by Perl operating platform M1SA software. There are 1728 sequences containing SSR, 1815 SSRs. There are 192 types of repetitive primitive (motif) patterns appearing in 1815 perfect SSR, of which each repeats the most frequent types and 100% of them are as follows. Two base repeats (AT/AT) n 56.22, three base repeats (AAT/ATT) n 13.06, four base repeats (TTTG/CAAA) n 15.07, five base repeats (TTTTC/GAAAA) n 13.33% and six base repeats (ATCCCT/AGGGAT) n 50.00g. The length of microsatellite ranged from 18bp to 84bp with an average length of 21.34bp. Sequences between 18 and 25 account for a large proportion of sunflower rust microsatellites, and only 42 microsatellites are larger than 25 in length. The results showed that microsatellites were associated with the average gene expression level. The average expression level of microsatellite gene was lower than that without microsatellite gene. Only 649 (37.56%) of the 1728 Unigene had the corresponding go classification number and 62.44% could not be successfully annotated. Of these, 587 sequences were annotated as molecular functional 264, and 423 in biological processes were annotated as cell components. The optimum conditions for SSR-PCR reaction of sunflower rust were investigated. According to the general PCR reaction conditions, the reaction system was optimized by changing one of the four factors (template DNA, primer concentration and Taq enzyme catalytic activity concentration) affecting the PCR reaction. Temperature gradient test was used to optimize the optimal system of primer annealing. The optimal system was: the concentration of template DNA was 20ng, the amount of primer was 0.6 渭 L (10mm), the dosage of primer was 1 渭 L (2.5mmol/L) Taq enzyme activity concentration was 2.0 渭 L (3U/L). The optimal amplification procedure was as follows: predenaturation at 94 鈩，
本文编号：2172879
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