基于向日葵銹菌轉(zhuǎn)錄組測序的SSR標(biāo)記及遺傳多態(tài)性分析
發(fā)布時(shí)間：2018-08-08 19:54
【摘要】：向日葵銹病(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測序技術(shù)獲得的向日葵銹菌轉(zhuǎn)錄組數(shù)據(jù),通過軟件進(jìn)行大規(guī)模轉(zhuǎn)錄組簡單重復(fù)序列(simple sequence repeat,SSR)標(biāo)記的發(fā)掘,同時(shí)對其組成、分布及特征進(jìn)行分析。使用primer5.0軟件,對挖掘的包含SSR序列的轉(zhuǎn)錄本設(shè)計(jì)引物探究向日葵銹菌SSR-PCR反應(yīng)的最佳條件。使用UPGMA法聚類40個(gè)向日葵銹菌菌樣SSR分析結(jié)果,并進(jìn)行了遺傳多態(tài)性分析和組群劃分。1.根據(jù)向日葵銹菌轉(zhuǎn)錄組數(shù)據(jù),使用Perl操作平臺M1SA軟件對向日葵銹菌轉(zhuǎn)錄組序列進(jìn)行高通量微衛(wèi)星SSR位點(diǎn)發(fā)掘。包含SSR的序列有1728條,共1815個(gè)SSR。有192種重復(fù)基元(motif)模式出現(xiàn)在1815條完美型SSR中共,其中各個(gè)重復(fù)基元出現(xiàn)頻率最多類型和百分百如下,二堿基重復(fù)(AT/AT)n 56.22%、三堿基重復(fù)(AAT/ATT)n 13.06%、四堿基重復(fù)(TTTG/CAAA)n 15.07%、五堿基重復(fù)(TTTTC/GAAAA)n 13.33%和六堿基重復(fù)(ATCCCT/AGGGAT)n 50.00%。微衛(wèi)星長度從18bp到84bp不等,平均長21.34bp。長度在18到25之間的序列在向日葵銹菌微衛(wèi)星中占極大比例,長度大于25的微衛(wèi)星僅占微衛(wèi)星共有42條。研究結(jié)果顯示,微衛(wèi)星與基因平均表達(dá)水平存在關(guān)聯(lián),含微衛(wèi)星基因的平均表達(dá)水平低于不含微衛(wèi)星基因的平均表達(dá)水平。1728條Unigene中只有649個(gè)(37.56%)得到了相應(yīng)的GO分類號,62.44%不能成功注釋,其中587條序列被注釋為分子功能,264條被注釋到生物過程,423條被注釋到細(xì)胞組分。2.設(shè)計(jì)試驗(yàn)探索適宜向日葵銹菌SSR-PCR反應(yīng)的最佳條件。根據(jù)普通PCR反應(yīng)條件,改變影響PCR反應(yīng)的4個(gè)因素(模板DNA、引物濃度、dNTPs和Taq酶催化活性濃度)之一,進(jìn)行反應(yīng)體系優(yōu)化,并通過溫度梯度試驗(yàn)優(yōu)化引物退火溫度。PCR最佳優(yōu)化體系:25μL體系中,模板DNA質(zhì)量濃度為20ng,引物用量為0.6μL(10mm),dNTPs用量為1μL(2.5mmol/L),Taq酶催化活性濃度為2.OμL(3U/L)。最佳擴(kuò)增程序:預(yù)變性94℃ 5min,變性94℃ 1min,退火Tm℃(退火溫度隨引物不同而定)1min20s,延伸72℃ 1 min,35個(gè)循環(huán)后72 ℃延伸10min;25μL反應(yīng)體系。3.設(shè)計(jì)了 170對向日葵銹菌SSR引物,30對引物可以在向日葵銹菌中有效擴(kuò)增。選取10對引物作為核心引物分析40份向日葵銹菌樣品遺傳多態(tài)性。10條引物共擴(kuò)增出35條帶,32條條帶具有多態(tài)性,多態(tài)性條帶比率為91.42%,每個(gè)引物可擴(kuò)增2～4條多態(tài)性條帶。以遺傳相似性系數(shù)GS=0.81456為閾值,40份向日葵銹菌樣品按照地理位置大致聚類到三個(gè)組群,分別是群1:內(nèi)蒙古東部、吉林、黑龍江;群2:內(nèi)蒙古中部、山西、河北;群3:內(nèi)蒙古西部、新疆、寧夏。按照地理緯度分布,向日葵銹菌的三個(gè)組群分別對應(yīng)濕潤、半濕潤大陸性季風(fēng)氣候、干旱或半干旱內(nèi)陸氣候和半濕潤半干旱內(nèi)陸高原氣候。
[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，
本文編號：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，
本文編號：2172879
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