本文關(guān)鍵詞： 賽葵黃脈病毒 云南賽葵黃脈病毒 群體遺傳變異 準種 雜草　出處：《西南大學》2017年碩士論文　論文類型：學位論文

【摘要】：賽葵黃脈病毒(Malvastrum yellow vein virus,MaYVV)和云南賽葵黃脈病毒(Malvastrum yellow vein Yunnan virus,MaYVYV)是雙生病毒科(Geminiviridae)菜豆金色花葉病毒屬(Begomovirus)的單組份病毒,并伴隨β衛(wèi)星分子(MaYVB和MaYVYB),屬不同致病類型。這兩種病毒主要侵染錦葵科和菊科中的雜草,且常常與其它雙生病毒發(fā)生復合侵染,但鮮有在作物上引起危害的報道。本實驗室前期對危害作物的雙生病毒的遺傳變異機制進行了系統(tǒng)的研究,為了進一步了解雙生病毒在作物和雜草上的遺傳變異機制及進化軌跡,本研究以主要侵染雜草的MaYVV和MaYVYV為研究對象,對兩種病毒的寄主范圍、發(fā)生分布以及遺傳變異規(guī)律進行了研究。從四川、福建、廣西、廣東和云南等地區(qū)采集了賽葵、勝紅薊、番茄、辣椒和甘薯等雜草和作物共計181份田間樣品,利用檢測雙生病毒及其伴隨β衛(wèi)星的通用引物分別對樣品進行PCR檢測。結(jié)果表明,在賽葵和勝紅薊等雜草上檢測到雙生病毒及其伴隨β衛(wèi)星,檢出率分別為69.06%和40.33%,在作物上沒有檢測到雙生病毒。進一步利用檢測MaYVV/MaYVB和MaYVYV/MaYVYB的特異性引物對樣品進行特異性檢測,結(jié)果表明,在雙生病毒及β衛(wèi)星檢測呈陽性的樣品中,MaYVV和MaYVB的檢出率分別為31.20%和53.42%;MaYVYV和MaYVYB的檢出率分別為41.60%和61.64%;這兩種病毒復合侵染的檢出率為32.26%。MaYVV和MaYVYV在賽葵和勝紅薊等雜草上發(fā)生普遍,且在賽葵上這兩種病毒的復合侵染檢出率高達31.18%,但在作物上未檢測到。相比MaYVV,MaYVYV的地理分布更廣泛。選取采自不同地區(qū)不同寄主的代表性樣品,通過PCR擴增、克隆和測序獲得四川賽葵SC933、廣西賽葵GX125和GX175共3條MaYVV分離物全序列,以及四川賽葵SC933和SC939、廣西賽葵GX125和GX199共4條MaYVYV分離物全序列。從GenBank數(shù)據(jù)庫下載已登錄的MaYVV和MaYVYV不同分離物的全基因組序列,對其群體遺傳變異進行分析。結(jié)果表明,采自不同地區(qū)、不同寄主及不同年份的MaYVV遺傳變異水平之間具有明顯的遺傳差異,這說明MaYVV群體的遺傳變異受到地理因素、寄主因素及時間變遷的強烈影響,而MaYVYV群體的遺傳變異則主要受到地理因素影響。對病毒群體進行中性分析,發(fā)現(xiàn)MaYVV群體的中性檢測參數(shù)D值為0.87616~1.13887,均為正值,而MaYVYV群體的中性檢測參數(shù)D值為-2.48095~-2.05184,均為負值且差異顯著,表明,MaYVV群體呈現(xiàn)出平衡或穩(wěn)定的態(tài)勢,而MaYVYV群體則呈現(xiàn)正在擴張的明顯趨勢。這兩種病毒在種內(nèi)均未檢測到重組現(xiàn)象。遺傳多樣性分析發(fā)現(xiàn),MaYVV群體的單倍型多樣性(Hd)和核苷酸多樣性(Pi)分別為0.944±0.070和0.02433±0.00315,相應的MaYVYV群體的Hd和Pi值分別為0.971±0.033和0.00414±0.00046,表明MaYVV和MaYVYV群體均具有豐富的遺傳多樣性。其IR區(qū)的Pi值分別為0.06467±0.00912和0.00567±0.00177,大于編碼區(qū)AC1、AC4及AV1的Pi值。說明,MaYVV和MaYVYV群體的IR區(qū)的遺傳變異最大,受到選擇壓力較弱;編碼區(qū)AC1、AC4及AV1區(qū)較為保守,所受到的選擇壓力較強。以接種MaYVV Y47A分離物(Y47A)及其伴隨β衛(wèi)星(Y47β)的本氏煙為材料分別構(gòu)建MaYVV的實驗種群B60(60dpi)和B120(120dpi),同時以四川賽葵樣品為材料構(gòu)建MaYVV的自然種群SC906和SC915,以四川賽葵、廣西賽葵及廣西勝紅薊為材料構(gòu)建MaYVYV的自然種群SC933、GX149和GX161,對MaYVV和MaYVYV實驗種群及自然種群的遺傳結(jié)構(gòu)及變異進行分析。結(jié)果表明,兩種病毒的種群遺傳結(jié)構(gòu)均符合病毒“準種”特征。MaYVV實驗種群B60的突變克隆百分率和突變頻率分別為22.72%和2.03×10~(-4),種群B120的突變克隆百分率和突變頻率分別為51.52%和5.18×10~(-4),表明MaYVV的實驗種群遺傳變異水平隨著時間的推移呈升高趨勢。MaYVV自然種群SC906和SC915的突變頻率分別為2.54×10~(-4)和8.55×10~(-4),略高于實驗種群,而MaYVYV的自然種群SC933、GX149和GX199的突變頻率分別為9.26×10~(-4)、1.52×10~(-3)和9.59×10~(-4),均高于MaYVV自然種群的突變頻率。進一步分析發(fā)現(xiàn),各種群在IR區(qū)的突變較集中,該區(qū)域變異最強,突變頻率在10~(-3)數(shù)量級水平。在MaYVV實驗種群中,IR區(qū)突變位點主要集中在IR區(qū)3′端的2635位(主要是堿基G突變?yōu)閴A基A);在MaYVYV自然種群中,除種群GX161外,其余種群的IR區(qū)突變位點主要集中在IR區(qū)3′端的2669位(主要是堿基C突變?yōu)閴A基T)。推測這兩個位點可能是突變熱點。AC1和AC4重疊區(qū)比AC1非重疊區(qū)的變異水平低,表明MaYVV和MaYVYV種群在編碼區(qū)的突變分布不均衡。兩種病毒的變異類型以堿基C突變?yōu)閴A基T為主,有少量堿基插入和缺失突變類型。
[Abstract]:Malvastrum yellow vein virus (Malvastrum yellow vein virus, MaYVV) and Yunnan Malvastrum yellow vein virus (Malvastrum yellow vein Yunnan virus, MaYVYV) is Geminiviridae (Geminiviridae) begomovirus (Begomovirus) virus with single component, and beta satellite molecules (MaYVB and MaYVYB), belonging to different pathogenic types these two kinds of virus infection. The main weeds in Malvaceae and Compositae, and often mixed infection with other geminiviruses, but rarely cause harm report on crops. The genetic variation mechanism in the previous crop harm to twin virus were studied, in order to further understand the mechanism of genetic variation in crop and the weeds and the evolutionary trajectory of geminivirus, this study mainly infected weed MaYVV and MaYVYV as the research object, the host range of two of the virus, and the occurrence and distribution of genetic variation in For the study. From Sichuan, Fujian, Guangxi, Guangdong and Yunnan areas were collected Malvastrum, ageratum, tomato, pepper and sweet potato and other crops and weeds for a total of 181 field samples were detected by PCR, the samples were detected by geminiviruses and associated beta satellite universal primers. The results showed that to geminivirus and with the beta satellite detection in Malvastrum a.conyzoides and weeds, detection rates were 69.06% and 40.33%, did not detect geminiviruses on crops. The further use of specific primers for detection of MaYVV/MaYVB and MaYVYV/MaYVYB for specific detection of the samples, the results show that the satellite in geminiviruses and beta test positive samples in the MaYVV, and the detection rate of MaYVB were 31.20% and 53.42%; MaYVYV and MaYVYB were detected in 41.60% and 61.64%; the detection of the two viruses mixed infection rate of 32.26%.MaYVV and MaYVYV in Malvastrum and wins The widespread occurrence of Ageratum weed, and mixed infection of the two viruses in Malvastrum on detection rate as high as 31.18%, but the crop is not detected. Compared to MaYVV, the wider geographical distribution of MaYVYV. Were collected from different regions of different host representative samples by PCR amplification, cloning and sequencing Sichuan Malvastrum SC933 Guangxi and GX175 GX125, Malvastrum a total of 3 isolates of MaYVV sequence, and Sichuan SC933 Malvastrum and SC939, GX125 and GX199 Guangxi Malvastrum a total of 4 isolates of MaYVYV sequences. The whole genome sequence of MaYVV and MaYVYV of different isolates download logged in from the GenBank database, the genetic the variation of population was investigated. The results showed that collected from different regions, there are obvious genetic differences between different hosts and different MaYVV levels of genetic variation, indicating that the genetic variation of MaYVV populations by geographic factors, host factors and time change The strong influence of genetic variation of MaYVYV population is mainly affected by geographical factors. Neutral analysis of the virus group, D MaYVV group found that neutral detection parameters value of 0.87616~1.13887, and D were all positive, neutral detection parameters of group MaYVYV was -2.48095~ -2.05184, were negative and significant difference, show that the MaYVV group showing a balance or stability of the situation, while the MaYVYV group showed a obvious trend is expanding. The two viruses were not detected in intraspecific recombination. Genetic diversity analysis showed that MaYVV population haplotype diversity (Hd) and nucleotide diversity (Pi) were 0.944 + 0.070 and 0.02433 + 0.00315 and the corresponding MaYVYV groups Hd and Pi were 0.971 + 0.033 and 0.00414 + 0.00046, indicating that MaYVV and MaYVYV populations had abundant genetic diversity. The IR values of Pi were 0.06467 + 0.00912 and 0.00567 More than + 0.00177, encoding AC1 AC4 and AV1 Pi value. MaYVV and MaYVYV groups, IR area of greatest variation under weaker selection pressure; encoding area AC1, AC4 and AV1 are more conservative, have strong selection pressure. With the inoculation of MaYVV Y47A isolate (Y47A) and its accompanying satellite beta (Y47 beta) experimental population of B60 benthamiana as materials were constructed MaYVV (60dpi) and B120 (120dpi), at the same time to Sichuan Malvastrum samples for material in the construction of natural populations of MaYVV SC906 in Sichuan and SC915, Malvastrum, Guangxi and Guangxi Malvastrum a.conyzoides as the material of natural population SC933. The construction of MaYVYV, GX149 and GX161, the genetic structure and variation of MaYVV and MaYVYV of experimental population and natural populations were analyzed. The results showed that the population genetic structure of two viruses are in line with the virus quasi species characteristics of experimental population of.MaYVV B60 mutant clone percentage and mutation frequency were 22.7 2%鍜，

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