赛葵黄脉病毒和云南赛葵黄脉病毒的群体遗传变异研究

发布时间：2018-01-20 17:12

  本文关键词： 赛葵黄脉病毒 云南赛葵黄脉病毒 群体遗传变异 准种 杂草　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：赛葵黄脉病毒(Malvastrum yellow vein virus,MaYVV)和云南赛葵黄脉病毒(Malvastrum yellow vein Yunnan virus,MaYVYV)是双生病毒科(Geminiviridae)菜豆金色花叶病毒属(Begomovirus)的单组份病毒,并伴随β卫星分子(MaYVB和MaYVYB),属不同致病类型。这两种病毒主要侵染锦葵科和菊科中的杂草,且常常与其它双生病毒发生复合侵染,但鲜有在作物上引起危害的报道。本实验室前期对危害作物的双生病毒的遗传变异机制进行了系统的研究,为了进一步了解双生病毒在作物和杂草上的遗传变异机制及进化轨迹,本研究以主要侵染杂草的MaYVV和MaYVYV为研究对象,对两种病毒的寄主范围、发生分布以及遗传变异规律进行了研究。从四川、福建、广西、广东和云南等地区采集了赛葵、胜红蓟、番茄、辣椒和甘薯等杂草和作物共计181份田间样品,利用检测双生病毒及其伴随β卫星的通用引物分别对样品进行PCR检测。结果表明,在赛葵和胜红蓟等杂草上检测到双生病毒及其伴随β卫星,检出率分别为69.06%和40.33%,在作物上没有检测到双生病毒。进一步利用检测MaYVV/MaYVB和MaYVYV/MaYVYB的特异性引物对样品进行特异性检测,结果表明,在双生病毒及β卫星检测呈阳性的样品中,MaYVV和MaYVB的检出率分别为31.20%和53.42%;MaYVYV和MaYVYB的检出率分别为41.60%和61.64%;这两种病毒复合侵染的检出率为32.26%。MaYVV和MaYVYV在赛葵和胜红蓟等杂草上发生普遍,且在赛葵上这两种病毒的复合侵染检出率高达31.18%,但在作物上未检测到。相比MaYVV,MaYVYV的地理分布更广泛。选取采自不同地区不同寄主的代表性样品,通过PCR扩增、克隆和测序获得四川赛葵SC933、广西赛葵GX125和GX175共3条MaYVV分离物全序列,以及四川赛葵SC933和SC939、广西赛葵GX125和GX199共4条MaYVYV分离物全序列。从GenBank数据库下载已登录的MaYVV和MaYVYV不同分离物的全基因组序列,对其群体遗传变异进行分析。结果表明,采自不同地区、不同寄主及不同年份的MaYVV遗传变异水平之间具有明显的遗传差异,这说明MaYVV群体的遗传变异受到地理因素、寄主因素及时间变迁的强烈影响,而MaYVYV群体的遗传变异则主要受到地理因素影响。对病毒群体进行中性分析,发现MaYVV群体的中性检测参数D值为0.87616~1.13887,均为正值,而MaYVYV群体的中性检测参数D值为-2.48095~-2.05184,均为负值且差异显著,表明,MaYVV群体呈现出平衡或稳定的态势,而MaYVYV群体则呈现正在扩张的明显趋势。这两种病毒在种内均未检测到重组现象。遗传多样性分析发现,MaYVV群体的单倍型多样性(Hd)和核苷酸多样性(Pi)分别为0.944±0.070和0.02433±0.00315,相应的MaYVYV群体的Hd和Pi值分别为0.971±0.033和0.00414±0.00046,表明MaYVV和MaYVYV群体均具有丰富的遗传多样性。其IR区的Pi值分别为0.06467±0.00912和0.00567±0.00177,大于编码区AC1、AC4及AV1的Pi值。说明,MaYVV和MaYVYV群体的IR区的遗传变异最大,受到选择压力较弱;编码区AC1、AC4及AV1区较为保守,所受到的选择压力较强。以接种MaYVV Y47A分离物(Y47A)及其伴随β卫星(Y47β)的本氏烟为材料分别构建MaYVV的实验种群B60(60dpi)和B120(120dpi),同时以四川赛葵样品为材料构建MaYVV的自然种群SC906和SC915,以四川赛葵、广西赛葵及广西胜红蓟为材料构建MaYVYV的自然种群SC933、GX149和GX161,对MaYVV和MaYVYV实验种群及自然种群的遗传结构及变异进行分析。结果表明,两种病毒的种群遗传结构均符合病毒“准种”特征。MaYVV实验种群B60的突变克隆百分率和突变频率分别为22.72%和2.03×10~(-4),种群B120的突变克隆百分率和突变频率分别为51.52%和5.18×10~(-4),表明MaYVV的实验种群遗传变异水平随着时间的推移呈升高趋势。MaYVV自然种群SC906和SC915的突变频率分别为2.54×10~(-4)和8.55×10~(-4),略高于实验种群,而MaYVYV的自然种群SC933、GX149和GX199的突变频率分别为9.26×10~(-4)、1.52×10~(-3)和9.59×10~(-4),均高于MaYVV自然种群的突变频率。进一步分析发现,各种群在IR区的突变较集中,该区域变异最强,突变频率在10~(-3)数量级水平。在MaYVV实验种群中,IR区突变位点主要集中在IR区3′端的2635位(主要是碱基G突变为碱基A);在MaYVYV自然种群中,除种群GX161外,其余种群的IR区突变位点主要集中在IR区3′端的2669位(主要是碱基C突变为碱基T)。推测这两个位点可能是突变热点。AC1和AC4重叠区比AC1非重叠区的变异水平低,表明MaYVV和MaYVYV种群在编码区的突变分布不均衡。两种病毒的变异类型以碱基C突变为碱基T为主,有少量碱基插入和缺失突变类型。
[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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