我国禾谷缢管蚜微卫星位点扩增稳定性及遗传多样性

为筛选用于我国禾谷缢管蚜种群遗传学研究的微卫星位点,从8个省(市)共9个地区采集282头试虫,采用微卫星PCR产物荧光标记与自动扫描分型方法,研究了8个欧洲禾谷缢管蚜微卫星位点在试虫个体中的扩增稳定性和遗传多样性。结果显示:位点R1.35在9个种群中均不能扩增;位点R5.29b只在7个种群的少数样本中能扩增;位点R2.73、R3.171、R5.10、R5.138、R5.50和R6.3在各种群中均能稳定扩增,这6个位点的无效等位基因频率为0.0044~0.2663,平均等位基因数为2.9~9.3个,观测杂合度为0.047~0.912,其中位点R6.3具有较低的观测杂合度(0.047)和等位基因数(2.9),不适合用于种群遗传多样性研究,而位点R2.73、R3.171、R5.10、R5.138和R5.50均具有较高的杂合度和等位基因数,可用于我国禾谷缢管蚜的种群遗传学研究。     

苹果小吉丁虫微卫星开发及其种群遗传结构分析

为研究苹果小吉丁虫Agrilus mali Matsumura的种群遗传结构,采用磁珠富集法以生物素标记探针(AC)_(12)和(AG)_(12)构建苹果小吉丁虫微卫星文库,根据阳性克隆测序获得的微卫星侧翼序列设计引物,筛选和开发苹果小吉丁虫多态性微卫星标记,并利用开发的微卫星标记对其不同地理种群的遗传结构进行分析。结果显示,从微卫星文库中的300个阳性克隆中筛选得到248个含有微卫星片段的克隆子,阳性克隆率为82.7%,其中完美型微卫星131个,占52.8%;不完美型90个,占36.3%;混合型27个,占10.9%,表明磁珠富集法开发新微卫星标记的效率较高。从获得的248个苹果小吉丁虫微卫星中筛选获得7个多态性微卫星位点,这7个位点在苹果小吉丁虫8个地理种群样本中的等位基因数为10~23个,有效等位基因数为1.674~12.218个,多态信息含量为0.304~0.796。8个苹果小吉丁虫种群的观测杂合度为0.095~0.676,期望杂合度为0.469~0.755,Shannon指数为0.791~1.621,遗传距离为0.071~0.788。采自新疆维吾尔自治区的7个苹果小吉丁虫种群之间遗传相似度较高,但其与辽宁省种群的遗传相似度较低。     

烟蚜茧蜂基因组中微卫星分布特点及分子标记开发

为明确寄生性天敌烟蚜茧蜂Aphidius gifuensis基因组中的微卫星分布特点并开发其分子标记,基于烟蚜茧蜂基因组草图,利用生物信息学方法分析其微卫星的分布特点并开发微卫星位点,采用PCR技术对获得的位点进行验证,获得高分辨率的分子标记位点。结果表明,烟蚜茧蜂基因组中共有360 199个微卫星,平均分布密度为1 787.93个/Mb,单碱基微卫星数量最多,占微卫星总数的81.46%,其中片段短于20 bp的微卫星居多,而数量最多的是单碱基（A）_n,其次是二碱基的（AT）_n和（AC）_n,以及三碱基的（AAT）_n和（ATC）_n。选取设计的45对微卫星引物进行PCR扩增,其中有21对引物能稳定扩增出清晰的条带。基于烟蚜茧蜂山东泰安种群24个个体的遗传多样性检测结果显示,筛选出的21个微卫星位点的平均观测杂合度、期望杂合度和多态性信息含量分别为0.48、0.57和0.50,其中位点S6的多态性最低,多态性信息含量为0.04,另有4个位点S12、S20、S29和S35显著偏离哈迪-温伯格平衡。结合PCR验证结果和遗传多样性分析结果筛选和开发出16个烟蚜茧蜂的有效微卫星位点,可作为有效的分子标记用于烟蚜茧蜂种群遗传学研究。     

新疆天山马鹿(Cervus elaphus songaricus)南山种群遗传多样性研究

为了解天山马鹿南山种群的遗传多样性,选用5对微卫星标记引物,利用PCR扩增,用非变性聚丙稀酰胺凝胶电泳、银染法显色,对天山马鹿南山山区冬季采集的255份马鹿粪便进行了检测,统计了该种群的基因频率(P)、有效等位基因数(Ne)、群体杂合度(He)、多态信息含量(PIC)。结果表明:255份粪便DNA分属111个体;种群平均等位基因4.40±0.90;平均有效等位基因数3.7391±0.1998;平均多态信息含量0.6825±0.0083;平均期望杂合度0.7257±0.0001;平均观察杂合度0.894±0.0214。整个种群没显著偏离Hardy-Weinberg平衡,种群固定系数Fis为-0.232。从此可见,天山马鹿南山种群具有比较丰富的遗传多样性。     

禾谷丝核菌基因组SSR标记的开发及评价

群体遗传学研究对于了解病原菌的流行、变异及进化规律具有重要意义。但是到目前为止,对小麦纹枯病菌禾谷丝核菌群体遗传结构的了解并不深入,缺乏有效的SSR标记是主要原因。本研究根据禾谷丝核菌全基因组序列进行了微卫星位点搜索并设计SSR引物,通过电泳筛选和测序验证,最终筛选出12个多态性较好且稳定可靠的SSR标记。利用这些标记对收集自我国安徽、江苏、河南三省的23个禾谷丝核菌菌株进行了多态性分析,结果发现禾谷丝核菌基因组中长片段微卫星位点较少。12对引物扩增出的等位基因数平均为6.1个,期望杂合度平均为0.651,观测杂合度平均为0.508,表明这些标记具有较高的多态性,能够满足禾谷丝核菌群体遗传学研究需要。本研究为进一步进行禾谷丝核菌的多样性、群体遗传结构分析及进化学研究提供了有效工具。     

甘肃省苹果蠹蛾不同地理种群遗传多样性分析

为探明苹果蠹蛾不同地理种群的遗传变异、种群分化及传播扩散情况,利用9对微卫星引物对甘肃9个不同苹果蠹蛾地理种群的遗传多样性进行了研究。应用POPGENE 1.32和NTSYSpc 2.1软件分析表明:9对引物共检测到21个多样性位点,多态性百分比为95.45%;9个不同地理种群的观测等位基因为1.954 5,有效等位基因为1.741 3,Nei氏指数为0.411 3,Shannon指数为0.592 7±0.147 2;遗传相似度在0.325 8~0.817 0范围内。UPGMA聚类分析表明甘州、金塔种群聚为一支,与肃州种群合为一支;临泽种群与总寨种群聚为一支;七里河与敦煌种群聚为一支;高台与民勤种群聚为一支,各分支间的遗传分化系数Fst分别为0.795 0、0.531 4、0.857 9、0.598 0。Mantel检测表明,种群间的遗传距离与地理距离无显著相关性。研究表明,甘肃苹果蠹蛾种群的遗传多样性较低,遗传变异主要来自种群内部,种群之间的基因交流较少,在甘肃省的传播以人为被动传播扩散为主。     

新疆鹅喉羚叶尔羌亚种微卫星遗传多样性研究

为了深入了解国家二级保护动物新疆鹅喉羚叶尔羌亚种(Gazella subgutturosa yarkandensis)种群的遗传现状,运用非损伤取样技术,从主要分布区中收集的58份粪便样本和15份肌肉样品中提取基因组DNA,采用10个特异微卫星位点来评价该区域的鹅喉羚种群的遗传多样性。经多次重复提取和重复PCR扩增后,发现58份粪便样本来源于49只不同的鹅喉羚个体。10个微卫星位点的平均的个体识别率(DP)值为0.918,累积个体识别率(CDP)值为0.9992。总共检测到57个等位基因,平均等位基因数(A)为5.7±1.3375(5~8);平均有效等位基因数(Ne)为4.72±0.6903(3.68~5.76);平均期望杂合度(He)为0.685±0.0296(0.5862~0.7627);平均多态信息含量(PIC)为0.719±0.0583(0.661~0.819)。除了和静种群在0ar FCB304位点上和且末种群在BM1818位上偏离Hardy-Weinderg平衡之外都基本遵守此平衡。以上结果显示该分布区鹅喉羚种群具有较高的遗传多样性。     

中国柑橘大实蝇遗传多样性分析

利用4对微卫星引物对采自中国7省市的18个柑橘大实蝇地理种群进行标记,并进行遗传多样性分析。结果表明,从供试柑橘大实蝇地理种群检测到的等位基因数为19~30个,平均为23.06个,且4个微卫星位点(Bp125、BcuF 1.6、Bcac 5.10、Bcac 6.10)的等位基因数分别为7、14、9、8个。对18个柑橘大实蝇种群的哈迪—温伯格平衡测试结果显示,其卡方值的变化范围为5.582~∞,各种群间差异显著(变化范围为0.000~0.694)。表明中国柑橘大实蝇种群的遗传多样性较高,且不同种群间存在较大的遗传分化。     

刺槐叶瘿蚊群体微卫星分析中样本量对遗传多样性指标的影响

采用微卫星标记进行刺槐叶瘿蚊群体遗传研究时,群体的样本量对遗传多样性指标准确性有一定影响。鉴于此,本实验设置了12个样本量梯度,统计分析在刺槐叶瘿蚊微卫星分子标记研究中,不同样本量对遗传多样性参数的影响,探讨刺槐叶瘿蚊群体遗传结构研究中所需的最适样本量。结果表明,样本量与平均等位基因数、平均有效基因数呈正相关,与平均观测杂合度、平均期望杂合度及内氏遗传多样性指数呈中度相关;在进行刺槐叶瘿蚊群体遗传结构的研究时,最适参数为期望杂合度和内氏遗传多样性指数,最小样本量为20。     

亚洲玉米螟Bt抗性种群的遗传多样性分析

近20年来,在北美、西欧、南美、菲律宾等20多个国家大面积商业化应用Bt玉米防治欧洲玉米螟Ostrinia nubilalis(Hübner)等鳞翅目害虫的实践证明,其不可避免地胁迫靶标害虫产生遗传分化,出现新的抗性种群。研究精准有效的抗性检测方法,将为抗性监测和抗性治理策略的有效性提供科学依据。本研究基于高通量测序获得的亚洲玉米螟Ostrinia furnacalis(Guenée)转录组数据,应用MISA (MicroSAtellite)软件搜索SSR位点,从61 622条EST(Expressed Sequence Tag)序列中获得了3 467个SSR位点。通过设计、筛选,共获得3 316对特异性引物,从中挑选了150对引物进行PCR扩增,共有51对扩增出目的条带,对亚洲玉米螟敏感种群(ACB-BtS)及5个Bt毒素抗性种群(ACB-AbR、ACB-AcR、ACB-AhR、ACB-FR、ACB-IeR)进行多态性检测,最终得到20条高多态性引物。利用这20对微卫星引物共检测到126个等位基因,平均每个位点6.3个。不同Bt抗性种群间产生了一定程度的遗传分化,种群间的平均遗传分化系数(F_(st))为0.195 9,即说明种群间的遗传变异为19.6%。根据遗传距离建立了UPGMA系统发育树,显示6个种群的相似度,即ACB-AbR与ACB-AcR相似度高。种群变异相似度规律与已报道的亚洲玉米螟对各Bt毒素的交互抗性规律相一致。本研究发现的SSR位点可作为亚洲玉米螟不同Bt毒素抗性种群的分子检测方法。     

Development of a multiplex RT-PCR detection and identification system for <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> and <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis>

We isolated eight polymorphic microsatellite markers for the basidiomycete Armillaria cepistipes and characterised them by analysing 50 isolates representing two geographically distinct populations from Switzerland and the Ukraine. The number of alleles per locus and population varied from one to eight, resulting in 43 alleles over the eight loci and two populations. In both populations, no significant linkage disequilibrium was observed between pairs of loci. Significant (P < 0.05) deviations from Hardy-Weinberg equilibrium were observed at one locus in the Swiss population and at three loci in the Ukrainian population. Of the eight loci developed for A. cepistipes, six were also polymorphic in A. gallica, four in A. ostoyae, two in A. mellea, and one in A. borealis. Beside the potential to be used for population genetic studies on A. cepistipes, these microsatellites thus represent additional molecular markers for three of the four annulated Armillaria species occurring in Europe.     

Isolation of microsatellites from an enriched genomic library of the plant-parasitic nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> and their detection in other root-knot nematode species

The root-knot nematode Meloidogyne incognita is a polyphagous pest distributed from temperate to tropical regions. However, the lack of suitable markers leads to a poor knowledge of its population genetic structure and colonization process. Here we describe the first characterization of 15 microsatellite loci from this nematode, that were developed from an enriched genomic library. Although the variability of these microsatellites was generally low, three of them exhibited a significant level of intrapopulation polymorphism, with three to seven alleles detected. The observed and expected heterozygosities ranged from 0.025 to 0.385 and from 0.024 to 0.779, respectively. Thus, these new microsatellite markers have potential value for the implementation of genotyping experiments in this nematode. Furthermore, successful cross-amplification of the variable microsatellite loci in seven other Meloidogyne species provides the opportunity of using these markers for population genetic studies in these damaging plant-parasitic nematodes.     

Gene and Genotypic Diversity of Phytophthora cinnamomi in South Africa and Australia Revealed by DNA Polymorphisms

Phytophthora cinnamomi isolates from South Africa and Australia were compared to assess genetic differentiation between the two populations. These two populations were analysed for levels of phenotypic diversity using random amplified polymorphic DNAs (RAPDs) and gene and genotypic diversity using restriction fragment length polymorphisms (RFLPs). Sixteen RAPD markers from four decanucleotide Operon primers and 34 RFLP alleles from 15 putative loci were used. A few isolates from Papua New Guinea known to posses alleles different from Australian isolates were also included for comparative purposes. South African and Australian P. cinnamomi populations were almost identical with an extremely low level of genetic distance between them (D_m=0.003). Common features for the two populations include shared alleles, low levels of phenotypic/genotypic diversity, high clonality, and low observed and expected levels of heterozygosity. Furthermore, relatively high levels of genetic differentiation between mating type populations (D_m South Africa=0.020 and D_m Australia=0.025 respectively), negative fixation indices, and significant deviations from Hardy–Weinberg equilibrium, all provided evidence for the lack of frequent sexual reproduction in both populations. The data strongly suggest that both the South African and Australian P. cinnamomi populations are introduced.     

黄淮麦区小麦全蚀病菌群体的遗传多样性分析

为了解小麦全蚀病病菌的群体组成和遗传多样性,采用8对多态性较好的微卫星标记,对我国黄淮麦区的116个小麦全蚀病菌株进行了分析。8个SSR标记的平均等位基因数为4.25个,多态性信息含量的平均值为0.66。供试菌株的平均有效等位基因数和基因遗传多样性指数分别为1.46和0.27,漯河群体的遗传多样性水平最高,周口群体最低。不同群体间遗传距离均较小,为0.0199~0.1153,其中徐州和周口群体间的遗传距离相对较大,而周口和驻马店群体间的遗传距离相对较小。分子方差分析结果显示,小麦全蚀病菌群体间和群体内均存在着一定的遗传分化,群体间的遗传变异占总变异的10%,群体内遗传变异占90%。6个群体间的基因流为3.5。根据SSR多态性,对来源不同菌株的UPGMA聚类分析结果显示,小麦全蚀病菌群体结构与地理来源有一定的关系。     

Development and characterization of EST-derived SSR markers in the cereal cyst nematode <Emphasis Type="BoldItalic">Heterodera avenae</Emphasis>

The cereal cyst nematode Heterodera avenae is a pathogen of cereal crops and causes high yield losses worldwide. In this study, a collection of 37,348 expressed sequence tags (ESTs) of H. avenae was mined for EST-based simple sequence repeat (SSR) markers, which resulted in the identification of 5604 SSRs. A total of 210 pairs of SSR primers were further developed and used for validation of the amplification rate and assessment of the polymorphism. Eight SSR markers were finally identified and analyzed using 96 individual cysts of H. avenae sampled from three provinces in China. These loci were found to be moderately polymorphic with 3–8 alleles per locus. The observed and expected heterozygosity across the three populations ranged from 0.000 to 0.594 and from 0.000 to 0.731, respectively. The polymorphism information content (PIC) was medium and ranged from 0.080 to 0.562, with a mean of 0.409. The F _ST ranged from 0.1034 to 0.1550, indicating moderate genetic differentiation among the three H. avenae populations. These EST-SSR markers can be used to study population genetic diversity and the dispersal route of H. avenae in China.     

Cross species transferability and characterization of microsatellite markers in Prosopis cineraria,a multipurpose tree species of Indian Thar Desert

Prosopis cineraria (L.) Druce, family Fabaceae, is an important multipurpose tree species of the Indian Thar Desert. It is an extremely drought and heat tolerant plant and has ability to survive in highly alkaline and saline environments. Considering the commercial, horticultural and ecological importance of P. cineraria, there is a great need to develop genomic resources in the form of microsatellite markers for the genetic enhancement of this crop. We identified and characterized 10 microsatellite markers in P. cineraria by cross species amplification. Total 18 Simple Sequence Repeat (SSR) primer pairs developed in P. chilensis, P. alba, and P. flexuosa were used to amplify SSR loci in P. cineraria. Out of eighteen SSR markers tested, ten (55.5%) amplified recognizable amplicons. The number of alleles detected at each locus ranged from one to four, a total of 24 with an average of 2.4 alleles. Observed heterozygosity (Ho) and expected heterozygosity (He) values varied from 0.14 to 0.85 and 0.21 to 0.56 with an average of 0.47 and 0.37, respectively. The polymorphic information content (PIC) values ranged from 0.49 to 0.78 with an average of 0.66. Of the nine polymorphic markers, seven were highly informative and polymorphic (PIC > 0.5). These microsatellite markers are characterized for the first time in P. cineraria. All microsatellite markers identified in this study may be useful in comparative genomics and population genetics studies of P. cineraria.     

Microsatellite analysis of Icelandic populations of the poplar fungal pathogen Melampsora larici-populina shows evidence of repeated colonization events

The basidiomycete Melampsora larici-populina causes foliar rust on Populus species from the sections Aigeiros and Tacamahaca, causing reduction in biomass production and economic losses. In the present study, samples of Icelandic M. larici-populina were collected for analysis of genetic diversity and population structure. A total of 439 isolates, collected at 15 locations, and analysed using 22 microsatellite markers were compared to data from French M. larici-populina populations. Twenty-one of the loci analysed were polymorphic, with an average of 3.4 alleles per locus. The mean observed and expected heterozygosities for all populations were 0.35 and 0.38. Evidence was found for a substructure within the Icelandic population with three subpopulations being the most likely scenario with low levels of gene flow. The population structure seen here is most likely shaped by both isolation and genetic drift as well as repeated events of colonization. In the future it can therefore be expected that regional poplar rust genotypes in Iceland change by two different modes; on one hand by transport of spores within the country and on the other hand by repeated colonization events. The results reported here underline the importance of closely monitoring the development of fungal diseases in Iceland, and to carefully select for resistance in Icelandic plant breeding programs.