黄连木居群遗传多样性的SSR标记分析

为系统揭示黄连木天然居群在遗传多样性水平上的差异及遗传分化状况,采用SSR(Simple sequence repeats)分子标记对其8个居群进行遗传多样性分析.在建立良好的反应体系基础上,从已有的阿月浑子SSR引物中筛选出9对适合进行黄连木遗传分析的引物,在8个黄连木居群中共检测到43条等位基因,位点平均等位基因数为4.78,平均多态位点百分率达90.28%.各居群内平均有效等位基因数为2.08,平均期望杂合度(He)为0.472,说明各居群的遗传多样性处于中等水平.按检测到的有效等位基因数(Ne)和期望杂合度(He),各居群的遗传多样性由高至低依次为安康唐县顺平辉县略阳栾川林州涉县.居群间的遗传分化系数(FST)平均为0.319,说明居群内变异是黄连木变异的主要来源,并根据遗传距离将8个居群分为三大类.     

用SSR标记检测杂交籼稻三系亲本的遗传差异

随机选用分布于水稻(Oryza sativa L.)12条染色体上的110对引物,共筛选出26对具有多态性的SSR引物,对24份杂交籼稻三系亲本材料进行遗传多样性和亲缘关系分析.全部24份亲本中共检测出116对等位基因,每个SSR引物可检测到的等位基因数目为2～7个不等,平均每对引物可以检测到4.4个等位基因.多态信息量(PIC)的变动范围为0.068 9～0.803 6,平均PIC值为0.603 4;期望杂合度(He)的变动范围为0.081 6～0.829 8,平均值为0.616 9;多样性指数(1)变幅为0.173 2～1.791 3,平均为1.160 0.对供试亲本材料根据遗传距离进行UPGMA聚类分析,结果表明,SSR微卫星标记可以将24份亲本材料分为3个大群,各组亲本间的遗传差异较大.通过分子标记辅助育种,可以有效地鉴定供试亲本材料之间的亲缘关系,分析预测新种质材料的遗传差异,可作为选育强优势组合杂交水稻的重要参考指标之一.图2表3参24     

中国大麦育成品种(系)的遗传多样性

为了解我国大麦育成品种(系)的遗传基础现状,利用位于大麦7个连锁群上不同位置的25对SSR引物对来自国内不同区域的大麦推广和新育成品种(系)的遗传多样性及其亲缘关系进行分析.结果表明,25对SSR引物在73个大麦品种间均有多态性扩增,每一对引物检测到的等位基因数目在2-6之间,平均为3.92个.SSR引物的多态性信息含量(PIC)变幅为0.252-0.780,平均为0.569.遗传多样性分析发现,我国不同大麦产区的皮大麦品种(系)间遗传相似性系数(GS)变化范围为0.661-0.767(平均值0.714),青藏高原地区青稞品种(系)间遗传相似性系数变化范围为0.533-0.925(平均值0.699),表明目前国内皮大麦和青稞的遗传多样性均较差、品种遗传背景单一.通过聚类分析,在遗传相似系数0.671水平处,42份皮大麦材料聚为2大类;在遗传相似系数0.618水平处,31份青稞材料聚为5大类,来源地相同的材料通常聚在同一大类或亚类,材料聚类结果与其地理来源有一定相关性.本研究表明SSR标记具有较好的遗传变异检测能力,对大麦品种(系)间的遗传多样性评价可为我国大麦育种过程中亲本材料的选择和利用提供重要依据.     

大麦EST-SSR分子标记开发及特征分析

大麦是一种古老的栽培作物,也是分子遗传学研究中常用的模式植物,为满足大麦基因作图、遗传多样性、种质资源鉴定和分子辅助育种等研究的需求,拟在NCBI公共数据库基础上,开发基于表达序列标签(EST)的简单序列重复(SSR)分子标记.从NCBI数据库中获得了525 781条大麦EST序列,将这些序列进行聚类和去冗余后共获得61 902个Unigene,随后通过MISA软件搜索Unigene中的SSR位点,并设计引物9 659对,最后通过电子PCR(E–PCR)和普通PCR对引物进行验证.结果显示:(1)61 902个Unigene中含有SSR位点24 648个,其中复合SSR位点5 843个,约占3.42%,单纯SSR位点23 805个,约占96.58%;(2)经E-PCR验证后发现9 659对SSR引物中有1 060对(11%)能够成功扩增出产物;(3)随机选取的11对引物经普通PCR验证发现,这些引物都能在2份野生大麦品种、2份栽培大麦品种及1份小麦、硬粒小麦、荆州黑麦和节节麦中成功扩增.本研究表明日益丰富的EST公共数据库是大麦SSR分子标记的重要来源;利用EST公共数据库、SSR搜索软件,结合E–PCR验证是开发SSR分子标记省时高效的手段.     

长江上游地区杂交水稻亲本遗传多样性

利用表型鉴定以及47对均匀分布于水稻12条染色体的SSR标记,对长江上游138个杂交水稻亲本进行检测,并采用遗传相似系数及数学模型分析其群体结构,以了解其遗传多样性信息.结果显示,长江上游杂交水稻亲本的保持系、恢复系以及总体的表型性状遗传变异程度较大,适用于进行遗传多样性分析.利用47对SSR标记共检测到94个等位基因位点,平均每个位点2个;其中有效等位基因数67.05个,占71.33%,Nei氏遗传多样性指数变幅为0-0.51,平均值为0.26.供试材料可分为恢复系类群和保持系类群,与生产上利用的保持系和恢复系高度一致.本研究表明利用SSR标记能详细了解长江上游杂交水稻亲本遗传多样性信息并有效区分恢复系与保持系.     

用SSR标记检测同源四倍体与二倍体水稻的遗传差异

随机选用分布于水稻(Oryza stativa L.)12条染色体上的15对SSR(simplej sequence repeats)引物，对18种中科院成都生物所培育的四倍体水稻和9种大面积种植的二倍体水稻进行了SSR多态性分析．11对具多态性的引物共检测到33条多态性条带，平均每对引物检测到3个等位基因．研究结果发现，所用同源四倍体水稻的基因组与二倍体水稻基因组大部分相同，只是在某些位点上具有差异，并筛选出部分SSR标记来区分二倍体与其同源四倍体．本研究还对二倍体与同源四倍体之间遗传差异的原因进行了初步的探讨．图2表2参12     

基于山桐子转录组序列的SSR分子标记开发

为研究山桐子遗传多样性,对山桐子转录组数据进行分析,开发SSR分子标记技术.将山桐子高通量转录组测序获得的84 213条Unigene进行简单重复序列(SSR)位点挖掘和分析,并结合引物验证,初步证明其可行性.通过软件分析,共获得含SSR位点的序列数23 077,出现频率为27.40%,涉及序列数量为29 953条,发生频率为35%.SSR序列中包括1 620种重复基元类型,其中单核苷酸、二核苷酸和三核苷酸为优势重复类型,SSR位点数分别为9 782(32.67%)、6 921(23.11%)和5 420(18.10%).单核苷酸中A/T类型比例最高,为9 630(32.15%),二核苷酸和三核苷酸中以类型AG/CT(4 679;15.62%)和AAG/CCT(1 220;1.53%)为主.SSR位点重复次数差别较大,主要是3次(4 748;15.70%),其次为6次(3 707;12.25%)和5次(3 475;11.60%).运用多态性分析的方式初步验证SSR位点在不同地区山桐子标记中的可行性.同时,利用Primer 5.0进行引物设计,随机筛选100对引物进行验证,31对引物可以扩增出条带,19对引物扩增出预期大小的条带,8对引物能特异性区分宜宾、资阳、宁强、成都4个不同地区的山桐子.本研究通过分析山桐子高通量转录组序列的SSR信息,筛选出8对引物验证不同地区山桐子的遗传多样性,将有助于山桐子基因挖掘、分子标记育种和资源保护等后续工作的开展.     

基于中华猕猴桃“红阳”转录组序列开发EST-SSR分子标记(英文)

为开发猕猴桃EST-SSR标记,了解28个品种猕猴桃间的遗传多样性和遗传关系,对中华猕猴桃"红阳"的转录组序列进行分析,并根据分析结果设计SSR引物.之后采用CTAB法提取28个品种猕猴桃的DNA作为SSR-PCR的扩增模板,并根据扩增结果进行聚类分析.研究中共得到包含SSR的序列21 848条,其中重复单元为单碱基、双碱基、三碱基、四碱基、五碱基和六碱基的序列分别为1 642、15 965、3 141、248、368和484条,随机选择其中46条序列设计SSR引物.根据初步的PCR扩增,筛选出32对条带较少且明亮的引物分别对28个品种猕猴桃的DNA样本进行扩增,并对引物对应的SSR序列进行定位.结果显示,32对引物对应的序列中有19条能够得到完整的所在基因、染色体以及染色体中具体位置的信息.这些引物中有26对具有多态性,共统计到等位基因120个,每对引物得到1-11个等位基因,平均3.75个.28个猕猴桃品种之间的遗传相似性系数在0.53-0.97之间,在遗传相似系数为0.72的水平上,可将它们分为5大类,分类结果与传统形态学的划分基本一致.本研究揭示的各样本间的遗传关系可为未来猕猴桃的种质改良提供依据.图4表5参33     

杏鲍菇转录组数据SSR位点的生物信息学分析

为了开发杏鲍菇分子标记,利用前期高通量测序技术获得的杏鲍菇转录组数据,采用生物信息学软件Trinity拼接,然后通过MISA软件检索并分析杏鲍菇转录组简单重复序列(Simple sequence repeats,SSR)位点.共获得45 833条unigene,在其中的3 256条unigene中检测到3 811条符合软件参数的SSR序列,发生频率为8.31%.SSR位点平均长度为15 bp,平均分布频率是1/15.91 kb.在杏鲍菇转录组的SSRs中,单核苷酸与三核苷酸重复基元为主要重复类型,分别占总SSRs的45.24%和35.53%.SSR基元的重复次数为5-24次,其中5次或10次重复是发生频率最高的,SSR重复基元共有85个类型,其中A/T是最常见的重复基元,比例为38.97%,其次为AG/CT,比例为8.24%.对3 811个SSR位点设计出3 644对SSR引物,随机选择20对引物进行PCR扩增,其中9对在3个杏鲍菇品种中表现出多态性.本研究表明杏鲍菇转录组SSR位点多态性丰富,具有较高的应用潜力.     

基于EST-SSR标记的沙棘品种鉴定及指纹图谱构建

以沙棘(Hippophae rhamnoides)优良品种“实优1号”为材料,对其叶片进行转录组测序,利用微卫星识别软件(microsatellite identification tool, MISA)和Primer 3(version 2.3.4)对获得的序列进行简单重复序列(simple sequence repeat, SSR)位点挖掘和引物设计,以收集的42份沙棘品种为研究材料,开展聚合酶链式反应(PCR)和毛细管电泳检测,旨在开发一套多态性高、稳定性好和通用性强的表达序列标签微卫星(express sequence tags from simple sequence repeat, EST-SSR)引物,构建沙棘指纹图谱,从而实现沙棘品种的快速准确鉴定,并对沙棘品种间亲缘关系进行分析。“实优1号”转录组测序共获得6 196个SSR位点,其中,重复基元类型为182种,SSR基序长度主要分布在10～21 bp区间,占全部SSR的81.58%,主要SSR重复类型为单核苷酸重复(48.72%)、二核苷酸重复(22.68%)和三核苷酸重复(18.85%)。利用筛选出的28对引物在42...     

Isozyme, ISSR and RAPD profiling of genotypes in marvel grass (Dichanthium annulatum)

Genetic analysis of 30 accessions of marvel grass (Dichanthium annulatum Forsk.), a tropical range grass collected from grasslands and open fields of drier regions, was carried out with the objectives of identifying unique materials that could be used in developing the core germplasm for such regions as well as to explore gene (s) for drought tolerance. Five inter-simple sequence repeat (ISSR) primers [(CA)4, (AGAC), (GACA) 4; 27 random amplified polymorphic DNA (RAPD) and four enzyme systems were employed in the present study. In total, ISSR yielded 61 (52 polymorphic), RAPD 269 (253 polymorphic) and enzyme 55 isozymes (44 polymorphic) bands. The average polymorphic information content (PIC) and marker index (MI) across all polymorphic bands of 3 markers systems ranged from 0.419 to 0.480 and 4.34 to 5.25 respectively Dendrogram analysis revealed three main clusters with all three markers. Four enzymes namely esterase (EST), polyphenoloxidase (PPO), peroxidase (PRX) and superoxide dismutase (SOD) revealed 55 alleles from a total of 16 enzyme-coding loci. Of these, 14 loci and 44 alleles were polymorphic. The mean number of alleles per locus was 3.43. Mean heterozygosity observed among the polymorphic loci ranged from 0.406 (SOD) to 0.836 (EST) and accession wise from 0.679 (1G3108) to 0.743 (IGKMD-10). Though there was intermixing of few accessions of one agro-climatic region to another largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 iterations also showed large numbers of nodes (11 to 17) having strong clustering (> 50 bootstrap values) in all three marker systems. The accessions of the arid and drier regions forming one cluster are assigned as distinct core collection of Dichanthium and can be targeted for isolation of gene (s) for drought tolerance. Variations in isozyme allele numbers and high PIC (0.48) and MI (4.98) as observed with ISSR markers indicated their usefulness for germplasm characterization.     

Use of EST database markers from M. truncatula in the transferability to other forage legumes

In general tropical forage legumes lack microsatellites or simple sequence repeat (SSR) markers. Development of genic SSR markers from expressed sequence tagged (EST) database is an alternate and efficient approach to generate the standard DNA markers for genome analysis of such crop species. In the present paper a total of 816 EST-SSRs containing perfect repeats of mono (33.5%), di (14.7%), tri (39.3%), tetra (2.7%), penta (0.7%) and hexa (0.4%) nucleotides were identified from 1,87,763 ESTs of Medicago truncatula. Along with, 70 (8.5%) SSRs of a compound type were also observed. Seven primer pairs of tri repeats were tested for cross transferability in 19 accessions of forage legumes comprising 11 genera. At two different annealing temperatures (55 and 60 degreesC) all primer pairs except AJ410087 reacted with many accessions of forage legumes. Atotal of 51 alleles were detected with six M. truncatula EST-SSRs primer-pairs against DNAfrom 19 accessions representing 11 genera where number of alleles ranged from 2 to 13. The cross-transferability of these EST-SSRs was 40.6% at 55 degreesC and 32.3% at 60 degreesC annealing temperature. 24 alleles of the total 50 (48%) at 55 degreesC and 27 of 51 (53%) at 60 degreesC were polymorphic among the accessions. These 27 polymorphic amplicons identified could be used as DNA markers. This study demonstrates the developed SSR markers from M. truncatula ESTs as a valuable genetic markers and also proposes the possibility of transferring these markers between species of different genera of the legumes of forage importance. It was evident from the results obtained with a set of Desmanthus virgatus accessions where SequentialAgglomerative Hierarchical and Nested (SAHN) cluster analysis based on Dice similarity and Unweighted Pair Group Method with Arithmetic mean Algorithm (UPGMA) revealed significant variability (24 to 74%) among the accessions. High bootstrap values (>30) supported the nodes generated by dendrogram analysis of accessions.     

Effects of Population Size, Mating System, and Evolutionary Origin on Genetic Diversity in Spiranthes sinensis and S. hongkongensis

Hong Kong once supported more than 109 species of wild orchids, of which approximately 30% were endemic. Most of the local wild orchids have now become rare or endangered. I conducted a comparative study of genetic diversity in two closely related terrestrial orchids, an allotetraploid, Spiranthes hongkongensis , and its diploid progenitor, S. sinensis , to assess the effects of the population bottleneck associated with the origin of the polyploid and to investigate the relationships between number of breeding individuals, mating system, and level of isozyme variation in their populations. Nearly complete genetic uniformity was observed both within and among populations of S. hongkongensis . In contrast, S. sinensis had high levels of genetic variation for all of the genetic parameters examined. Regression analysis of population size and several components of genetic diversity in S. sinensis revealed that, among various measures of within-population variation, the proportion of polymorphic loci ( P ) and average number of alleles per locus ( A ) or per polymorphic locus ( A _p ) were the most sensitive to population size ( R ² = 0.942, p = 0.001; R ² = 0.932, p = 0.002; and R ² = 0.923, p = 0.002 respectively). The highly negative correlation ( r = −0.999, p < 0.01) between population size and the mean frequency of private alleles in pairwise population comparisons, p (1), indicated that population size may also be used to predict the extent of population differentiation caused by random genetic drift. Conservation of genetic diversity in S. sinensis could be maximized by protecting several of both large and small populations, whereas fewer populations may be needed to achieve this goal for S. hongkongensis.     

Genetic Diversity in a Threatened Wetland Species, Helonias bullata (Liliaceae)

Genetic variation was examined in Helonias bullata , a threatened perennial plant species that occurs in isolated wetland habitats. Fifteen populations representing the species' geographic range were sampled. Genetic diversity was low for the species ( H _es = 0.053) as well as within populations ( H _ep = 0.029). Of the 33 allozyme loci examined, 11 (33%) were polymorphic, while on average only 12.8% (4) of the loci were polymorphic within populations. The number of alleles per polymorphic locus was 2.36 for the species and averaged 2.09 across populations. For every genetic parameter calculated, variation in H. bullata was lower than that typically found for narrowly distributed plant species. The lowest levels of genetic diversity were found in northern areas that were colonized following the last glacial epoch. The number of genotypes detected per population ranged from three to 21, with a mean of 13 for this clonally reproducing species. We found a relatively high proportion of total genetic diversity (30.6%) among populations and a significant correlation (p < 0.002) between genetic distance and geographic distance. Genetic drift phenomena appear to play a major role in the population genetics of this species. Anomalously, several populations that appeared most limited in size and vigor were genetically most variable, perhaps because they represent older, relictual populations. Life-history characteristics of H. bullata coupled with low levels of genetic diversity and the degradation and disappearance of wetlands threaten the existence of this species.     

Genetic diversity of blackspot seabream (<Emphasis Type="Italic">Pagellus bogaraveo</Emphasis>) populations off Spanish Coasts: a preliminary study

Genetic diversity among four natural samples of Blackspot seabream (Pagellus bogaraveo, Brünnich, 1768) from different fishing grounds exploited by Spanish fisheries was analyzed through the use of 12 microsatellite markers. The samples were captured off the Spanish coasts from the Mediterranean Sea to the Cantabrian Sea within the same continental slope. High levels of genetic diversity were revealed for every population and every locus was polymorphic at the 0.95 level. The average number of alleles, average heterozygosity and PIC were found to be 15.75, 0.833 and 0.818, respectively. In general, population differentiation was not detected in these samples. Through AMOVA, a low level of variation between regions (Mediterranean vs. Atlantic samples) was observed, though this was not significant. A larger percentage of total variation was observed inside the ‘within populations’ class. Thus, AMOVA did not reveal any significant population substructure. Moreover, no correlation was found between geographical and F_ST estimates and the observed results did not allow the improvement of a model of isolation by distance. The high homogeneity revealed by means of these markers could indicate the absence of physical frontiers between the geographical areas analyzed in this survey, especially between Atlantic and Mediterranean areas.     

Genetic consequences of long larval life in the starfish Linckia laevigata (Echinodermata: Asteroidea) on the Great Barrier Reef

Gene flow between populations of the asteroid Linckia laevigata (Linnaeus) was investigated by examining over 1000 individuals collected from ten reefs throughout the Great Barrier Reef (GBR), Australia, for genetic variation at seven polymorphic enzyme loci. Despite geographic separations in excess of 1000 km, Nei's unbiased genetic distance (0 to 0.003) and standardised genetic variation between populations (F _ST) values (mean 0.0011) were small and not significant. Genetic homogeneity among L. laevigata populations is consistent with the long-distance dispersal capability of its 28 d planktonic larval phase, and is greater than that observed for other asteroid species, including another high-dispersal species, Acanthaster planci, which has a 14 d larval phase. Variation within populations was also higher than previously recorded for asteroids (mean heterozygosity=0.384; number of alleles per locus ranged from 5.1 to 6.0 in each population). Among asteroids, dispersal ability is positively correlated with gene flow and levels of variation, and negatively correlated with levels of differentiation.     

Transferability of STS markers in studying genetic relationships of marvel grass (Dichanthium annulatum)

Transferability of sequence-tagged-sites (STS) markers was assessed for genetic relationships study among accessions of marvel grass (Dichanthium annulatum Forsk.). In total, 17 STS primers of Stylosanthes origin were tested for their reactivity with thirty accessions of Dichanthium annulatum. Of these, 14 (82.4%) reacted and a total 106 (84 polymorphic) bands were scored. The number of bands generated by individual primer pairs ranged from 4 to 11 with an average of 7.57 bands, whereas polymorphic bands ranged from 4 to 9 with an average of 6.0 bands accounts to an average polymorphism of 80.1%. Polymorphic information content (PIC) ranged from 0.222 to 0.499 and marker index (MI) from 1.33 to 4.49. Utilizing Dice coefficient of genetic similarity dendrogram was generated through un-weighted pairgroup method with arithmetic mean (UPGMA) algorithm. Further, clustering through sequential agglomerative hierarchical and nested (SAHN) method resulted three main clusters constituted all accessions except IGBANG-D-2. Though there was intermixing of few accessions of one agro-climatic region to another, largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 scale also showed large number of nodes (11 to 17) having strong clustering (> 50). Thus, results demonstrate the utility of STS markers of Stylosanthes in studying the genetic relationships among accessions of Dichanthium.     

Genetic studies in marine fishes

Nine polymorphic loci were found among 42 presumptive protein-coding gene loci surveyed among 474 red drum (Sciaenops ocellatus) sampled in 1987 from 13 nearshore and 1 offshore localities from the Atlantic coast of the southeastern USA and the northern Gulf of Mexico. The mean number of alleles over the polymorphic loci was 3.8, and the average heterozygosity over all loci examined was estimated as 0.047. These data indicate that red drum have normal levels of genetic variability. Wright'sF1 _ST values (the standardized variance of allele frequencies between samples) over all polymorphic loci ranged from 0.009 to 0.027 (meanF1 _ST =0.019), and estimates of the effective number of migrants (N _e m) per generation using Wright's island model ranged from 9.0 to 27.5. High levels of gene flow among the red drum samples were also indicated by Slatkin's qualitative analysis using conditional average allele frequencies. Nei's estimates of genetic distance between pairs of samples ranged from 0.000 to 0.009, indicating a high degree of nuclear gene similarity among all samples. Highly significant heterogeneity in allele frequencies at the locus for adenosine deaminase was detected between red drum sampled from the Atlantic and those sampled from the Gulf and among red drum sampled from the Gulf.