Rapid estimation of genetic relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomic DNA samples

A procedure which involves the use of RAPD markers, obtained from bulked genomic DNA samples, to estimate genetic relatedness among heterogeneous populations is demonstrated in this study. Bulked samples of genomic DNA from several alfalfa plants per population were used as templates in polymerase chain reactions with different random primers to produce RAPD patterns. The results show that the RAPD patterns can be used to determine genetic distances among heterogeneous populations and cultivars which correspond to their known relatedness. The results also indicate that, by using ten primers with bulked DNA samples from ten individuals, 18–72 populations or cultivars can be distinguished from each other on the basis of at least one unique RAPD marker. We anticipate that DNA bulking and methods for comparing RAPD patterns will be very useful for identifying cultivars, for studying phylogenetic relationships among heterogeneous populations and for selecting parents to maximize heterosis in crosses.     

Inheritance and mapping of 2n-egg production in diploid alfalfa.

The production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cytokinesis after restitutional meiosis. The formation of 2n eggs through diplosporic apomeiosis has also been documented in a diploid mutant of M. sativa subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular tagging of 2n-egg formation appears to be an essential step towards marker-assisted breeding and map-based cloning strategies aimed at investigating and manipulating reproductive mutants of the M. sativa complex. We made controlled crosses between PG-F9 and three wild type plants of M. sativa subsp. coerulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F1 progenies with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa because of endosperm imbalance, and owing to the negligible selfing rate, seed set in 2x-4x crosses was used to discriminate the genetic capacity for 2n-egg production. F1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified fragment length polymorphism) markers was employed to identify a genetic linkage group related to the 2n-egg trait using one of the three F1 progenies. This approach enabled us to detect a paternal ISSR marker of 610 bp, generated by primer (CA)8-GC, located 9.8 cM from a putative gene (termed Tne1, two-n-eggs) that in its recessive form determines 2n eggs and a 30% recombination genomic window surrounding the target locus. Eight additional RAPD and AFLP markers, seven of maternal, and one of paternal origin, significantly co-segregated with the trait under investigation. The minimum number of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses was estimated by ANOVA and regression analysis. Four maternal and three paternal independent molecular markers significantly affected the trait. A paternal RAPD marker allele, mapped in the same linkage group of Tne1, explained 43% of the variation for seed set in 2x-4x crosses indicating the presence of a major QTL. A map of the PG-F9 chromosome regions carrying the minor genes that determine the expression level of 2n eggs was constructed using selected RAPD and AFLP markers. Two of these genes were linked to previously mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evidence support the involvement of at least five genes.     

香菇亲本菌株及其杂交后代的RAPD分析*

运用随机扩增多态性DNA（RandomamplifiedpolymorphicDNA,RAPD）技术对源于两个香菇（Lentinulaedodes）双核菌株的孢子单核体、原生质体单核体及其杂交后代进行了基因组DNA多态性分析。用9个随机引物共扩增出116条DNA片段,其中82．5％具有多态性。综合分析9个随机引物的扩增谱带,可将所有供试亲本单核体清楚地分开,且早核体聚类分析的结果与其来源及遗传背景相吻合。此外,用两个双核亲本菌株的各4个不同交配型的孢子单核体两两支配所得的所有杂交组合,也均可与双核亲本菌株明确地区分开来。因此,在杂交育种中,RAPD分析可为亲本的选配及杂种的鉴定提供可靠依据。     

香菇亲本菌株及其杂交后代的RAPD分析

运用随机扩增多态性DNA（RandomamplifiedpolymorphicDNA,RAPD）技术对源于两个香菇（Lentinulaedodes）双核菌株的孢子单核体、原生质体单核体及其杂交后代进行了基因组DNA多态性分析。用9个随机引物共扩增出116条DNA片段,其中82．5％具有多态性。综合分析9个随机引物的扩增谱带,可将所有供试亲本单核体清楚地分开,且早核体聚类分析的结果与其来源及遗传背景相吻合。此外,用两个双核亲本菌株的各4个不同交配型的孢子单核体两两支配所得的所有杂交组合,也均可与双核亲本菌株明确地区分开来。因此,在杂交育种中,RAPD分析可为亲本的选配及杂种的鉴定提供可靠依据。     

大白菜杂交种'冠春'杂交率的RAPD分析

从春大白菜品种‘冠春’及其亲本中提取基因组DNA,用320个随机引物进行RAPD扩增,从中筛选出5 个可将亲本和子代区分的引物S4、S47、S73、S134和S194。S4产生父本特征带S4-370;S47和S134产生母本特征带S47-700 和S134-1200;S73和S494产生亲本互补的特征带S73-660、S73-730和S494-400、S494-1770,上述谱带均在子代中出现。以这5个引物产生的特征谱带建立杂交种‘冠春’及其亲本的RAPD特异指纹。通过对134个‘冠春’的种子进行纯度鉴定,结果表明2个父本和4个母本与大田检测结果完全一致。进一步验证了4种鉴定大白菜杂交种方法的可行性。     

苹果属小金海棠的遗传多样性初步研究

用RAPD技术建立了苹果属小金海棠（Malus xiaojinensis）2个自然分布区的3个群体内随机选取的30株树（10株／每群体）及其相应的子代实生苗共6个群体、60个样本植株的分子标记。通过对15个3承机引物产生的81条RAPD这的统计,计算了不同群体RAPD多态性带的数目。用TREECON软件分析了不同群体及所有个体间的遗传关系,并用AMOVA技术分析了物种的遗传变异。结果是15个引物在全部分析个体中产生了58条多态性带,平均每引物3.8条。现有分布3个群体及其相应的子代实生苗群体的平均多态性带的数目都为1.5条左右。其中平均多态性带的数目最低的群体仅有0.7条,最高的群体也只有2.5条。遗传关系分析表明,2个自然分布区的不同群体间存在遗传分化现象。AMOVA分析显示小金海棠的遗传变异有相当一部分来源于群体间。     

DNA polymorphism in various goose lines by RAPD-PCR

RAPD markers often primers were used to assess the polymorphism among pooled DNA of eight goose lines. The number of bands amplified by each primer ranged from 1 to 8, within a mean of 2.86. Some bands appeared specific for the line or genetic background. RAPD technique is an effective method for generating the polymorphic DNA marker in the goose. RAPD patterns from mixed DNA samples can reflect the genetic information of populations. The present study indicated that 10 generations selected for egg production and body weight under various pressure, resulted in genetic variation among goose lines as detected by RAPD. Selection for meat traits caused greater genetic diversity than selection for egg production.     

Genetic variability and differentiation of Caragana microphylla populations as revealed by RAPD markers

Genetic variability in random amplified polymorphic DNA (RAPD) was studied in 90 individuals of Caragana microphylla, an outcrossing perennial shrub species, from five natural populations sampled in Inner Mongolia steppe of China on a small scale. Nineteen selected primers were used to amplify DNA samples, and totally 225 bands were detected. The percentage of polymorphic bands within populations ranged form 58.22% to 63.56%, with an average of 60% at the population level and 71.11% at the species level, indicating relatively high genetic variations in C. microphylla species. Shannon's information index (I) and Nei's gene diversity (h) showed the similar trend with each other. According to the analysis of Nei's gene diversity, the percentage of genetic variation among populations was 7.13%, indicating a low level of genetic differentiation among populations. There existed a strong gene flow (Nm = 3.26) among populations. Although AMOVA analysis also revealed most variation was within populations (phi(ST) = 4.1%), a significant proportion was observed among populations (P<0.001) in the present study, suggesting genetic differentiation occurred among populations at a certain extent. Based on Mantel's tests and the results of previous studies, the genetic structure pattern of C. microphylla accorded with the isolation-by-distance model on a very large scale, however, on a small scale, the significant genetic differentiation among populations might be enhanced by the micro-environmental divergence among the sampling sites, rather than by geographic factors. Analysis of the genetic variations of C. microphylla populations provided useful information for the adaptive strategy of Caragana species.     

Identification of F1 hybrids of artichoke by ISSR markers and morphological analysis

Five different artichoke hybrid populations (crosses between a male sterile artichoke (MS6) and three American accessions (AMA3, AMA7, AMB1) and two Spanish accessions (SP2, SPA2)) plus a commercial hybrid population (Opal F1, Nunhems, Netherlands) were used for molecular and morphological characterisation in order to identify the purity of the F1 populations. Molecular analysis was carried out using the inter-simple sequence repeat (ISSR) technique with twenty primers. Out of these, nine were polymorphic, producing 82 DNA bands per primer on average, 56 of which were polymorphic among the populations. A dendrogram, drawn on the basis of a similarity matrix using the UPGMA algorithm, revealed that the 66 samples (10 plants per F1 populations and their respective progenitors) could be classified into two major clusters at a Nei’s genetic distance of 0.13. The male parents were genetically quite similar to all their respective progenies. In order to confirm hybrid purity, only the primers which amplified bands specific to the male parent of each hybrid were considered and the inheritance of the single locus for each cross-combination was analysed. Three ISSR markers (857c, 857g and 878) were able to confirm the hybrid purity. The morphological trait analysis showed that eight of the 17 morphological characters were significantly different among the six F1 populations. When the Mantel test was applied to the matrix of the Nei’s genetic distances and the matrix of morphological traits, a significant degree of correlation was observed between them. A selection method using ISSR markers based on cluster analysis is suggested to confirm the purity of artichoke hybrids and to predict the characters expected by any F1 hybrid offspring.     

A directed search for DNA sequences tightly linked to cereal cyst nematode resistance genes in Triticum tauschii.

An improved system for identifying DNA sequences linked to a targeted region was developed by fractionating DNA sequences prior to polymerase chain reaction (PCR) analysis. In an attempt to identify DNA markers linked to a strong CCN resistance gene, Ccn-D1, in Triticum tauschii, DNA samples from individuals homozygous for resistance and susceptibility at the Ccn-D1 locus in a segregating progeny were bulked separately to produce "near isogenic" DNA pools. The polymerase chain reaction was employed to generate several DNA amplification products from each of the bulked DNA segregants using 240 random (RAPD) and 4 semirandom (consensus sequences of intron-splice junctions) primers. A DNA polymorphic fragment was apparent between the resistant and susceptible bulks using one of the semirandom primers. Hydroxylapatite chromatography of reannealed DNA (to Cot values > 100) was used to enrich low copy DNA sequences in the bulk DNA segregants (resistant and susceptible DNA pools). PCR analysis on the low copy enriched DNA pool increased the level of polymorphism detected between bulked segregants. One of the RAPD fragments present in only the resistant low copy DNA pool was cloned and mapped to the distal region of the long arm of chromosome 2D. By using the cloned RAPD fragment, csE20-2, to assay an RFLP locus in three independent F2 progenies, complete cosegregation was obtained with the Ccn-D1 locus. Joint segregation analysis from a genome-wide mapping of RFLP markers and a second CCN resistance in T. tauschii, Ccn-D2, showed this locus to be loosely linked to the proximal region of chromosome 2.     

Use of RAPD analyses to estimate population genetic parameters in the alfalfa leaf-cutting bee, Megachile rotundata.

RAPD analyses were performed on five geographically isolated populations of Megachile rotundata. We used haploid males of the alfalfa leaf-cutting bee, M. rotundata, to overcome the limitation of the dominance of RAPD markers in the determination of population genetic parameters. Sixteen primers gave rise to 130 polymorphic and 31 monomorphic bands. The unbiased estimators calculated in this study include within- and between-population heterozygosity, nucleotide divergence, and genetic distance. The genetic diversity (H = 0.32-0.35) was found to be about 10 times that of previous estimates (H = 0.033) based on allozyme data. Contrary to the data obtained at the protein level, our results suggest that Hymenoptera do not have a lower level of genetic variability at the DNA level compared with other insect species. Regardless of the different assumptions underlying the calculation of heterozygosity, divergence, and genetic distance, all five populations showed a parallel interrelationship for the three parameters. We conclude that RAPD markers are a convenient tool to estimate population genetic variation in haploid M. rotundata and that with an adequate sample size the technique is applicable to the evaluation of divergence in diploid populations. Key words : Megachile rotundata, RAPD, heterozygosity, genetic distance, nucleotide divergence.     

银鲫生殖方式多样性的转铁蛋白的同工酶标记的遗传分析

用银鲫克隆D,克隆A和鲤鱼的精子分别与银鲫克隆F的卵子受精产生了三种繁殖组合FD,FA和FL;再用转铁蛋白和同工酶标记对这三种组合的遗传模式进行比较研究。结果发现,FL组合的子代具有其母本完全相同的体形和电泳图谱,表现出雌核生殖银鲫的克隆品性。而在FD组合中则出现了体形的分化和酶谱的多态性;在一些个体的蛋白座位上同时检测到了父本和母本各自特有的谱带,证明FD生殖过程中有性重组的发生。同时,在所研究的蛋白的不同座位上存在着极端的连锁不平衡现象,可以推断在FD的重组过程中多个基因座位组成的连锁群（甚至是染色体组）可能作为一个整体参与基因的传递。此外,不同的蛋白座位上都未观察到重组的纯合表型,暗示在不同的基因连锁群之间还可能存在一种平衡致死的机制。FA组合的F1代具有类似母本克隆F的体形和蛋白表型,FA组合俱本近交产生的F2代却同时出现了克隆F和克隆A的体形和表型。即父本和母本的染色体组都能够通过有性生殖传递给FA子代,然而可能由于父母本基因的不相容性而使F1代父本的基因表达受到抑制。相对于雌核生殖的克隆遗传 ,本研究的揭示出来的有性生殖特性及伴随的有性质组能够使银鲫在一定程度上卸去积累的遗传负荷并从其它种群获取新的基因型,以维持其遗传多样性。多种可选择的生殖方式可能对于银鲫在自然条件下的生态适应有着重要意义,对于银鲫的遗传选育和养殖管理也有一定的参考价值。     

有性生殖对栗疫病菌群体结构的影响

采用RAPD方法对来源于栗疫病菌8个不同子囊壳的子囊孢子后代和无性生殖的对照群体各23个菌株进行了群体结构的比较。从RAPD随机引物中筛选出扩增多态性丰富的4条引物,共扩增出条带73条,多态性检测率为100%。研究结果表明,在8个子囊壳和无性生殖群体中的基因多样性,64.27%由群体内部引起,只有35.73%的多样性由群体之间的基因差异引起。各子囊壳群体间存在的基因流动很小(Nm=0.8994)。有性群体和无性群体之间的遗传距离为0.1389,基因流动值为3.4212,说明子囊壳群体和无性生殖群体之间存在一定的系统关系。分析表明栗疫病菌子囊孢子后代在自然界的传播对自然界的病菌的多样性起重要的作用。     

Inheritance of random amplified polymorphic DNA (RAPD-PCR) markers and their use in population genetic studies of Calomys musculinus (Rodentia,Muridae), the reservoir of Argentine hemorrhagic fever

In order to assess the reliability of RAPD markers in the estimation of the genetic structure of natural populations of the murid rodent Calomys musculinus (reservoir of Junin virus, ethiological agent of Argentine Hemorrhagic Fever), we have analyzed the heritability of RAPD bands in 10 parents and their offspring (33 individuals). Fourteen out of a total of 119 bands obtained were absent in the parental patterns, but consistently amplified in offspring from some families. These bands can be eliminated from analyses. Overall degree of band sharing between individuals, including non-parental bands, correctly grouped members of a family in the same cluster in a UPGMA tree, with a high bootstrap percentage. Results support the usefulness of RAPDs as hereditable markers. One hundred polymorphic RAPD loci were identified in three natural populations of C. musculinus. Mean expected heterozygosity in three natural populations ranged from 0.206 to 0.220. Allele frequency based and phenotype based measures of genetic differentiation among natural populations of C. musculinus gave similar results (Weir and Cockerham's theta = 0.133; Excoffier et al.'s phi = 0.127). These values were considerably higher than those found previously using allozymes as genetic markers, and are compatible with moderate to low levels of gene flow among populations.     

利用RAPD-PCR探讨天蓝喇叭虫生物群系分化

选择天蓝喇叭虫(Stentor coeruleus)作为研究对象,对武汉市南湖、月湖、关桥3个水体共5个样点天蓝喇叭虫(S.coeruleus)样本的总DNA进行随机扩增多态DNA聚类分析,以检测各个样本的遗传相似性和趋异程度,借以评估样本间的遗传变异度。结果如下:(1)从98条随机引物中筛选12条引物共扩增出89条大小为100～1500bp的清晰条带,平均每条引物扩增出7.4条片段。(2)用Rapdistance1.04分析显示,不同样点样本之间存在着一定的变异,其遗传距离在0.076～0.416之间。(3)构建的聚类图中,南湖3个样点的遗传距离较近,在聚类图上聚成一枝,应该为同一个种群。本试验将为探讨水体原生动物迁徙能力对生物群系分化的影响积累实例资料,更希望可以促进水体原生动物的研究和种间过渡区本质及物种扩散行为的研究。     

Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers

Genetic variation within and between five populations of Oryza granulata from two regions of China was investigated using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pairwise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. Our results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Received: 29 March 2000 / Accepted: 15 May 2000     

Conservation Genetics of Mexican Beech, Fagus grandifoliavar. mexicana

Fagus grandifolia var. mexicana(Fagaceae) is a Mexican endemic tree, currently threatened with extinction. In order to assess the level and structure of genetic variation in four remaining populations, leaf samples were analysed using random amplified polymorphic DNA (RAPD) and cpDNA PCR-RFLP markers. A sample of the more widespread congener, F. grandifoliavar. grandifolia from the USA was also analysed for comparison. Thirty-three polymorphic RAPD bands were produced using 18 10-mer primers. AMOVA of RAPD data indicated significant (P < 0.002) population differentiation, with 15.6% of variation recorded between Mexican populations. PCR-RFLP analysis enabled three cpDNA haplotypes to be identified, denoted types A, B, and C. Types A and B were each restricted to an individual Mexican population, whereas Type C was fixed for two Mexican populations, and the population from the USA. Within-population genetic variation, quantified as percentage polymorphic bands, Shannon's Diversity Index and Nei's gene diversity measure, was found to be lower in Mexican populations than in that from the USA, and was positively related to population size. These results suggest that an unexpectedly high degree of genetic variation exists within Mexican beech, and this variation should be considered in developing the conservation strategy that is urgently required if extinction of this taxon is to be prevented.     

Assignment of sockeye salmon (Oncorhynchus nerka) to spawning sites using DNA markers

Randomly amplified polymorphic DNA (RAPD) markers were used to assign individual adult sockeye salmon to their spawning sites using a genotype assignment test. Six primers were selected for use by screening bulked DNA samples for markers missing in fish from one or more of 5 sites in British Columbia or Alaska. Of 73 markers scored, 54 showed variation between or within sites among the sampled fish. Thirty-seven of the variable markers were not detected in any fish from one or more sites; 18 variable markers were detected in all fish from one or more other sites. Thus 25% of markers scored were found in all fish of some sites and in no fish of some other sites. An assignment test placed all 70 fish tested into their correct populations. Principal coordinate analysis of genetic variation produced clusters of fish corresponding to each sampling site. No sex-specific RAPD markers were detected among more than 1300 screened.     

Molecular differentiation of the Old World Culicoides imicola species complex (Diptera, Ceratopogonidae), inferred using random amplified polymorphic DNA markers

Samples of seven of the 10 morphological species of midges of the Culicoides imicola complex were considered. The importance of this species complex is connected to its vectorial capacity for African horse sickness virus (AHSV) and bluetongue virus (BTV). Consequently, the risk of transmission may vary dramatically, depending upon the particular cryptic species present in a given area. The species complex is confined to the Old World and our samples were collected in Southern Africa, Madagascar and the Ivory Coast. Genomic DNA of 350 randomly sampled individual midges from 19 populations was amplified using four 20-mer primers by the random amplified polymorphic DNA (RAPD) technique. One hundred and ninety-six interpretable polymorphic bands were obtained. Species-specific RAPD profiles were defined and for five species diagnostic RAPD fragments were identified. A high degree of polymorphism was detected in the species complex, most of which was observed within populations (from 64 to 76%). Principal coordinate analysis (PCO) and cluster analysis provided an estimate of the degree of variation between and within populations and species. There was substantial concordance between the taxonomies derived from morphological and molecular data. The amount and the different distributions of genetic (RAPD) variation among the taxa can be associated to their life histories, i.e. the abundance and distribution of the larval breeding sites and their seasonality.