Characterization of new microsatellite loci isolated from Santiria trimera (Burseraceae)

? Premise of the study: To study the genetic structure among three morphotypes of an African rainforest tree species, Santiria trimera, nuclear microsatellite markers were isolated and characterized. ? Methods and Results: Seven polymorphic loci were isolated using a pyrosequencing-based protocol and successfully amplified on three different morphotypes of S. trimera. For six of the seven loci, there is at least one private allele for one of the three morphotypes. The mean effective number of alleles is about four for each of the three morphotypes. ? Conclusions: These microsatellite markers are promising to explore the genetic delimitation among sympatric morphotypes found in Gabonese forests and to study the spatial genetic structure within each gene pool.     

濒危灌木长叶红砂遗传多样性的RAPD分析

应用RAPD分子标记对濒危灌木长叶红砂(Reaumuria trigyna)种群遗传多样性进行了分析.应用18条随机引物对长叶红砂5个种群的95个个体进行扩增,检测到118个位点,其中多态位点105个.结果表明:长叶红砂种群的多态位点比率(P)为88.98%,显示出长叶红砂种群存在较高的遗传多样性.Shannon多样性指数(0.4966)、Nei基因多样性指数(0.3303)和基因分化系数(Gst=0.1425)的分析结果显示,长叶红砂种群遗传变异大多存在于种群内,种群间的遗传分化占14.25%.聚类分析表明,长叶红砂种群遗传距离与地理距离之间无直接相关关系.遗传多样性水平与物种特性和所处不同群落有关,濒危植物并不一定表现为遗传变异水平的降低.     

Microsatellite primers in Oenothera harringtonii (Onagraceae), an annual endemic to the shortgrass prairie of Colorado

? Premise of the study: Microsatellite markers were developed in the annual herb, Oenothera harringtonii, to investigate patterns of genetic diversity, gene flow, and parentage within and among populations of this Colorado endemic. ? Methods and Results: Ten polymorphic loci were identified in O. harringtonii and tested in four populations sampled across the range of the species. These loci contained trinucleotide repeats with 7-29 alleles per locus. Nine of the 10 loci also amplified in O. caespitosa subsp. macroglottis, O. caespitosa subsp. marginata, and O. caespitosa subsp. navajoensis. In addition, we optimized three markers developed for O. biennis and provide reports of their effectiveness in all four taxa. ? Conclusions: These results indicate the utility of these markers in O. harringtonii for future studies of genetic structure, gene flow, and parentage as well as their applicability in other members of the O. caespitosa species complex.     

Development of EST-SSR markers for Elaeocarpus photiniifolia (Elaeocarpaceae), an endemic taxon of the Bonin Islands

? Premise of the study: Expressed sequence tag (EST)-derived microsatellite markers were developed for Elaeocarpus photiniifolia, an endemic taxon of the Bonin Islands. ? Methods and Results: Initially, a complementary DNA (cDNA) library was constructed by de novo pyrosequencing of total RNA extracted from a seedling. A total of 267 primer pairs were designed from the library. Of the 48 tested loci, 25 loci were polymorphic among 41 individuals representing the entire geographical range of the species, with the number of alleles per locus and expected heterozygosity ranging from two to 14 and 0.09 to 0.86, respectively. Most loci were transferable to a related species, E. sylvestris. ? Conclusions: The developed markers will be useful for evaluating the genetic structure of E. photiniifolia.     

Genetic variation,population structure and identification of yellow catfish, <Emphasis Type="Italic">Mystus nemurus</Emphasis> (C&;V) in Thailand using RAPD,ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.     

Genetic structure of island populations of <Emphasis Type="Italic">Prunus lannesiana</Emphasis> var. <Emphasis Type="Italic">speciosa</Emphasis> revealed by chloroplast DNA,AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.     

DNA allelic variations at the loci putatively implicated in seed oil formation among Brassica oilseed cultivars

Knowledge about the genes implicated in lipid biosynthesis acquired from the model plant Arabidopsis is useful in understanding the formation of seed oil in Brassica oilseeds. In this paper, we report the screening of polymorphic markers at the loci putative for the seed oil formation between two geographically different genotypes: the Chinese cultivar Ningyou-7 and the European cultivar Tapidor. These primer pairs (150) were designed based on 75 Brassica genes that were Arabidopsis orthologues implicated in the oil formation. A total of 52 out of the 150 primer pairs associated with 47 of the 75 genes showed polymorphisms between the two genotypes. The type of polymorphisms that could be detected on capillary electrophoresis images and their respective visual futures are described. Further, we selected 34 polymorphic markers to scan allelic variations and found rich DNA polymorphisms among the 54 Brassica oilseed cultivars. On the average, each primer pair resulted in 5.6 alleles at the region that was covered. The correlation between the alleles and seed quality traits revealed that the alleles of BnFAD7 were related to the variation of linolenic acid (C_18:3) contents among the cultivars. The allele FAD7-ics11170 (3/4)-b that was significantly correlated with high linolenic acid content can be used as an efficient marker for the selection of breeding materials with high linolenic acid content.     

Genetic variation and cultivar identification in Cymbidium ensifolium

As a popular flowering species with many cultivars, Cymbidium ensifolium (L.) is commercially important in horticulture. However, so far little has been known about genetic diversity and conservation genetics of this species. Understanding of the genetic variation and relationships in cultivars of C.?ensifolium is a prerequisite for development of future germplasm conservation and cultivar improvement. Here we report assessment of genetic variations in C.?ensifolium cultivars using the DNA fingerprinting technique of inter-simple sequence repeats (ISSR). A total of 239 ISSR loci were identified and used for evaluation of genetic variation with a selection of 19 ISSR primers. Among these ISSR loci, 99.16% were polymorphic with wide genetic variation as shown by Nei??s gene diversity (H?=?0.2431) among 85 tested cultivars. ISSR fingerprinting profiles showed that each cultivar had its characteristic DNA pattern, indicating unequivocal cultivar identification at molecular level. Eighteen cultivar-specific ISSR markers were identified in seven cultivars. The cultivar Sijiwenhan was confirmed as hybrid by four ISSR primers. Several cultivars with same name but different geographical origins were distinguished based on their ISSR profiles. A dendrogram generated with ISSR markers could group 73 of 85 cultivars into four major clusters. Further analysis of ISSR variation revealed that about 69% of total genetic variation in this species is due to genetic divergence inside geographical groups. Our results suggest that both germplasm collection and in?situ conservation are important for future planning of C.?ensifolium species conservation.     

Development of microsatellite markers for Anadenanthera colubrina (Leguminosae), a neotropical tree species

? Premise of the study: We developed and characterized nuclear microsatellite markers for Anadenanthera colubrina, a tropical tree species widely distributed in South America. ? Methods and Results: Leaf samples of mature A. colubrina trees, popularly called "angico," were collected from an area that is greatly impacted by agricultural practices in the region of Ribeir?o Preto in S?o Paulo State in southeastern Brazil. Twenty simple sequence repeat (SSR) markers were developed, 14 of which had polymorphic loci. A total of 96 alleles were detected with an average of 6.86 alleles per polymorphic locus. The expected heterozygosity, calculated at polymorphic loci, ranged from 0.18 to 0.83. Finally, we demonstrated that 18 loci were cross-amplified in A. peregrina. ? Conclusions: A total of 14 polymorphic markers suggest a high potential for genetic diversity, gene flow, and mating system analyses in A. colubrina.     

Isolation and identification of 21 microsatellite loci in the Copper redhorse (Moxostoma hubbsi; Catostomidae) and their variability in other catostomids

Catostomidae represent an important family of freshwater fishes mainly distributed in North America, but also found in Eurasia. This paper describes the development of microsatellite DNA markers for a highly threatened member of this family, the Copper redhorse (Moxostoma hubbsi), as well as cross‐catostomids amplifications. 168 tetra‐nucleotide loci were screened to develop 21 polymorphic markers, with an average number of 8.5 alleles per locus and observed heterozygosity ranging between 0.52 and 1.00. Successful amplification was obtained for 12 other members of the family at between seven to 19 loci, with between two to 18 loci being polymorphic per species.     

地被植物鹅绒委陵菜的遗传多样性研究

采用RAPD方法对不同海拔和植被盖度下的鹅绒委陵菜（Potentilla anserinaL.）6个种群的遗传多样性水平进行了研究。结果表明,13条随机引物共扩增出132条带,多态性位点为117;鹅绒委陵菜物种水平的遗传多样性较高,且种群间分化明,多态位点比率（88.64%）、Nei’s基因多样性（0.3180）、Shannon信息多样性指数（0.4732）均远高于多数克隆植物。6个种群内的遗传多样性水平变化较大,多态位点在各种群中分布不均衡,种群间分化系数（GST）为47.7%,基因流较低（Nm=0.5482）,多态位点比率在31.06%～74.24%之间,Shannon信息指数在0.1711～0.3625之间,Nei基因多样性指数在0.1164～0.2425之间。多样性水平的变化与海拔没有明显的相关性,而与生境盖度呈显著正相关。从而推断在盖度低、资源丰富的环境中,该物种可能更倾向于克隆繁殖。     

Microsatellite DNA markers for three toad-headed lizard species (Phrynocephalus vlangalii, P. przewalskii and P. guttatus)

To assess the impact of natural landscapes on the population structure of lizards, 10 polymorphic microsatellite DNA markers were developed for the Qinghai toad-headed lizard, Phrynocephalus vlangalii. The number of alleles at these informative loci ranged from four to 28. The novel markers and those previously developed for Phrynocephalus przewalskii were cross-tested among three toad-headed lizard species P. vlangalii, P. przewalskii and P. guttatus. A high cross-utility rate of more than 58% was observed among these three species. These markers are expected to be useful tools for taxonomic considerations as well as population genetic analysis and future conservation management.     

Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers

Abstract.— We examined genetic variation at 21 polymorphic allozyme loci, 15 nuclear DNA loci, and mitochondrial DNA in four spawning populations of sockeye salmon ( Oncorhynchus nerka ) from Cook Inlet, Alaska, to test for differences in the patterns of divergence among different types of markers. We were specifically interested in testing the suggestion that natural selection at allozyme loci compromises the effectiveness of these markers for describing the amount and patterns of gene flow among populations. We found concordance among markers in the amount of genetic variation within and among populations, with the striking exception of one allozyme locus ( sAH ), which exhibited more than three times the amount of among-population differentiation as other loci. A consideration of reports of discordance between allozymes and other loci indicates that these differences usually result from one or two exceptional loci. We conclude that it is important to examine many loci when estimating genetic differentiation to infer historical amounts of gene flow and patterns of genetic exchange among populations. It is less important whether those loci are allozymes or nuclear DNA markers.     

动物种群遗传多态性研究中的PCR技术

基因组DNA的变异是种群遗传多态性研究的基础。PCR技术可以在反应管内经济快速地扩增特定DNA序列,在动物种群遗传多态性研究中的应用主要包括三个方面：(1)种群遗传多态位点的检测;(2)基因定位或利用已经定位的单拷贝基因设计染色体位点特异的分子标记;(3)与DNA测序技术相结合,高效经济地获取特定基因座位的全部遗传变异。     

Genetic mapping in Eucalyptus urophylla and Eucalyptus grandis using RAPD markers.

Two single-tree linkage maps were constructed for Eucalyptus urophylla and Eucalyptus grandis, based on the segregation of 480 random amplified polymorphic DNA (RAPD) markers in a F1 interspecific progeny. A mixture of three types of single-locus segregations were observed: 244 1:1 female, 211 1:1 male, and 25 markers common to both, segregating 3:1. Markers segregating in the 1:1 ratio (testcross loci) were used to establish separate maternal and paternal maps, while markers segregating in the 3:1 ratio were used to identify homology between linkage groups of the two species maps. An average of 2.8 polymorphic loci were mapped for each arbitrary decamer primer used in the polymerase chain reaction. The mean interval size beween framework markers on the maps was 14 cM. The maps comprised 269 markers covering 1331 cM and 236 markers covering 1415 cM, in 11 linkage groups, for E. urophylla (2n = 2x = 22) and E. grandis (2n = 2x = 22), respectively. A comparative mapping analysis with two other E. urophylla and E. grandis linkage maps showed that RAPDs could be reliable markers for establishing a consensus species map. RAPD markers were automatically and quantitatively scored with an imaging analyzer. They were classified into four categories based on their optical density. A fragment intensity threshold is proposed to optimize the selection of reliable RAPD markers for future mapping experiments. Key words : genetic linkage map, Eucalyptus urophylla, Eucalyptus grandis, random amplified polymorphic DNA, RAPD, automated data collection.     

基于RAPD数据探讨中华蓑藓(Macromitrium cavaleriei Card.& Thér.)形态变异的遗传基础

中华蓑藓(Macromitrium cavaleriei Card.&;Thér.)的形态变异强烈,为了解形态变异是否具有遗传学背景,本研究以采自浙南凤阳山、浙中金华北山、浙西北的天目山和江西庐山4个种群的中华蓑藓为材料,运用随机扩增多态性DNA(RAPD)探讨了中华蓑藓的遗传多样性。从100条随机引物中筛选出了15条有效引物,利用它们共获得183个条带,其中多态性条带占79.69%,中华蓑藓各种群间的Dice遗传距离为0.0732~0.1514。POPGENE32软件分析得到种的Nei基因多样性指数(H)为0.3378,Shannon指数(I)为0.5126,遗传分化系数(G_st)为0.1670;基因流(N_m)为2.4947;种群内遗传多样性指数为0.4055,占种群总的遗传多样性的79.11%,反映出研究区域内的中华蓑藓遗传变异大多数存在于种群内,中华蓑藓形态变异并没有多少遗传背景,很可能是生态环境因素引起的可塑性变异。聚类分析表明,中华蓑藓种群遗传距离与地理距离之间无直接相关关系,遗传多样性水平与物种特性和所处不同群落有关。虽然4个种群内的形态变异丰富,但是属于同一物种的范围。     

Development of SNP-based CAPS and dCAPS markers in eight different genes involved in starch biosynthesis in rice

Single nucleotide polymorphisms (SNPs), which are inexhaustible, highly stable, and simply detectable sequence polymorphisms, can lead to phenotypic variations by affecting protein composition changes. Here, we report development of 25 new cleaved amplified polymorphic sequence or derived cleaved amplified polymorphic sequence markers that have discrete band sizes in relation to the SNP genotypes in eight putative gene regions. The average frequency of DNA polymorphisms was 1 per 175 bp (SNPs, 1 per 217 bp; In/dels, 1 per 906 bp). In primary statistical analysis of each marker on 55 diverse rice accessions, including different ecotypes, the mean value of the major allele frequency was 0.658 (0.509–0.927). The average polymorphism information content was 0.326 (0.126–0.375). The mean value of the inbreeding coefficient (f) was 0.950 and was positive (heterozygote deficiency) at all loci, corresponding to the inbreeding system in rice. In cluster analysis, all rice accessions clustered mainly into three groups according to the ecotypes. The association analysis showed that the SNP of Granule-bound starch synthase I and ADP-glucose pyrophosphorylase small subunit (ADPase-S) genes were highly associated with apparent amylose content variation than the others. These new SNP markers may be useful in genotyping rice germplasm, in marker-assisted selection for improving starch quality and content, and in linkage as well as association studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Mapping and Character Tagging in Brassica juncea—I. Degree,Nature and Linkage Relationship of RFLPs and Their Association With Quantitative Traits

For molecular mapping and character tagging in Indian mustard (Brassica juncea), cultivar Varuna and exotic collection BEC 144 were chosen on the basis of morphological and molecular differences. High degree of RFLP was detected between them using genomic DNA clones of Pst! subgenomic library as probes. Of the 48 probe — enzyme combinations, 89.5% were polymorphic. Majority of the probes revealed duplicate loci and a high frequency of null alleles (71.4%). Segregation analysis in the F² population revealed significant deviation from expected 1:2:1 ratio for 32% of the markers. Using the computer package MAPMAKER, 15 markers could be placed in six linkage groups which covered a total length of 173.9 eM. Based on single factor analysis of variance, three significant marker-quantitative trait associations viz. BJG 59a primary branches/plant, BJG 42Gb - secondary branches/plant and BJG 433-days to f1owering could be identified. The BEC 144 alleles at the quantitative trait loci (QTL) in the marked genomic regions enhanced the trait expression. The putative gene action at these loci was found to be non-additive.     

Monomorphic species specific DNA, detected by polymerase chain reaction with random primers

Intra- and interspecific variability of total DNA isolated from haploid megagametophytes of coniferous species was examined using polymerase chain reaction with random primers. Based on this technique, one can with certainty detect heterozygosity at gene loci carrying null alleles and thus reveal cryptic intraspecific genetic variation. Large population samples were used. Along with random amplified polymorphic DNA, i.e., widely known fragments (amplicons) polymorphic within a species, we found invariant loci lacking individual or geographic variability but differentiating species within genera and other taxa. This DNA was termed RAMD (random amplified monomorphic DNA) to distinguish it from polymorphic DNA. Our findings suggest that genetic monomorphism of species and the dual structure of the eukaryotic genome can be detected at the DNA level as was previously shown for protein gene markers.