Genetic similarity among Arabidopsis thaliana ecotypes estimated by DNA sequence comparison

DNA polymorphisms among Arabidopsis thaliana ecotypes are widely used as genetic markers in map-based cloning strategies. New PCR-based molecular markers do not only facilitate molecular mapping, but can also be used to obtain reliable sequence information for cladistic analyses. We have used CAPS (cleaved amplified polymorphic sequences) markers and a direct sequencing strategy to estimate genetic similarity among eighteen Arabidopsis ecotypes. Sequences at four loci, two from the nuclear and two from a non-nuclaar genome, were analysed. For each ecotype more than 1000pb of sequence information was obtained, and genetic similarity was calculated from a total of 35 polymorphic sites using a character-based approach. Divergence ranged from zero up to 50 discordant characters among the 72 characters defined by the polymorphisms. Separate calculations based on the nuclear and the non-nuclear sequences were performed and revealed a number of common features, including the existence of small clusters of very closely related ecotypes separated from each other by extensive sequence divergence. Our results provide information useful especially to investigators setting up crosses for chromosome landing strategies.     

Genetic relationships and variation among ecotypes of seashore paspalum (Paspalum vaginatum) determined by random amplified polymorphic DNA markers.

Random amplified polymorphic DNA (RAPD) markers were used to assess genetic relationships and variation among ecotypes of the turfgrass seashore paspalum (Paspalum vaginatum Swartz). Vegetative tissues or seeds of 46 seashore paspalum ecotypes were obtained from various locations in the United States, Argentina, and South Africa. Leaf DNA extracts were screened for RAPD markers using 34 10-mer random primers. A total of 195 reproducible RAPD fragments were observed, with an average of six fragments per primer. One hundred and sixty-nine fragments (87% of the total observed) were polymorphic, among which 27 fragments (16%) were present in three or less ecotypes, indicating the occurrence of a high level of genetic variation among the examined accessions of this species. Cluster analysis (UPGMA) and principal coordinates analysis were performed on the RAPD data set. The results illustrate genetic relationships among the 46 ecotypes, and between ecotypes and their geographical origins. Ecotypes from southern Africa could be differentiated from the U.S. and most of the Argentinean ecotypes. With a few exceptions, ecotypes collected from Argentina, Hawaii, Florida, and Texas were separated into distinct clusters.     

Haplotypic Divergence Coupled with Lack of Diversity at the Arabidopsis Thaliana Alcohol Dehydrogenase Locus: Roles for Both Balancing and Directional Selection?

We designate a region of the alcohol dehydrogenase locus (Adh) of the weedy crucifer, Arabidopsis thaliana, as ``hypervariable'''' on the basis of a comparison of sequences from ecotypes Columbia and Landsberg. We found eight synonymous and two replacement mutations in the first 262 nucleotides of exon 4, and an additional two mutations in the contiguous region of intron 3. The rest of the sequence (2611 bp) has just three mutations, all of them confined to noncoding regions. Our survey of the hypervariable region among 37 ecotypes of A. thaliana revealed two predominant haplotypes, corresponding to the Columbia and Landsberg sequences. We identified five additional haplotypes and 4 additional segregating sites. The lack of haplotype diversity is presumably in part a function of low rates of recombination between haplotypes conferred by A. thaliana''s tendency to self-fertilize. However, an analysis in 32 ecotypes of 12 genome-wide polymorphic markers distinguishing Columbia and Landsberg ecotypes indicated levels of outcrossing sufficient at least to erode linkage disequilibrium between dispersed markers. We discuss possible evolutionary explanations for the coupled observation of marked divergence within the hypervariable region and a lack of haplotype diversity among ecotypes. The sequence of the region for closely related species argues against the possibility that one allele is the product of introgression. We note (1) that several loss of function mutations (both naturally and chemically induced) map to the hypervariable region, and (2) the presence of two amino acid replacement polymorphisms, one of which causes the mobility difference between the two major classes of A. thaliana Adh electrophoretic alleles. We argue that protein polymorphism in such a functionally significant part of the molecule may be subject to balancing selection. The observed pattern of extensive divergence between the alleles is consistent with this explanation because balancing selection on a particular site maintains linked neutral polymorphisms at intermediate frequencies.     

Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana

Konieczny and Ausubel have described a technique whereby Arabidopsis thaliana loci can be rapidly mapped to one of the ten chromosome arms using a small number of F2 progeny from crosses between the ecotypes Landsberg erecta and Columbia. The technique involves the use of 18 co-dominant, cleaved amplified polymorphic sequence (CAPS) markers which are evenly distributed throughout the Arabidopsis genome. We have mapped these 18 markers using recombinant inbred (RI) lines generated in our laboratory. These data enable a better integration of loci mapped relative to the CAPS markers into the restriction fragment length polymorphism (RFLP) map generated using Arabidopsis RI lines.     

Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)

The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73.     

Population genomics of the killer whale indicates ecotype evolution in sympatry involving both selection and drift

The evolution of diversity in the marine ecosystem is poorly understood, given the relatively high potential for connectivity, especially for highly mobile species such as whales and dolphins. The killer whale (Orcinus orca) has a worldwide distribution, and individual social groups travel over a wide geographic range. Even so, regional populations have been shown to be genetically differentiated, including among different foraging specialists (ecotypes) in sympatry. Given the strong matrifocal social structure of this species together with strong resource specializations, understanding the process of differentiation will require an understanding of the relative importance of both genetic drift and local adaptation. Here we provide a high‐resolution analysis based on nuclear single‐nucleotide polymorphic markers and inference about differentiation at both neutral loci and those potentially under selection. We find that all population comparisons, within or among foraging ecotypes, show significant differentiation, including populations in parapatry and sympatry. Loci putatively under selection show a different pattern of structure compared to neutral loci and are associated with gene ontology terms reflecting physiologically relevant functions (e.g. related to digestion). The pattern of differentiation for one ecotype in the North Pacific suggests local adaptation and shows some fixed differences among sympatric ecotypes. We suggest that differential habitat use and resource specializations have promoted sufficient isolation to allow differential evolution at neutral and functional loci, but that the process is recent and dependent on both selection and drift.     

Genetic Diversity of Persian Ecotypes of Indian Walnut (<Emphasis Type="Italic">Aeluropus littoralis</Emphasis> (Gouan) Pari.) by AFLP and ISSR Markers

Indian walnut Aeluropus littoralis (Gouan) Pari, well known for its salt tolerance and forage quality, is a perennial monocot grass from Chlorideae tribe that shows a high potential to become an important genetic model. Although, Aeluropus germplasm is very rich in Iran; but is still unexplored. In this study, 20 ecotypes of A. littoralis were gathered from different geographic zones and a set 16 primers (ISSRs-AFLPs combined) was used for genetic diversity evaluation. A total of 635 fragments and 594 polymorphic fragments were produced (93.5%) and modest levels of genetic similarity were found (ranging from 0.38 to 0.71) among ecotypes. The results showed an equal competition between the two markers on polymorphism detection, so both molecular techniques were able to distinguish the A. littoralis ecotypes. Furthermore, Dendrogram based on both the Bayesian and PCoA analysis showed a clear discrimination and significant variation among ecotypes; however, molecular clustering indicated a weak concordance with geographical grouping. The amount of molecular diversity revealed by molecular markers for A. littoralis in this study can be exploited for pasture conservation programs and new forage crop development.     

用分子标记技术分析不同生态型芦苇的遗传多样性

用分子标记技术对河西走廊4种不同生态型芦苇进行了遗传多态性分子标记分析。从30条ISSR引物和45条RAPD引物中分别筛选出适合4种不同生态型芦苇分析的9条和13条引物,前者共扩增出99条带,多态性位点数为51;后者共扩增出195条带,多态性位点数为87。两种分子标记分析所得的遗传相似系数呈显著正相关(r=0.845,P<0.05)。4种生态型芦苇表现出由水生芦苇经盐渍芦苇向沙丘芦苇逐渐演化的趋势。     

Generation of co-dominant PCR-based markers by duplex analysis on high resolution gels

Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high-performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready-to-use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single-nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co-dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36 Arabidopsis thaliana loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half ( n = 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co-dominant PCR-based molecular markers for fine-scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.     

红檵木的3′端延长随机引物扩增DNA(3′-ERPAD)标记

为了对116个10－mer随机引物的RAPD分子标记进行深入了解,探讨分子标记与园艺性状的关系,筛出多态性明显、重复性好、亮度大、分子量也较大（1031bp以上）的10－mer随机引物,在其3'端添加一任意碱基,对14个红木材料和2个来源不同的生态型木材料进行3'-ERPAD标记分析。发现在S2和S35的延伸引物中能够得到最佳扩增结果的引物对是S2－G＋S2－C和S35－A＋S35－C,它们的扩增结果都含有与各自基本引物相同的差异扩增带,这些差异带分别是S21500、S21031和S353000、S35900、S35560。通过与园艺性状比较,发现S21500片段可能与控制叶色的基因有关。     

pARMS,multiple marker-containing plasmids for easy RFLP analysis inArabidopsis thaliana

A simplified way is presented for the analysis of 13 RFLP markers, each of them detecting solely a pair of polymorphic bands afterEcoR I digestion of genomic DNA fromArabidopsis thaliana ecotypes Landsbergerecta and Columbia. Five plasmids (pARMS) containing multiple marker inserts were constructed that score 13 loci, almost evenly distributed on the five chromosomes (Fabri and Schäffner, 1994). Since their polymorphic signals do not interfere with each other, pARMS2,?6, and?7 can be combined to score eight RFLP marker in a single Southern experiment. Two additional plasmids—pARMS5 and?8— allow the joint analysis of another five markers. If pARMS are used for rough mapping of a mutation, which will be the major application, this procedure reduces processing and handling to just five plasmids and also results in more uniform signal intensities due to a more even labeling.     

Development of SSR Markers for Genetic Analysis of Silverleaf Nightshade (Solanum elaeagnifolium) and Related Species

Silverleaf nightshade (2n?=?2x?=?24) is a serious weed in the Solanaceae, for which no specific molecular markers are currently available. In order to investigate the extent and distribution of genetic diversity among accessions of silverleaf nightshade, we developed 23 simple sequence repeat (SSR) markers from publicly available nucleotide and EST databases for silverleaf nightshade. Eleven of them were single-locus polymorphic markers. The number of alleles among these loci ranged from 2 to 4. The observed and expected heterozygosity ranged from 0 to 0.97 and 0.07 to 0.64, respectively. Fourteen SSR markers enabled to amplify alleles in morphologically similar species quena. These results proved that the SSR markers that we developed could be useful for (1) determining genetic diversity and structure among natural populations of silverleaf nightshade and (2) identifying silverleaf nightshade and quena ecotypes. This is the first set of species-specific SSR markers identified in silverleaf nightshade, which could contribute to the better understanding of genetic diversity of silverleaf nightshade and related species.     

毛乌素沙地中间锦鸡儿群体繁育系统的变化

中间锦鸡儿在毛乌素沙地这个介于典型草原和典型荒漠之间的生态过渡带广泛分布 ,是当地灌丛生态系统的一个优势种或建群种[1,2 ] 。我们曾研究表明 ,中间锦鸡儿群体内高度的遗传多样性以及不同景观生态类型群体之间有强大的基因流存在[3 ,4 ] ;特别是同一植株不同种子显示的不同的种子蛋白组成进一步证明该植物的异交性[4 ,5] 。上述群体遗传研究还表明 ,中间锦鸡儿和柠条锦鸡儿共享 1个基因库 ,与徐朗然等 ① 报道的种间杂交结果一致。ＲＡＰＤ研究发现 ,少数多态ＤＮＡ在群体里的频率与所在地区的水分状况有关 :滩地为零或很低 ,生境越干…     

Variation of Breeding Systems in Populations of Caragana intermedia (Leguminosae ) in Maowusu Sandy Grassland

Maowusu sandy grassland locating at an ecotone between typical desert and typical grassland contained several landscape ecotypes or elements where Caragana intermedia Kuang et H.C.Fu naturally distributed as a dominant species in shrub ecosystems. Based on a phenomenon of gene segregation of open-pollinated seeds within each plants similar to Mendel's segregation in F2, a study on testing the breeding systems of populations under 4 landscape ecotypes was conducted. Statistical data showed its availability in estimation of breeding system parameters when isozymes were used as genetic markers. Nei's genetic differentiation GST among 4 ecotypes in Maowusu was estimated at 0.07 from lap loci close to a GST =0.076 reported in the authors' laboratory. The results indicated that breeding systems of populations gradually changed from total outbreeding to partial inbreeding when water conditions worsened. Therefore, the former RAPD data, especially the cline of frequencies for a few polymorphic DNAs in different landscapes can be partially explained by gene fixation caused by selfing or inbreeding probably induced by drought. It was difficult to assess breeding system parameters by using one dimensional SDS polyacrylamide gel electrophoresis of seed proteins of C. intermedia simply due to a difficulty of genetic analysis of seed protein subunits.     

Linking adaptation,delimitation of evolutionarily significant units (ESUs), and gene function: a case study using hemlock looper ecotypes

Developing genetic markers for the identification of recently diverged groups, such as ecotypes or species complexes, remains difficult due to challenges with incomplete lineage sorting, hybridization and introgression. Genome‐wide scans of single nucleotide polymorphisms (SNPs) have proven useful for inferring patterns of genetic differentiation at the population level. In combination with a new analytical technique, the discriminant analysis of principal components (DAPC), and within the framework of iterative taxonomy, it may also be possible to extract a combination of SNPs as markers for the delimitation of closely related groups. In addition, since DAPC identifies the loci contributing the most to group clustering, it may be possible to link putative biological function to differences that define group boundaries. We tested this technique on two ecotypes of the hemlock looper (Lambdina fiscellaria), which differ in terms of number of larval stadia, developmental rate and fecundity. It was possible to separately cluster the two ecotypes with 95% correct assignment using 27 SNPs. We also determined that a storage hexamerin carried eight of these SNPs, including the two highest contributing loci, of which the top contributor was nonsynonymous. Other studies have found this protein to be highly expressed just before metamorphosis, pointing to a possible connection between its role in clustering ecotypes and its biological function. These SNP markers can now be further developed for high throughput delimitation of individuals of unknown ecotype identity.     

Site-specific genetic divergence in parallel hybrid zones suggests nonallopatric evolution of reproductive barriers

The evolution of reproductive isolation in the presence of gene flow is supported by theoretical models but rarely by data. Empirical support might be gained from studies of parallel hybrid zones between interbreeding taxa. We analysed gene flow over two hybrid zones separating ecotypes of Littorina saxatilis to test the expectation that neutral genetic markers will show site-specific differences if barriers have evolved in situ. Distinct ecotypes found in contrasting shore habitats are separated by divergent selection and poor dispersal, but hybrid zones appear between them. Swedish islands formed by postglacial uplift 5000 years ago provide opportunities to assess genetic structure in a recently evolved system. Each island houses a discrete population containing subpopulations of different ecotypes. Hybrid zones between ecotypes may be a product of ecological divergence occurring on each island or a consequence of secondary overlap of ecotypes of allopatric origin that have spread among the islands. We used six microsatellite loci to assess gene flow and genetic profiles of hybrid zones on two islands. We found reduced gene flow over both hybrid zones, indicating the presence of local reproductive barriers between ecotypes. Nevertheless, subpopulations of different ecotypes from the same island were genetically more similar to each other than were subpopulations of the same ecotype from different islands. Moreover, neutral genetic traits separating the two ecotypes across hybrid zones were site-specific. This supports a scenario of in situ origin of ecotypes by ecological divergence and nonallopatric evolution of reproductive barriers.     

A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers

A set of mapping markers have been designed for Arabidopsis thaliana that correspond to DNA fragments amplifed by the polymerase chain reaction (PCR). The ecotype of origin of these amplified fragments can be determined by cleavage with a restriction endo-nuclease. Specifically, 18 sets of PCR primers were synthesized, each of which amplifies a single mapped DNA sequence from the Columbia and Landsberg erecta ecotypes. Also identifed was at least one restriction endonuclease for each of these PCR products that generates ecotype-specific digestion patterns. Using these co-dominant cleaved amplified polymorphic sequences (CAPS), an Arabidopsis gene can be unambiguously mapped to one of the 10 Arabidopsis chromosome arms in a single cross using a limited number of F₂ progeny.     

Efficient protocols for CAPS-based mapping inArabidopsis

Positional cloning continues to be an essential method for gene identification and characterisation. The introduction of PCR-based techniques such as Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Length Polymorphisms (SSLP) and Cleaved Amplified Polymorphic Sequences (CAPS) has greatly increased the efficiency of gene mapping in arabidopsis. To develop the CAPS marker approach further, we have altered several critical mapping parameters. Efficiency was improved by using a small volume of dry seed for DNA extraction instead of the commonly used vegetative tissue. Reproducibility of PCR reactions was enhanced by faster and reduced protocols for PCR and restriction enzyme digestion and optimisation of PCR conditions for over 50 CAPS primer pairs. Finally, the density of genetic markers was increased by providing polymorphic information for all CAPS markers in arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col) and Cape Verde Islands (Cvi).     

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.     

Distribution of the genetic diversity of the Siberian stone pine,Pinus sibirica Du Tour,along the latitudinal and longitudinal profiles

The Siberian stone pine (Pinus sibirica Du Tour) is one of the main forest-forming coniferous species of the boreal ecosystems of Western Siberia. We used the isozyme method to analyze 11 ecotypes representing the latitudinal and longitudinal profiles within the species range, including samples from the geographic boundaries of the distribution. The genetic structure of the ecotypes is described on the basis of the variability for 26 isozyme loci encoding for 16 enzyme systems. The greatest genetic diversity was observed in the taiga ecotypes in the central part of the studied area, while the ecotypes along the species range boundaries were shown to be genetically depauperized. Approximately 8.1 % of the observed genetic diversity is attributed to differences between the studied ecotypes. We detected high levels of genetic diversity for the Fest-2, Pgm-1, Sod-4, and a few other loci, as well as a correlation between allele frequencies and geographical locations of the populations. The results of multivariate analysis of allelic frequencies as well as cluster analysis allowed us to discriminate three major groups of ecotypes: north-eastern, central and south-western. In view of our results, we compare two hypotheses: one which attributes the spatial distribution of genetic variations to the selectivity for some of the polymorphic allozyme loci, and the other based in the history of the formation of the range of the Siberian stone pine.