Single Sequence Repeat Research Articles

Transcriptome resources for the white‐footed mouse (Peromyscus leucopus): new genomic tools for investigating ecologically divergent urban and rural populations

Genomic resources are important and attainable for examining evolutionary change in divergent natural populations of nonmodel species. We utilized two next-generation sequencing (NGS) platforms, 454 and SOLiD 5500XL, to assemble low-coverage transcriptomes of the white-footed mouse (Peromyscus leucopus), a widespread and abundant native rodent in eastern North America. We sequenced liver mRNA transcripts from multiple individuals collected from urban populations in New York City and rural populations in undisturbed protected areas nearby and assembled a reference transcriptome using 1 080 065 954 SOLiD 5500XL (75 bp) reads and 3 052 640 454 GS FLX + reads. The reference contained 40 908 contigs with a N50 = 1044 bp and a total content of 30.06 Megabases (Mb). Contigs were annotated from Mus musculus (39.96% annotated) Uniprot databases. We identified 104 655 high-quality single nucleotide polymorphisms (SNPs) and 65 single sequence repeats (SSRs) with flanking primers. We also used normalized read counts to identify putative gene expression differences in 10 genes between populations. There were 19 contigs significantly differentially expressed in urban populations compared to rural populations, with gene function annotations generally related to the translation and modification of proteins and those involved in immune responses. The individual transcriptomes generated in this study will be used to investigate evolutionary responses to urbanization. The reference transcriptome provides a valuable resource for the scientific community using North American Peromyscus species as emerging model systems for ecological genetics and adaptation.

Correction of the misclassification of species in the Portuguese collection of Cucurbita pepo L. using DNA markers

In this study, the genetic variability among 130 accessions of the Portuguese germplasm collection of Cucurbita pepo L. maintained at the Banco Português de Germoplasma Vegetal was assessed using AFLP (amplified fragment length polymorphism) and RAPD (random amplified polymorphic DNA) techniques for the identification of a genetically diverse core group of accessions for field phenotypic analysis. The surprisingly completely different molecular patterns exhibited by multiple accessions was later confirmed in the distribution of the putative C. pepo plants into two clusters drastically separated at a very low level of genetic similarity (DICE coefficient = 0.37). Additional analyses with RAPD and ISSR (inter single sequence repeat) markers and the introduction of standard genotypes of C. maxima L. and C. moschata L. into the analyses allowed the identification of multiple accessions of the last species wrongly included in the C. pepo collection. This study is a good example of the usefulness of DNA markers in the establishment and management of plant germplasm collections.

The application of high resolution melting in the analysis of simple sequence repeat and single nucleotide polymorphism markers in a pea (Pisum sativum L.) population

The aim of this study was to verify the high resolution melting (HRM) method in the analysis of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in pea (Pisum sativum L.). A recombinant inbred line population, Carneval × MP1401, was tested for three SNP and 103 SSR markers. HRM analysis was conducted on a LightScanner 96 instrument with LC Green dye. The melting curve shape permitted two polymorphic genotypes to be distinguished. The results were confirmed by gel electrophoresis. Three SSR markers were sequenced and analysed by the melting prediction software. The results confirmed the presence of one polymerase chain reaction (PCR) product with two melting domains. Sequence tagged site (STS) markers produced specific products: Psat_EST_00189_01_1 (300 bp), Pis_GEN_18_2_1 (400 bp), Pis_GEN_7_1-2_1 (600 bp). Amplicons contained one, four and seven single nucleotide polymorphisms, respectively. Melting curve differences enabled the population genotyping except for Psat_EST_00189_01_1 where resolution was too low. Primers for Psat_EST_00189_01_1 were redesigned to obtain a shorter (100 bp) PCR product which increased the resolution. The number of SNPs and amplicon length are crucial for HRM resolution. The HRM method is fast and has a high throughput. The melting analysis of 96 samples takes less than 10 min. Agarose gel analysis confirmed the reliability of HRM, which eliminates laborious post-PCR analysis.

De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification.

BackgroundRed clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress.ResultsPlants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing ‘transcript tags’. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress.ConclusionsThis de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-453) contains supplementary material, which is available to authorized users.

DNA extraction protocols for molecular studies of Asiatic black bears

Abstract Optimization of DNA extraction and amplification techniques increases the reliability of genetic studies used to estimate population size and assess genetic diversity, particularly for non-invasively collected samples. We evaluated the effectiveness of 4 DNA extraction procedures—the Roboscreen and Qiagen kit methods and the Organic and Chelex methods, as applied to invasively and non-invasively collected samples from Asiatic black bears (Ursus thibetanus). We assessed effectiveness based on quantity and purity of the extracts by method, and their ability to amplify genomic DNA via polymerase chain reaction (PCR) amplification of a single simple sequence repeat (SSR) marker. For each method, the greatest average DNA quantity and purity index (86.25%) was from blood, while muscle showed greatest DNA quantity and purity index using the Qiagen DNA extraction method. The Roboscreen kit and Chelex method provided a 57% and 58% PCR success rate, respectively, for hairs, demonstrating the value of such ...

Genetic diversity in East African finger millet (Eleusine coracana (L.) Gaertn) landraces based on SSR markers and some qualitative traits

In this study, genetic diversity in 340 finger millet accessions from Kenya, Tanzania and Uganda and 15 minicore accessions was assessed using 23 single-sequence repeat markers and five qualitative traits. Nineteen markers were polymorphic with a mean polymorphic information content value of 0.606 and a range of 0.035–0.889, with allele size ranging from 148 to 478. A total of 195 alleles were detected (range of 3–23 and average of 10.3 alleles per locus), with 57.7% being rare and 17.4% being private. Differentiation between the accessions of the three countries was weak, with most of the genetic diversity being explained by variability within the countries and subregions than by that among the countries and subregions. The highest genetic diversity was observed in the Kenyan accessions (0.638 ± 0.283) and the least in the Ugandan accessions (0.583 ± 0.264). The highest differentiation based on Wright's fixation index was observed between the Ugandan and Tanzanian accessions (FST= 0.117; P< 0.001). There was no association between the morphological traits assessed and the genetic classes observed. The low variability between the countries could be attributed to a shared gene pool, as the crop originated from the East African region. Farmers' selection for adaptation and end use could have contributed to the high diversity within the countries. Concerted efforts need to be made to characterize the large germplasm stocks in East Africa for their effective conservation and utilization. The lack of representation of accessions from the three countries in all global minicore diversity clusters points to the need to explore the East African germplasm to identify the diversity not captured earlier to be included in the global repository.

Comparative ampelographic and genetic analysis of grapevine cultivars from Algeria and Morocco

Background and Aims North Africa has a long history of viticulture and a wide diversity of grape cultivars. Ampelographic studies have been made of grapevine cultivars grown all over the world, but only a few describe those of Algeria and Morocco. Many Maghrebi cultivars held in germplasm banks or found growing wild in this region have recently been subjected to microsatellite profiling by different researchers, though little comparative analysis has been undertaken. The aim of the present work was to clarify the identity of the grapevine cultivars growing in the Maghreb via ampelographic and single-nucleotide polymorphism analysis. Methods and Results Seventy-one accessions were studied through the ampelographic construction of their mean leaves, via genotypic analysis using single-nucleotide polymorphism markers, and the comparison of these results with previously reported single sequence repeat marker profiles and ampelographic data for other grapevine material from the Maghreb. Conclusion New synonyms and homonyms were detected between Maghrebi cultivars. Some misinterpretations and errors of identification made during the making of the studied germplasm collections were identified. Significance of the Study This study helps clarify the confusion over the identity of Algerian and Moroccan grapevine cultivars and provides a general picture of grapevine diversity in the Maghreb.

Production and molecular characterization of somatic hybrids between Pleurotus florida and Lentinula edodes.

Nine inter-generic somatic hybrids named as pfle were produced through PEG-mediated protoplast fusion between Pleurotus florida and Lentinula edodes using double selection method. Hybridity of the newly developed strains was established on the basis of colony morphology, mycelial growth, hyphal traits, fruit-body productivity and inter single sequence repeat (ISSR) marker profiling. Hybrid population was assessed with different phenotypic variables by one-way analysis of variance. Principal component matrices were analyzed for the six phenotypic variables in scatter plot showing maximum positive correlation between each variable for all strains examined. Six ISSR primers generated 66 reproducible fragments with 98.48 % polymorphism. The dendrogram thus created based on unweighted pair-group method with mathematic averages method of clustering and Euclidean distance which exhibited three major groups between the parents and pfle hybrids. Though P. florida parent remained in one group but it showed different degrees of genetic distance with all the hybrid lines belonging to the other two groups while L. edodes was most distantly related to all the hybrid lines. L. edodes specific sequence-rich ISSR amplicon was recorded in all the hybrid lines and in L. edodes but not in P. florida. All the fruit body generating pfle hybrid lines could produce basidiocarp on paddy straw in sub-tropical climate and showed phenotypic resemblance to the P. florida parent.

Use of VeraCode 384-plex assays for watermelon diversity analysis and integrated genetic map of watermelon with single nucleotide polymorphisms and simple sequence repeats

Watermelon (Citrullus lanatus var. lanatus) is one of the most important vegetable crops in the world. Molecular markers have become the tools of choice for resolving watermelon taxonomic relationships and evolution. Increased numbers of single nucleotide polymorphism (SNP) markers together with simple sequence repeat (SSR) markers would be useful for phylogenetic analyses of germplasm accessions and for linkage mapping for marker-assisted breeding with quantitative trait loci and single genes. We aimed to construct a genetic map based on SNPs (generated by Illumina Veracode multiplex assays for genotyping) and SSR markers and evaluate relationships inferred from SNP genotypes between 130 watermelon accessions collected throughout the world. We incorporated 282 markers (232 SNPs and 50 SSRs) into the linkage map. The genetic map consisted of 11 linkage groups spanning 924.72 cM with an average distance of 3.28 cM between markers. Because all of the SNP-containing sequences were assembled with the whole-genome sequence draft for watermelon, chromosome numbers could be readily assigned for all the linkage groups. We found that 134 SNPs were polymorphic in 130 watermelon accessions chosen for diversity studies. The current 384-plex SNP set is a powerful tool for characterizing genetic relatedness and for developing medium-resolution genetic maps.

Spud DB: A Resource for Mining Sequences, Genotypes, and Phenotypes to Accelerate Potato Breeding

Potato is the world's third most important crop, and is becoming increasingly important in developing countries. Cultivated potato is a highly heterozygous tetraploid (2n = 4x = 48) and suffers from significant inbreeding depression when selfed. As potato can be vegetatively propagated, breeding has been based primarily on phenotypic selection in F1 populations. However, recent advances in genome sequencing and genotyping methods have resulted in the development of large genomic, genetic, and phenotypic datasets that will enable more efficient and rapid breeding approaches. We have developed Spud DB (http://potato.plantbiology.msu.edu/) for the community to access the potato genome sequence and associated annotation datasets, along with phenotypic and genotypic data from a diversity panel of 250 potato clones. The Breeder's Assistant is a web tool to retrieve pertinent phenotypic and genotypic data in a user‐guided manner, and query polymorphic markers such as single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) to identify custom sets of markers for a gene or region of interest. To browse and query the potato genome, a genome browser with 94 tracks of genome annotation, sequence variants, and expression abundance has been deployed. Spud DB also provides a comprehensive search page to data mine the potato genome through tools that query sequence identifiers, functional annotation, gene ontology (GO), InterPro domains, and basic local alignment search tool (BLAST) databases. Collectively, this resource links potato genomic data with phenotypic and genotypic data from a large collection of potato lines for use by the potato community, especially breeders and geneticists.

Paternity identification in sugarcane polycrosses by using microsatellite markers.

Although polycrosses have been used to test the potential of cross-combination of a large number of sugarcane parents, the male parent of the half-sib progenies produced is unknown. The present study aimed to integrate the molecular marker technology to the sugarcane polycross approach by the application of microsatellite markers to identify the male parent of 41 elite clones derived from polycross families. Ten microsatellite [single sequence repeats (SSRs)] primer pairs were used to identify the most likely male parent considering markers present in the selected clone but absent in the female parent. The number of alleles generated by the 10 microsatellite primer pairs ranged from 102 (cross-pollination lantern 4) to 120 (cross-pollination lantern 2) with an average of 113.25 alleles per SSR. The average genetic similarity among the involved parents in the polycrosses was 45.9%. The results of the analysis of the SSR markers absent in the female parent and present only in the selected clone as well as the genetic similarity values allowed the identification of the most likely male parent in 73% of the total clones evaluated and also to detect probable contaminations. The obtained results highlight the importance of using molecular marker technology in the identification and confirmation of the male parent of high-performance clones derived from polycrosses in the sugarcane breeding programs.

Genetic variation in and spatial structure of natural populations of Dipterocarpus alatus (Dipterocarpaceae) determined using single sequence repeat markers.

Dipterocarpus alatus (Dipterocarpaceae) is widely distributed in lowland forests in central and southern Vietnam, Cambodia, Laos, Myanmar, Philippines, Thailand, and India. Due to over-exploitation and habitat destruction, the species is now threatened. The genetic variation within and among populations of D. alatus was investigated on the basis of 9 microsatellite (single sequence repeat, SSR) loci. In all, 268 sampled trees from 10 populations in central and southern Vietnam were analyzed in this study. The SSR data showed a high genetic variability within populations with an average of HO = 0.209 and HE = 0.239. Genetic differentiation among populations was high (FST = 0.266), indicating limited gene flow (Nm = 0.69). Analysis of molecular variance showed that most genetic variation was within populations (74.96%). This study highlights the importance of conserving the genetic resources of D. alatus species.

Sequence divergence of microsatellites for phylogeographic assessment of Moroccan Medicago species

Six Medicago species were investigated to characterize and valorize plant genetic resources of pastoral interest in Morocco. Samples were obtained from the core collection of the South Australian Research and Development Institute (SARDI). The transferability of single sequence repeat markers of Medicago truncatula was successful with 97.6% efficiency across the five species. A total of 283 alleles and 243 genotypes were generated using seven SSR markers, confirming the high level of polymorphism that is characteristic of the Medicago genus, despite a heterozygosity deficit (HO = 0.378; HE = 0.705). In addition, a high level of gene flow was revealed among the species analyzed with significant intra-specific variation. The unweighted pair group method with arithmetic mean dendrogram generated by the dissimilarity matrix revealed that M. polymorpha and M. orbicularis are closely related, and that M. truncatula is likely the ancestral species. The Pearson correlation index revealed no significant correlations between the geographic distribution of the Moroccan species and genetic similarities, indicating local adaptation of these species to different ecological environments independent of their topographical proximities. The substantial genetic variation observed was likely due to the predominance of selfing species, the relative proximity of prospected sites, human impacts, and the nature of the SARDI core collections, which are selected for their high genetic diversity. The results of this first report on Moroccan Medicago species will be of great interest for establishing strategies aiming at reasonable management and selection programs for local and Mediterranean germplasm in the face of increasing environmental change.

A novel dominant glossy mutation causes suppression of wax biosynthesis pathway and deficiency of cuticular wax in Brassica napus

BackgroundThe aerial parts of land plants are covered with cuticular waxes that limit non-stomatal water loss and gaseous exchange, and protect plants from ultraviolet radiation and pathogen attack. This is the first report on the characterization and genetic mapping of a novel dominant glossy mutant (BnaA.GL) in Brassica napus.ResultsTransmission electron microscopy revealed that the cuticle ultrastructure of GL mutant leaf and stem were altered dramatically compared with that of wide type (WT). Scanning electron microscopy corroborated the reduction of wax on the leaf and stem surface. A cuticular wax analysis of the GL mutant leaves further confirmed the drastic decrease in the total wax content, and a wax compositional analysis revealed an increase in aldehydes but a severe decrease in alkanes, ketones and secondary alcohols. These results suggested a likely blockage of the decarbonylation step in the wax biosynthesis pathway. Genetic mapping narrowed the location of the BnaA.GL gene to the end of A9 chromosome. A single-nucleotide polymorphism (SNP) chip assay in combination with bulk segregant analysis (BSA) also located SNPs in the same region. Two SNPs, two single sequence repeat (SSR) markers and one IP marker were located on the flanking region of the BnaA.GL gene at a distance of 0.6 cM. A gene homologous to ECERIFERUM1 (CER1) was located in the mapped region. A cDNA microarray chip assay revealed coordinated down regulation of genes encoding enzymes of the cuticular wax biosynthetic pathway in the glossy mutant, with BnCER1 being one of the most severely suppressed genes.ConclusionsOur results indicated that surface wax biosynthesis is broadly affected in the glossy mutant due to the suppression of the BnCER1 and other wax-related genes. These findings offer novel clues for elucidating the molecular basis of the glossy phenotype.

Development of mapped simple sequence repeat markers from common bean (Phaseolus vulgaris L.) based on genome sequences of a Chinese landrace and diversity evaluation

Microsatellite or single sequence repeat (SSR) markers have been commonly used in genetic research in many crop species, including common bean (Phaseolus vulgaris L.). A limited number of existing SSR markers have been designed from high-throughput sequencing of the genome, warranting the exploitation of new SSR markers from genomic regions. In this paper, we sequenced total DNA from the genotype Hong Yundou with a 454-FLX pyrosequencer and found numerous SSR loci. Based on these, a large number of SSR markers were developed and 90 genomic-SSR markers with clear bands were tested for mapping and diversity detection. The new SSR markers proved to be highly polymorphic for molecular polymorphism, with an average polymorphism information content value of 0.44 in 131 Chinese genotypes and breeding lines, effective for distinguishing Andean and Mesoamerican genotypes. In addition, we integrated 85 primers of the 90 polymorphism markers into the bean map using an F2 segregating population derived from Hong Yundou crossed with Jingdou. The distribution of SSR markers among 11 chromosomes was not random and tended to cluster on the linkage map, with 14 new markers mapped on chromosome Pv01, whereas only four loci were located on chromosome Pv04. Overall, these new markers have potential for genetic mapping, genetic diversity studies and map-based cloning in common bean.

Head-blighting populations of Fusarium culmorum from Germany, Russia, and Syria analyzed by microsatellite markers show a recombining structure

Fusarium culmorum is a haploid, worldwide occurring phytopathogenic fungus causing seedling blight, foot rot, and head blight of cereals and producing the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) associated with health hazards in human and animals. The fungus reproduces asexually by conidiospores, a teleomorph is not known. We analyzed for the first time naturally occurring F. culmorum populations collected randomly in the field from infected wheat heads. A total of 186 isolates, from three populations from Germany (GER), Russia (RUS), Syria (SYR), as well as an international collection (INT) for comparison, were genotyped by 10 microsatellite (SSR, single sequence repeat) markers. A high genetic diversity within the three natural populations and the INT population as well was detected. About 90 % of multi-locus haplotypes (MLH) were unique across populations. The largest part of variance (81 %) was found within populations. Accordingly, population subdivision was low, fixation indices were significant only in one out of six comparisons, while estimates of gene flow (Nm) ranged from 0.8–4.8. Linkage equilibrium was revealed by the index of multi-locus association and the quotient of observed and expected variance when two linked markers were deleted. DON and NIV chemotypes grouped closely together in a principle coordinate analysis. SYR isolates were partly separated from GER and RUS populations. All population-genetic parameters were in a similar range compared to those for the sexually propagating species F. graminearum. In conclusion, results support the hypothesis of a recombining structure in F. culmorum as revealed by the high genetic variation within populations, a low fixation index and low gametic phase disequilibrium within populations.

How does a Mycobacterium change its spots? Applying molecular tools to track diverse strains of Mycobacterium avium subspecies paratuberculosis

Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne's disease research. These data can now be used to define the extent of strain diversity on the farm. However, to perform these important tasks, researchers must have a way to distinguish the many MAP isolates/strains that are present in the environment or host to enable tracking over time. Recent studies have described genetic diversity of the Mycobacterium avium complex (MAC), of which MAP is a member, through pulsed-field gel electrophoresis, single sequence repeats, variable-number tandem repeats, genome rearrangements, single nucleotide polymorphisms and genomewide comparisons to identify insertions and deletions. Combinations of these methods can now provide discrimination sufficient for dependable strain tracking. These molecular epidemiology techniques are being applied to understand transmission of Johne's disease within dairy cattle herds as well as identify which strains predominate in wildlife.

Diversidade genética de clones de bananeira 'Prata-Anã' (AAB) por meio de marcadores SSR

Diversidade genética de 20 clones de 'Prata-Anã Gorutuba', quatro clones de 'Prata-Rio', quatro clones de 'Prata-Catarina' e as cultivares Caipira, Thap Maeo, Tropical, Maçã e Prata-Anã Comum foi avaliada por meio de marcadores moleculares Simple Sequence Repeats. De um total de 19 pares de primers SSRs utilizados, 57,8% deles amplificaram bandas polimórficas e distintas, 26,3% não produziram produtos específicos e 15,7% apresentaram falhas na amplificação de alguns indivíduos. O dendrograma indicou a formação de dois grupos. O primeiro grupo com a cultivar triploide Caipira, genoma exclusivamente A; enquanto o segundo (formado por sete subgrupos) agrupou todas as cultivares resultantes da hibridação natural ou artificial entre Musa acuminata e M. balbisiana, o subgrupo II, Tropical (AAAB) e o subgrupo III, Maçã (AAB). Os subgrupos IV, V, VI e VII foram formados, respectivamente, por: 'Prata-Catarina' clones 1 e 2; 'Prata-Rio' clones 1 e 2; 'Prata-Catarina' clone 3; 'Prata-Gorutuba' clones 12 e 17, 'Prata-Catarina' clone 4, 'Prata-Rio' clone 4, 'Thap Maeo' e 'Prata-Anã'. O subgrupo VIII foi formado exclusivamente pelos clones de' Prata-Anã Gorutuba'. Os resultados indicam a eficiência dos marcadores microssatélites na discriminação e na caracterização dos clones da 'Prata-Anã Gorutuba' da cultivar Prata-Anã.

Genotypic diversity and genetic structure of populations of the distylous aquatic plantNymphoides peltata(Menyanthaceae) in China

AbstractNymphoides peltatais a distylous aquatic plant. In order to explore the relative importance of clonal growth and sexual reproduction within populations and also to reveal the origin of the commonly occurring one style morph populations in China, we studied the clonal diversity and population genetic structure of this species. By using seven single sequence repeat primer pairs, a total of 192 multilocus genotypes (MLGs) were identified among the 472 samples from the 21 populations analyzed; each population had two or more MLGs. The number of MLGs was lower for the one style morph populations than distylous populations. Analysis of molecular variance showed that a smaller proportion of genetic variation resided among populations (36.8%). A neighbor‐joining tree indicated that there was no significant geographical structure of the genetic variation among populations ofN. peltata. The Bottleneck tests indicated that there had been significant bottlenecks in most of the studied populations. The successful sexual recruitment from seeds may have contributed to the high clonal diversity in distylous populations ofN.peltatain China. Demographic stochasticity or bottleneck should account for the one style morph population with several MLGs. And also the bottleneck effects or demographic changes caused by human activities occurring randomly in populations should account for the finding of no significant geographical structure of the genetic variation among the investigated populations ofN.peltata.

Population genetics structure of glyphosate‐resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism.