Diversity of Sinorhizobium Meliloti and S. medicae Nodulating Medicago Truncatula According to Host and Soil Origins

Summary The model legume Medicago truncatula was used to trap natural populations of Sinorhizobium meliloti and Sinorhizobium medicae in Tunisian soils to explore their genetic diversity. About 155 Sinorhizobium isolates were trapped from a combination of three soils and four Medicago truncatula populations in order to analyse soil and plant population effects on nodulating Sinorhizobium diversity. The species assignment was done according to the restriction fragment length polymorphism analysis of polymerase chain reaction (PCR/RFLP) of 16S rRNA genes and their infraspecific genetic diversity was assessed with the repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) technique. It appeared that the trapped bacteria were clustered according to the soil of origin, particularly Sinorhizobium medicae isolates. However, regarding the plant population effect, it appeared that no major clustering tendency could be suggested even if the Bulla Regia and Soliman Medicago truncatula populations appeared to nodulate together specific Sinorhizobium medicae genotypes.     

Low levels of quantitative and molecular genetic differentiation among natural populations of Medicago ciliaris Kroch. (Fabaceae) of different Tunisian eco-geographical origin

In this paper, we analyze the genetic variability in four Tunisian natural populations of Medicago ciliaris using 19 quantitative traits and six polymorphic microsatellite loci. We investigated the amplification transferability of 30 microsatellites developed in the model legume M. truncatula to M. ciliaris. Results revealed that about 56.66% of analyzed markers are valuable genetic markers for M. ciliaris. The most genetic diversity at quantitative traits and microsatellite loci was found to occur within populations (>80%). Low differentiations among populations at quantitative traits Q _ST  = 0.146 and molecular markers F _ST  = 0.18 were found. The majority of measured traits exhibited no significant difference in the level of Q _ST and F _ST . Furthermore, significant correlations established between these traits and eco-geographical factors suggested that natural selection should be invoked to explain the level of phenotypic divergence among populations rather than drift. There was no significant correlation between population differentiation at quantitative traits and molecular markers. Significant spatial genetic structure consistent with models of isolation by distance was detected within all studied populations. The site-of-origin environmental factors explain about 9.07% of total phenotypic genetic variation among populations. The eco-geographical factors that influence more the variation of measured traits among populations are the soil texture and altitude. Nevertheless, there were no consistent pattern of associations between gene diversity (He) and environmental factors.     

SSR analysis of the Medicago truncatula SARDI core collection reveals substantial diversity and unusual genotype dispersal throughout the Mediterranean basin

The world’s oldest and largest Medicago truncatula collection is housed at the South Australian Research and Development Institute (SARDI). We used six simple sequence repeat (SSR) loci to analyse the genetic diversity and relationships between randomly selected individuals from 192 accessions in the core collection. M. truncatula is composed of three subspecies (ssp.): ssp. truncatula, ssp. longeaculeata, and ssp. tricycla. Analysis at the level of six SSR loci supports the concept of ssp. tricycla, all the samples of which showed unique alleles at two loci. Contingency Chi-squared tests were significant between ssp. tricycla and ssp. truncatula at four loci, suggesting a barrier to gene flow between these subspecies. In accessions defined as ssp. longeaculeata, no unique allelic distribution or diagnostic sizes were observed, suggesting this apparent ssp. is a morphological variant of ssp. truncatula. The data also suggest M. truncatula that exhibits unusually wide genotype dispersal throughout its native Mediterranean region, possibly due to animal and trade-related movements. Our results showed the collection to be highly diverse, exhibiting an average of 25 SSR alleles per locus, with over 90% of individuals showing discrete genotypes. The rich diversity of the SARDI collection provides an invaluable resource for studying natural allelic variation of M. truncatula. To efficiently exploit the variation in the SARDI collection, we have defined a subset of accessions (n=61) that maximises the diversity.     

Morphological and microsatellite diversity associated with ecological factors in natural populations of Medicago laciniata Mill. (Fabaceae)

Genetic variability in 10 natural Tunisian populations of Medicago laciniata were analysed using 19 quantitative traits and 12 polymorphic microsatellite loci. A large degree of genetic variability within-populations and among-populations was detected for both quantitative characters and molecular markers. High genetic differentiation among populations for quantitative traits was seen, with Q _ST = 0.47, and F _ST = 0.47 for microsatellite markers. Several quantitative traits displayed no statistical difference in the levels of Q _ST and F _ST . Further, significant correlations between quantitative traits and eco-geographical factors suggest that divergence in the traits among populations may track environmental differences. There was no significant correlation between genetic variability at quantitative traits and microsatellite markers within populations. The site-of-origin of eco-geographical factors explain between 18.13% and 23.40% of genetic variance among populations at quantitative traits and microsatellite markers, respectively. The environmental factors that most influence variation in measured traits among populations are assimilated phosphorus (P₂0₅) and mean annual rainfall, followed by climate and soil texture, altitude and organic matter. Significant associations between eco-geographical factors and gene diversity, H _e , were established in five-microsatellite loci suggesting that these simple sequence repeats (SSRs) are not necessarily biologically neutral.     

Genetic diversity in European chestnut populations by means of genomic and genic microsatellite markers

Microsatellite or simple sequence repeats (SSRs) are one of the most used markers in population genetic studies. SSR markers developed from expressed sequence tags (EST) have proved useful to examine functional diversity in relation to adaptive variation. The information provided by both genomic and genic microsatellite markers could offer more accurate indication on the distribution of the genetic diversity among and within populations assuming different evolutionary drivers. This is the first study on chestnut (Castanea sativa Mill.) in which the genetic diversity was evaluated by means of genomic (SSRs) and genic (EST-SSRs) microsatellite markers. We genotyped nine natural European chestnut populations distributed throughout representative areas of contrasting climatic conditions in the Mediterranean basin. Genomic SSRs showed significantly higher levels of diversity in terms of number of alleles, effective number of alleles, expected heterozygosity and level of polymorphism. Furthermore, there were significant differences in the level of differentiation among populations. The UPGMA analysis revealed different clustering pattern between populations, being the grouping according to geographic distances in the case of genomic SSRs and two differentiated groups based on the northern–southern distribution of the populations for EST-SSRs. Furthermore, the EST-SSR transferability among related Castanea and Quercus species was stated. Our results confirm that combining genomic SSRs and EST-SSRs is a useful tool to give complementary information to explain the genetic and adaptive diversity in chestnut.     

A comparison of genetic variation among wild and cultivated Morus Species (Moraceae: Morus) as revealed by ISSR and SSR markers

In this study, inter-simple sequence repeats (ISSR) ans simple sequence repeat (SSR) markers were used to investigate genetic diversity of 27 mulberry accessions including 19 cultivated accessions (six M. multicaulis, three M. alba, two M. atropurpurea, two M. bombycis, one M. australis, two M. rotundiloba, one M. alba var. pendula, one M. alba var. macrophylla, and one M. alba var. venose) and 8 wild accessions (two M. cathayana, two M. laevigata, two M. wittiorum, one M. nigra and one M. mongolica). ISSRs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 27 mulberry genotypes. SSRs presented a higher level of polymorphism and greater information content. All index values of genetic diversity both markers analyzed using Popgene 32 software indicated that within wild species had higher genetic diversity than within cultivated species. Cultivation may caused the lose of genetic diversity of mulberry compared with wild species revealed by ISSR and SSR markers. The mean genetic similarity coefficients among all mulberry genotypes ascribed by ISSR and SSR matrices were 0.7677 and 0.6131, respectively. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. Cluster analysis of ISSR and SSR using UPGMA method revealed that the wild species are genetically distant from the domesticated species studied here. The correlation coefficients of similarity were statistically significant for both marker systems used. Principal coordinates analysis (PCA) for ISSR and SSR data also supports their UPGMA clustering. These results have an important implication for mulberry germplasm characterization, improvement, molecular systematics and conservation.     

Spatial pattern of nucleotide polymorphism indicates molecular adaptation in the bryophyte Sphagnum fimbriatum

In organisms with haploid-dominant life cycles, natural selection is expected to be especially effective because genetic variation is exposed directly to selection. However, in spore-producing plants with high dispersal abilities, among-population migration may counteract local adaptation by continuously redistributing genetic variability. In this study, we tested for adaptation at the molecular level by comparing nucleotide polymorphism in two genes (GapC and Rpb2) in 10 European populations of the peatmoss species, Sphagnum fimbriatum with variability at nine microsatellite loci assumed to be selectively neutral. In line with previous results, the GapC and Rpb2 genes showed strikingly different patterns of nucleotide polymorphism. Neutrality tests and comparison of population differentiation based on the GapC and Rpb2 genes with neutrally evolving microsatellites using coalescent simulations supported non-neutral evolution in GapC, but neutral evolution in the Rpb2 gene. These observations and the positions of the replacement mutations in the GAPDH enzyme (coded by GapC) indicate a significant impact of replacement mutations on enzyme function. Furthermore, the geographic distribution of alternate GapC alleles and/or linked genomic regions suggests that they have had differential success in the recolonization of Europe following the Last Glacial Maximum.     

Signatures of diversifying selection at EST‐SSR loci and association with climate in natural Eucalyptus populations

Understanding the environmental parameters that drive adaptation among populations is important in predicting how species may respond to global climatic changes and how gene pools might be managed to conserve adaptive genetic diversity. Here, we used Bayesian F_ST outlier tests and allele–climate association analyses to reveal two Eucalyptus EST‐SSR loci as strong candidates for diversifying selection in natural populations of a southwestern Australian forest tree, Eucalyptus gomphocephala (Myrtaceae). The Eucalyptus homolog of a CONSTANS‐like gene was an F_ST outlier, and allelic variation showed significant latitudinal clinal associations with annual and winter solar radiation, potential evaporation, summer precipitation and aridity. A second F_ST outlier locus, homologous to quinone oxidoreductase, was significantly associated with measures of temperature range, high summer temperature and summer solar radiation, with important implications for predicting the effect of temperature on natural populations in the context of climate change. We complemented these data with investigations into neutral population genetic structure and diversity throughout the species range. This study provides an investigation into selection signatures at gene‐homologous EST‐SSRs in natural Eucalyptus populations, and contributes to our understanding of the relationship between climate and adaptive genetic variation, informing the conservation of both putatively neutral and adaptive components of genetic diversity.     

Assessment of genetic diversity and population structure of Chinese wild almond, Amygdalus nana, using EST- and genomic SSRs

Amygdalus nana L., commonly known as wild almond, is an endangered wild relative of cultivated almond, which has great potential in almond crop breeding. In this study, we used microsatellite (SSR) loci derived from both expressed sequence tag (EST) and anonymous genomic sequence to explore the genetic diversity and population structure of A. nana in Xinjiang of China. Seven natural populations were collected across the whole distribution of A. nana in China, including populations from both inside (four populations) and outside (three populations) the established protected areas. A total of 22 and 19 alleles were detected from the seven pairs of EST and genomic SSR loci, respectively. Generally, the genomic SSRs showed lower levels of variation than EST-SSRs, which may partially due to the higher cross-species transferability in EST-SSRs than in genomic SSRs. The population-level genetic diversity (A = 1.84, P = 50.00%, H_o = 0.3491, H_E = 0.2271) was lower than cultivated almond and several wild fruit species with similar breeding system. Most of the genetic variation (82.16%) was partitioned within populations. In particular, the population collected from Tacheng County (outside the protected areas) had the highest levels of genetic diversity and had significantly different genetic constitution from other populations.     

Genetic diversity and structure of natural fragmented Chamaecyparis obtusa populations as revealed by microsatellite markers

The genetic diversity and population structure of hinoki (Chamaecyparis obtusa) in Japan were investigated by examining the distribution of alleles at 13 microsatellite loci in 25 natural populations from Iwaki in northern Japan to Yakushima Island in southern Japan. On average, 26.9 alleles per locus were identified across all populations and 4.0% of the genetic variation was retained among populations (G _ST = 0.040). According to linkage disequilibrium analysis, estimates of effective population size and detected evidence of bottleneck events, the genetic diversity of some populations may have declined as a result of fragmentation and/or over-exploitation. The central populations located in the Chubu district appear to have relatively large effective population sizes, while marginal populations, such as the Yakushima, Kobayashi and Iwaki populations, have smaller effective population sizes and are isolated from the other populations. Microsatellite analysis revealed the genetic uniqueness of the Yakushima population. Although genetic differentiation between populations was low, we detected a gradual cline in the genetic structure and found that locus Cos2619 may be non-neutral with respect to natural selection.     

High levels of genetic divergence and inbreeding in populations of cupuassu (Theobroma grandiflorum)

Theobroma grandiflorum (cupuassu) is an important fruit tree native to the Brazilian Amazon. Establishing the genetic diversity and structure of populations is critical to define long-term strategies for cupuassu conservation presently threatened by rapid deforestation. Three natural populations collected at the putative center of diversity, three groups of accessions established at a germplasm collection, and one derived from commercial plantings were analyzed. The genetic diversity was assessed using 21 polymorphic microsatellite loci originally developed for Theobroma cacao, disclosing a total of 113 alleles. The estimated genetic diversity parameters averaged over cupuassu populations (A = 3.53 alleles per locus; H _e = 0.426; H _o = 0.346) were lower than the values reported for other Neotropical tree species. The three natural populations presented a positive and significant fixation index (f), ranging from 0.133 to 0.234. Cupuassu apparently adhered to a general pattern of genetic diversity structure of some Neotropical tree species occurring at low densities, with a low intrapopulation genetic diversity and important levels of endogamy, possibly due to biparental inbreeding derived from the presence of spatial genetic structure in the populations. A high level of genetic divergence was detected among the natural populations (θ _p = 0.301), a strong differentiation caused by limited gene flow, and suggesting that human interference in spreading and/or stimulating plantings might have had a smaller effect than expected. The approximate location of the T. grandiflorum center of diversity could not be confirmed by analyzing natural populations from the putative region.     

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.Communicated by F. Salamini     

Single‐nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single‐nucleotide polymorphism (SNP) markers in M. truncatula through large‐scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.     

Genetic diversity assessment in greek Medicago truncatula genotypes using microsatellite markers

In this study we examined the genetic diversity and geographic scale of genotype distribution within the model legume species Medicago truncatula widely distributed in pasture and marginal agricultural lands in Greece and other Mediterranean countries. Thirty one Medicago truncatula and Medicago littorialis accessions were chosen on the basis of their geographical distributions and studied using 9 polymorphic simple sequence repeats (SSR) markers. The number of alleles per locus varied between 3 and 7. A total of 42 alleles were detected with a mean value of 4.66 alleles per locus. Geographic origin was not related with genotypic similarity among accessions. However, there were instances of close genetic relatedness between accessions from neighboring locations in a geographic compartment. In conclusion, the presented data revealed extensive M. truncatula genotype dispersal in Greece pointing to the significance of preserving local genetic resources in their natural environment.     

Assessment of the genetic diversity among Glyptosternum maculatum, an endemic fish of Yarlung Zangbo River, Tibet, China using SSR markers

The genetic diversity of Glyptosternum maculatum populations from Nyang River, Lhasa River, and Shetongmon Reach of Yarlung Zangbo River was assessed using six microsatellite markers. Overall, the genetic diversity across the three populations was low. The Shetongmon population exhibited the highest level of genetic diversity in terms of number of alleles and effective alleles, heterozygosity, and polymorphic information content value, followed by the Nyang population and Lhasa population. The analysis of molecular variance demonstrated that almost the variation (86.64%) occurred within populations. The differentiation among populations was not significant, and population structure was weak. These results revealed that three natural populations of G. maculatum are not genetically differentiated and the large disparity of living altitude did not caused genetic differentiation between different populations. Our observations will help identify the genetic relationship among populations to understand the genetic diversity of G. maculatum.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

An expressed sequence tag SSR map of tetraploid alfalfa (Medicago sativa L.)

A genetic map constructed from a population segregating for a trait of interest is required for QTL identification. The goal of this study was to construct a molecular map of tetraploid alfalfa (Medicago sativa.) using simple sequence repeat (SSR) markers derived primarily from expressed sequence tags (ESTs) and bacterial artificial chromosome (BAC) inserts of M. truncatula. This map will be used for the identification of drought tolerance QTL in alfalfa. Two first generation backcross populations were constructed from a cross between a water-use efficient, M. sativa subsp. falcata genotype and a low water-use efficient M. sativa subsp. sativa genotype. The two parents and their F₁ were screened with 1680 primer pairs designed to amplify SSRs, and 605 single dose alleles (SDAs) were amplified. In the F₁, 351 SDAs from 256 loci were mapped to 41 linkage groups. SDAs not inherited by the F₁, but transmitted through the recurrent parents and segregating in the backcross populations, were mapped to 43 linkage groups, and 44 of these loci were incorporated into the composite maps. Homologous linkage groups were joined to form eight composite linkage groups representing the eight chromosomes of M. sativa. The composite maps consist of eight composite linkage groups with 243 SDAs from M. truncatula EST sequences, 38 SDAs from M. truncatula BAC clone sequences, and five SDAs from alfalfa genomic SSRs. The total composite map length is 624 cM, with average marker density per composite linkage group ranging from 1.5 to 4.4 cM, and an overall average density of 2.2 cM. Segregation distortion was 10%, and distorted loci tended to cluster on individual homologues of several linkage groups. Electronic Supplementary Material Supplementary material is available for this article at     

Potential adaptive divergence between subspecies and populations of snapdragon plants inferred from QST–FST comparisons

Phenotypic divergence among natural populations can be explained by natural selection or by neutral processes such as drift. Many examples in the literature compare putatively neutral (F_ST) and quantitative genetic (Q_ST) differentiation in multiple populations to assess their evolutionary signature and identify candidate traits involved with local adaptation. Investigating these signatures in closely related or recently diversified species has the potential to shed light on the divergence processes acting at the interspecific level. Here, we conducted this comparison in two subspecies of snapdragon plants (eight populations of Antirrhinum majus pseudomajus and five populations of A. m. striatum) in a common garden experiment. We also tested whether altitude was involved with population phenotypic divergence. Our results identified candidate phenological and morphological traits involved with local adaptation. Most of these traits were identified in one subspecies but not the other. Phenotypic divergence increased with altitude for a few biomass‐related traits, but only in A. m. striatum. These traits therefore potentially reflect A. m. striatum adaptation to altitude. Our findings imply that adaptive processes potentially differ at the scale of A. majus subspecies.     

Effect of regeneration procedures on the genetic integrity of Brassica oleracea accessions

A Brassica oleracea collection of landraces collected in the northwest of Spain is kept at the Gene Bank placed at ‘Misión Biológica de Galicia’. Landraces of the collection are regenerated from time to time to restore the viability of the seed and to carry on field trials. The objective of this work is to study the effect of regeneration on the genetic integrity of three accessions of this collection, and to investigate the possible causes of the genetic changes observed. After characterizing the original populations and their following regenerated populations by 25 SSRs we concluded that there were significant changes in the population structure and the allelic frequency of individual loci due to the action of genetic drift, directional selection and probably assortative mating. Protocols to store and regenerate the accessions should be improved in order to avoid the effect of these forces in the genetic integrity of the collection. Research supported by the project PIE20064-01-089 and the Excma. Diputación Provincial de Pontevedra, Spain.