Analysis of molecular genetic diversity and population structure in Amaranthus germplasm using SSR markers

We assessed the molecular genetic diversity and population structure of Amaranthus species accessions using 11 simple sequence repeat markers. A total of 122 alleles were detected, and the number of alleles per marker (N_A) ranged from 6 to 21 with an average of 11.1 alleles. The frequency of major alleles per locus ranged from 0.148 to 0.695, with an average value of 0.496 per marker. The overall polymorphic information content values were 0.436–0.898, with an average value of 0.657. The observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged from 0.056 to 0.876 and from 0.480 to 0.907, with average values of 0.287 and 0.698, respectively. The average H_O (0.240) was lower than the H_E and gene flow (Nm), and showed substantial genetic variability among all populations of amaranth accessions. The sample groupings did not strictly follow the geographic affiliations of the accessions. A similar pattern was obtained using model-based structure analysis without grouping by species type. Knowledge of the genetic diversity and population structure of amaranth can be used to select representative genotypes and manage Amaranthus germplasm breeding programs.     

Evaluation of Lespedeza germplasm genetic diversity and its phylogenetic relationship with the genus Kummerowia

The genetic diversity of the genus Lespedeza is not well known and the phylogenetic relationship of Lespedeza with the genus Kummerowia is unclear. We report the first study in which polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from Medicago, cowpea and soybean were used to assess the genetic diversity of the USDA Lespedeza germplasm collection and clarify its phylogenetic relationship with the genus Kummerowia. Phylogenetic analysis partitioned 44 Lespedeza accessions into three main groups some of which were species-specific and eight subgroups. This data set revealed some misidentified accessions, and indicated that the two species in the genus Kummerowia are closely related to the genus Lespedeza. Morphological reexamination was used to correct the misidentified accessions within the genus Lespedeza. Our results demonstrated that phylogenetic analysis with morphological reexamination provides a more complete approach to classify accessions in plant germplasm collection and conservation.     

Genetic diversity in and phylogenetic relationships of the Brazilian endemic cotton,Gossypium mustelinum (Malvaceae)

Gossypium mustelinum, one of five tetraploid species in the cotton genus, is geographically restricted to a few states in NE Brazil. Allozyme analysis was used to assess levels and patterns of genetic diversity inG. mustelinum and its relationship to the other tetraploid species. Genetic variation was low, with only 6 of 50 loci examined being polymorphic, a mean of 1.14 alleles per locus and a mean panmictic heterozygosity of 0.08. These estimates are low relative to other tetraploid cotton species, but are typical of island endemics. Interpopulational genetic identities were uniformly high, lending support to the concept of there being only one wild species of Brazilian cotton. The limited allelic diversity observed was correlated with geographical distribution, although variability is so limited in the species that geographically marginal populations are electrophoretically ordinary. Phylogenetic and phenetic analyses demonstrate thatG. mustelinum is isolated among polyploid cotton species, occupying one of the three basal clades resulting from an early radiation of polyploid taxa subsequent to polyploid formation. We suggest thatG. mustelinum represents a paleoendemic that presently exists as a series of widely scattered, relictual populations. Despite several centuries of sympatric cultivation ofG. barbadense andG. hirsutum, there was little evidence of interspecific introgression of alleles from cultivated cottons intoG. mustelinum.     

Microsatellite diversity in tetraploid <Emphasis Type="Italic">Gossypium</Emphasis> germplasm: assembling a highly informative genotyping set of cotton SSRs

A series of 320 mapped simple sequence repeats (SSRs) have been used to screen the allelic diversity of tetraploid Gossypium species. Fourty-seven genotypes were analyzed representing (i) the wide spectrum of diversity of the cultivated pool and of the primitive landraces of species G. hirsutum (‘marie-galante’, ‘punctatum’, ‘richmondi’, ‘morrilli’, ‘palmeri’, and ‘latifolium’, and ‘yucatanense’), and (ii) species G. barbadense, G. darwinii and G. tomentosum. The polymorphism of 201 SSR loci revealed 1128 allelic variants ranging from 3 to 17 per locus. Neighbor-joining (NJ) method based on genetic dissimilarities produced groupings consistent with the assignments of accessions both at species and at race level. Our data confirmed the proximity of the Galapagos endemic species G. darwinii to species G. barbadense. Within species G. hirsutum, and as compared to the other 6 races, race yucatanense appeared as the most distant from cultivated genotypes. Race yucatanense also exhibited the highest number of unique alleles. The important informative heterogeneity of the 201 SSR loci was exploited to select the most polymorphic ones that were assembled into three series of genome-wide (i.e. each homoeologous AD chromosome pair being equally represented) and mutliplexable (× 3) SSRs. Using one of these ‘genotyping set’, consisting of 39 SSRs (one 3-plex for each of the 13 AD chromosomes pairs) or 45 loci, we were able to assess the relationships between accessions and the topology in the genetic diversity sampled. Such genotyping set of highly informative SSR markers assembled in PCR-multiplex, while increasing genotyping throughput, will be applicable for molecular genetic diversity studies of large germplasm collections. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Assessment of genetic diversity patterns in Chilean quinoa (Chenopodium quinoa Willd.) germplasm using multiplex fluorescent microsatellite markers

Quinoa (Chenopodium quinoa Willd.) is a staple seed crop in the Andean region of South America. Improving quinoa productivity is a primary food-security issue for this region, and has been part of the impetus for the establishment of several new quinoa breeding programs throughout the Andean region. Chilean quinoa has been characterized as morphologically diverse and bifurcated into coastal and highland ecotypes. The success of emerging breeding programs will rely heavily on the development of core germplasm collections and germplasm evaluation—especially of the coastal quinoa ecotypes that are often neglected in traditional breeding programs. Thus, the objective of this study was to characterize and quantify the genetic diversity within 28 Altiplano and 31 coastal Chilean accessions of quinoa using microsatellite markers. To facilitate the analysis, we also report the development of seven sets of fluorescent multiplexed microsatellite PCR reactions that result in genetic information for 20 highly polymorphic microsatellite loci. A total of 150 alleles were detected among the quinoa accession, ranging from 2 to 20 alleles per locus and an average 7.5 allele/locus. Both cluster (UPGMA) and principal component analyses separated the accessions into two discrete groups. The first group contained quinoa accessions from the north (Andean highlands) and the second group consisted of accessions from the south (lowland or coastal). Three accessions from Europe were classified into the southern quinoa group. The data obtained in the diversity analyses highlights the relationships within and among northern and southern Chilean quinoa accessions and provides the quinoa scientific community with a new set of easy to use and highly informative genetic markers.     

Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. Here we evaluated the use of microsatellite markers to quantify the genetic diversity within as well as among accessions sampled from the world germplasm collection of sorghum. Considerable variation was found at the five microsatellite loci analysed, with an average number of alleles per locus equal to 2.4 within accessions and 19.2 in the overall sample of 25 accessions. The collection of sorghum appeared highly structured genetically with about 70% of the total genetic diversity occurring among accessions. However, differentiation among morphologically defined races of sorghum, or among geographic origins, accounted for less than 15% of the total genetic diversity. Our results are in global agreement with those obtained previously with allozyme markers. We were also able to show that microsatellite data are useful in identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Received: 10 August 1999 / Accepted: 27 August 1999     

SSRs transferability and genetic diversity of Tunisian Festuca arundinacea and Lolium perenne

SSR primers specific to Lolium perenne generated a total of 96 alleles and 124 genotypes within Festuca arundinacea and Lolium perenne accessions. Their highly transferability (100 %) across genera was evidenced. Six alleles specific to loci H01F02, H02C11 and K01A03 and only 5/96 common alleles between both species (60, 140, 144, 190 and 192) expressed the differentiation between species. Besides, based on the Wrights fixation indices, the genetic variation within each species was attributable to differences within populations with a significant deficiency of heterozygous. The unweighted pair group method with arithmetic averaging dendrogram based on the Nei’s distances and the principal coordinate analysis based on Jaccard coefficient similarity distinguished each genus independently of the geographical origin. However, typically continuous genetic diversity and a low level of gene flow (N_m: 0.29–2.47) expressed the relatively closely relationships of both genera and suggest a possible hybridization in nature.     

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.     

Phylogenetic relationships and genetic diversity among members of theFestuca-Lolium complex (Poaceae) based on ITS sequence data

There have been few DNA sequencebased studies of phylogenetic relationships within theFestuca-Lolium complex. Here we infer the phylogeny of 31Festuca-Lolium taxa with a dataset of 116 ITS sequences. The results are consistent with previous studies that resolved two majorFestuca clades: one clade of fine fescues and another clade that containsLolium and associatedFestuca species. This study is unique in suggesting a third, basalFestuca clade, but support for the basal position of this group is low. Extensive sampling permitted investigation of the effects of lineage sorting and reticulate events on the evolution of the complex. Roughly half of the taxa show evidence of lineage sorting or reticulation, and the monophyly ofLolium has likely been obscured by reticulate events. Overall, polyploid species harbor higher levels of ITS sequence diversity than diploids; ITS sequence variants may provide clues to the identity of allopolyploid parents.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Analysis of diversity and relationships among mango cultivars using Start Codon Targeted (SCoT) markers

Genetic variation and relationships among 47 mango germplasm and 3 relative species from Guangxi province in China, were analyzed using Start Codon Targeted (SCoT) markers. Using 33 selected SCoT primers 273 bands were generated with an average of 8.27 bands per primer among the 50 accessions, of which 208 (76.19%) were polymorphic. Genetic relationships estimated using the SM similarity coefficient generated values between different pairs of accessions that varied from 0.531 to 0.923 with an average of 0.782. These coefficients were utilized to construct a dendrogram using the UPGMA. All 50 accessions were basically classified into six clusters and correspond well with their recorded pedigrees. The results will provide much more useful information for the management of germplasm and will also be useful to improve the current breeding strategies. The results also demonstrate that the SCoT marker system is useful for identification and genetic diversity analysis of mango cultivars.     

Genetic diversity analysis of 60 ginger germplasm core accessions using ISSR and SSR markers

Molecular techniques play a critical role in studies of phylogeny and, thus, have been applied to understand the distribution and extent of genetic variation within and between species. In the present study, a genetic analysis was undertaken using molecular markers (9 ISSR and 13 SSR) on 60 ginger cultivars from different regions of the eastern coast of India (Odisha). The data obtained with 22 polymorphic markers revealed moderate to high diversity in the collection. Both ISSR and SSR markers were efficient in distinguishing all the 60 ginger cultivars. A total of 42 and 160 polymorphic bands were observed with ISSR and SSR markers, respectively. However, SSR markers were observed to be better at displaying average polymorphism (63.29%) than ISSR markers (55%). Analysis of molecular variance results showed that 52 and 66% of the variation occurred among different ginger populations, whereas 48 and 34% of the variation was found within populations, respectively, using ISSR and SSR markers, indicating that ginger cultivars display significant genetic diversity at the population level. Principal coordinates analysis and the dendrogram constructed out of combined data of both markers showed grouping of ginger accessions to their respective area of collection, indicating geographical closeness due to genetic similarity irrespective of the relationship that exists at the morphological level.     

Assessing genetic diversity among six populations of Gossypium arboreum L. using microsatellites markers

Among the four cultivated cotton species, G. hirsutum (allotetraploid) presently holds a primary place in cultivation. Efforts to further improve this primary cotton face the constraints of its narrow genetic base due to repeated selective breeding and hence demands enrichment of diversity in the gene pool. G. arboreum (diploid species) is an invaluable genetic resource with great potential in this direction. Based on the dispersal and domestication in different directions from Indus valley, different races of G. arboreum have evolved, each having certain traits like drought and disease resistance, which the tetraploid cotton lack. Due to lack of systematic, race wise characterization of G. arboreum germplasm, it  has not been explored fully. During the present study, 100 polymorphic SSR loci were  used to genotype 95 accessions belonging to 6 races of G. arboreum producing 246 polymorphic alleles; mean number of effective alleles was 1.505. AMOVA showed 14 % of molecular variance among population groups, 34 % among individuals and remaining 52 % within individuals. UPGMA dendrogram, based on Nei’s genetic distance, distributed the six populations in two major clusters of 3 populations each; race ‘bengalense’ was found more close to ‘cernuum’ than the others. The clustering of 95 genotypes by UPGMA tree generation as well as PCoA analysis clustered ‘bengalense’ genotypes into one group along with some genotypes of ‘cernuum’, while rest of the genotypes made separate clusters. Outcomes of this research should be helpful in identifying the genotypes for their further utilization in hybridization program to obtain high level of germplasm diversity.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-015-0326-y) contains supplementary material, which is available to authorized users.     

Genetic diversity in a world germplasm collection of tall fescue

Festuca arundinacea Schreb., commonly known as tall fescue, is a major forage crop in temperate regions. Recently, a molecular analysis of different accessions of a world germplasm collection of tall fescue has demonstrated that it contains different species from the genus Festuca and allowed their rapid classification into the three major morphotypes (Continental, Mediterranean and Rhizomatous). In this study, we explored the genetic diversity of 161 accessions of Festuca species from 29 countries, including 28 accessions of INTA (Argentina), by analyzing 15 polymorphic SSR markers by capillary electrophoresis. These molecular markers allowed us to detect a total of 214 alleles. The number of alleles per locus varied between 5 and 24, and the values of polymorphic information content ranged from 0.627 to 0.840. In addition, the accessions analyzed by flow cytometry showed different ploidy levels (diploid, tetraploid, hexaploid and octaploid), placing in evidence that the world germplasm collection consisted of multiple species, as previously suggested. Interestingly, almost all accessions of INTA germplasm collection were true hexaploid tall fescue, belonging to two eco-geographic races (Continental and Mediterranean). Finally, the data presented revealed an ample genetic diversity of tall fescue showing the importance of preserving the INTA collection for future breeding programs.     

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.     

Analysis of the genetic diversity and relationships among and within species of Hippophae (Elaeagnaceae) based on RAPD markers

RAPD markers were used to assess the genetic diversity and inter- and intra-specific relationships of the genus Hippophae L. and to study the correlation between genetic distances and geographic distances among populations of H. rhamnoides ssp. sinensis. The results analyzed by the percentage of polymorphic loci and Shannon information index indicated that a high level of genetic diversity existed both among and within species of the genus Hippophae. In the UPGMA dendrogram, the species or subspecies were clustered into two main groups but not strictly grouped according to sect. Hippophae and sect. Gyantsensis Lian. The multiple regression analysis and Mantel test both indicated a significant correlation between genetic distance and altitude distance among populations of H. rhamnoides ssp. sinensis, and the cluster analysis suggested that the genetic variation among populations of H. rhamnoides ssp. sinensis was linked to their monophyletic origin. Moreover, some degree of genetic differentiation was found among samples collected at different times.     

Effects of fusicoccin on intact cotton plants (Gossypium hirsutum L.)

Fusicoccin (FC) was applied as a spray to shoots of intact field- and glasshouse-grown cotton plants. Distortions of shoot morphology resulted. Stems and petioles of FC-treated plants were irregular in diameter and twisted, whereas leaf laminae were curled and crinkled. Shoot elongation was inhibited by FC; the effect was dependent upon the concentration and timing of the applications.Abbreviation FC fusicoccin     

Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs

The objective of the present study was to identify favourable exotic Quantitative Trait Locus (QTL) alleles for the improvement of agronomic traits in the BC₂DH population S42 derived from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). QTLs were detected as a marker main effect and/or a marker × environment interaction effect (M × E) in a three-factorial ANOVA. Using field data of up to eight environments and genotype data of 98 SSR loci, we detected 86 QTLs for nine agronomic traits. At 60 QTLs the marker main effect, at five QTLs the M × E interaction effect, and at 21 QTLs both the effects were significant. The majority of the M × E interaction effects were due to changes in magnitude and are, therefore, still valuable for marker assisted selection across environments. The exotic alleles improved performance in 31 (36.0%) of 86 QTLs detected for agronomic traits. The exotic alleles had favourable effects on all analysed quantitative traits. These favourable exotic alleles were detected, in particular on the short arm of chromosome 2H and the long arm of chromosome 4H. The exotic allele on 4HL, for example, improved yield by 7.1%. Furthermore, the presence of the exotic allele on 2HS increased the yield component traits ears per m² and thousand grain weight by 16.4% and 3.2%, respectively. The present study, hence, demonstrated that wild barley does harbour valuable alleles, which can enrich the genetic basis of cultivated barley and improve quantitative agronomic traits.     

A rapid and high yielding DNA miniprep for cotton (Gossypium spp.)

A rapid DNA minipreparation method was developed for cotton and yields 500–600 μg DNA from 1.0 g fresh leaf tissue. Cotton DNA extracted using this method is completely digested with restriction enzymes, supports PCR and Southern DNA analyses and was used successfully in these applications. An erratum to this article is available at .