Assessment of genetic diversity among African cassava Manihot esculenta Grantz accessions resistant to the cassava mosaic virus disease using SSR markers

A study was conducted to determine the extent of genetic diversity among African cassava (Manihot esculenta Crantz) accessions resistant to the cassava mosaic virus disease (CMD), using simple sequence repeat (SSR) markers. The accessions included a breeding stock (clone 58308), five improved lines, 62 CMD resistant and 10 CMD susceptible landraces. Genetic diversity was assessed among accessions in five cluster groups derived from UPGMA analysis on data from 18 SSR primer pairs. Average gene diversity, He, was high in all cluster groups, with an average heterozygosity of 0.591 ± 0.061. The estimator of inbreeding Fis revealed a low level of inbreeding within groups and averaged −0.262 ± 0.142. Gene diversity among all accessions was 51.4% and gene diversity within cluster groups was 46.6%, while 4.8% was due to diversity between the different cluster groups. The amount of genetic differentiation measured by Gst and Fst were 9.6% and 12.1% respectively, indicating a weak genetic structure.     

Wild melon diversity in India (Punjab State)

We present here the first comprehensive genetic characterization of wild melon accessions from northern India. The genetic diversity among 43 wild melon accessions collected from the six agro-ecological regions of the Punjab State of India was assessed by measuring variation at 16 Simple Sequence Repeat (SSR) loci, morphological traits of plant habit and fruit morphological traits, two yield-associated traits, root nematode resistance and biochemical composition (ascorbic acid, carotenoids, titrable acidity). Variation among accessions was observed in plant habit and fruit traits and wild melon germplasm with high acidity and elevated carotenoid content and possessing resistance to Meloidogyne incognita was identified in the collection. A high level of genetic variability in wild melon germplasm was suggested by SSR analysis. Comparative analysis using SSRs of the genetic variability between wild melons from the north and other melons from the south and east regions of India and also reference accessions of cultivated melon from Spain, Japan, Korea, Maldives, Iraq and Israel, showed regional differentiation among Indian melon accessions and that Indian germplasm was not closely related to melon accessions from other parts of the world. A highly drought tolerant accession belonging to var. agrestis Naud. was also identified.     

Characterization of Seashore Paspalum (Paspalum vaginatum Swartz) Germplasm by Transferred SSRs from Wheat, Maize and Sorghum

One hundred and thirty SSR markers from wheat, maize and sorghum were screened for the transferability to Paspalum. The transfer rate was 67.5, 49.0 and 66.8% respectively. This would be a very efficient approach for DNA marker development for species which are not well studied molecularly. The polymorphism level for transferred SSR markers was 51.5% within species (Paspalum vaginatum) and 87.1% among Paspalum species. The high level of polymorphism is directly related to the high degree of heterozygosity maintained by its way of reproduction, i.e. self-incompatibility. Forty transferred polymorphic SSR markers were selected and used for characterization and evaluation of seventy-three Paspalum accessions. In total, 209 polymorphic bands were detected from these 40 SSR markers, with an average of five polymorphic bands per marker. The Paspalum accessions clustered into three major groups. Two very similar dendrograms can be generated from either 109 or 209 polymorphic bands. This led us to determine that 18 of the transferred SSR markers were sufficient for genetically differentiating the investigated germplasm accessions. The number of SSR markers required for germplasm characterization and evaluation is discussed. This is the first report of the transfer of SSR markers from major field crops to newly emerged environmental turfgrasses.     

A new collection of wild populations of Capsicum in Mexico and the southern United States

An exploration and collection mission for wild populations of Capsicum was carried out in the fall of 2006 and 2007, in 13 Mexican states and in the U.S. states of Arizona and Texas. The aim of this collection was to expand the number of accessions of wild chile pepper (Capsicum annuum var. glabriusculum and Capsicum frutescens) that are publicly available for research in plant improvement and for subsequent use in an inquiry into the domestication of C. annuum. While Mexico and the United States National Plant Germplasm System both have germplasm repositories INIFAP—Instituto Nacional de Investigaciones Forestales—Agrícolas y Pecuarias and USDA GRIN—United States Department of Agriculture’s Genetic Resources Information Network) with accessions of C. annuum var. glabriusculum, the very limited number available, their age, and/or validity of the information attached to many accessions do not allow for extensive research. Four hundred and sixty-six plants were sampled over two field seasons, of which copies of the collection reside in both UAA and at UC Davis. Given the current environment with the intellectual property of varieties of crop plants and, particularly, the extreme restrictions affecting explorations and the official procuring and sharing of germplasm across national borders, this U.S.—Mexico collaboration is one of the few examples of joint U.S.—Mexico germplasm collection efforts.     

Characterisation of germplasm accessions of Napier grass (pennisetum purpureum and P. purpureum×P. glaucum Hybrids) and comparison with farm clones using RAPD

Fifty six germplasm accessions of the important East African fodder crop Napier grass, Pennisetum purpureum, and its hybrids with P. glaucum, were characterised using 67 random amplified polymorphic DNA (RAPD) fragments. No or very low intra-accession variation was found for 49 of the accessions examined, confirming field observations that this species is predominantly clonally propagated. Comparison of intra and inter-accession variation identified several groups of identical/similar accessions that could be targeted if the collection is to be rationalised, and also highlighted two misplantings of germplasm material during transfer to a field trial site. A neighbour joining dendrogram of Jaccard's similarity estimates, clearly separated 50 accessions of P. purpureum from three P. glaucum individuals, and placed six hybrid accessions in an intermediate position. These groupings were well supported by a nested AMOVA (P<0.001; 29.5% of total variance due to taxonomic delineation). The main group of P. purpureum individuals could be further differentiated into five sub-groups (designated East Africa, Southern Africa, USA1, USA2 and Miscellaneous, to reflect the majority membership of sub-groups) and examination of the within P. purpureum component of the nested AMOVA, found them to be significantly different (P<0.001; 18.8% of variance). Genetic diversity across all accessions was found to be fairly high (Shannon's diversity index 0.306) and thus the collection probably represents a wide genetic base for this species. In addition to germplasm accessions, 25 Kenyan farm clones were also analysed. A principal coordinate analysis found that all but one of the clones clustered with the main P. purpureum group of accessions, indicating that the majority are probably not interspecific hybrids. The origin and pedigree of clones is discussed based on genetic similarity amongst clones and to germplasm accessions.     

Genetic analysis of wheat (Triticum aestivum L.) and related species with SSR markers

Genetic diversity among 19 Triticum aestivum accessions and 73 accessions of closely related species was analyzed using simple sequence repeat (SSR) markers. Forty-four out of 497 SSR markers were polymorphic. In total 274 alleles were detected (mean 6.32 alleles per locus). The polymorphic information content (PIC) of the loci ranged from 0.3589 to 0.8854 (mean 0.7538). The D genome contained the highest mean number of alleles (6.32) followed by the A and B genomes (6.13 and 5.94, respectively). The correlation between PIC and allele number was significant in all genome groups (0.7540, 0.7361 and 0.7482 for A, B and D genomes, respectively). Among the seven homologous chromosome groups, genetic diversity was lowest in group 7 and highest in group 5. In cluster and principal component analyses, all accessions grouped according to their genomes were consistent with their taxonomic classification. Accessions with the A and D genomes were clustered into two distinct groups, and AABB accessions showed abundant genetic diversity and a close relationship. Triticum durum and T. turgidum were clustered together, consistent with their morphological similarity. Cluster analysis indicated emmer is closely related to hexaploid wheat. Compared with common wheat, higher genetic variation was detected in spelt, T. aestivum subsp. yunnanense and subsp. tibetanum. In addition, a close genetic relationship between T. polonicum and T. macha was observed. The results of the clustering and principal component analyses were essentially consistent, but the latter method more explicitly displayed the relationships among wheat and closely related species.     

Genetic diversity and origin of North American green foxtail [<Emphasis Type="Italic">Setaria viridis</Emphasis> (L.) Beauv.] accessions

Setaria viridis (L.) P. Beauv. and its domesticated form, S. italica (L.) P. Beauv., have been developed over the past few years as model systems for C₄ photosynthesis and for the analysis of bioenergy traits. S. viridis is native to Eurasia, but is now a ubiquitous weed. An analysis of the population structure of a set of 232 S. viridis lines, mostly from North America but also comprising some accessions from around the world, using 11 SSR markers, showed that S. viridis populations in the US largely separate by latitude and/or climatic zone. S. viridis populations from the Northern US and Canada (north of 44°N) group with accessions from Western Europe, while populations in the Mid and Southern US predominantly group with accessions from Turkey and Iran. We hypothesize that S. viridis in the US was most likely introduced from Europe, and that introductions were competitive only in regions that had climatic conditions that were similar to those in the regions of origins. This hypothesis is supported by the fact that Canadian S. viridis lines were fast cycling and undersized when grown in the Mid-Western and Southern US compared to their morphology in their native environment. A comparison of the population structure obtained with 11 SSR markers and ~40,000 single nucleotide polymorphisms (SNPs) in a common set of S. viridis germplasm showed that both methods essentially yielded the same groupings, although admixture was identified at a higher frequency in the SNP analysis. Small numbers of SSR markers can thus be used effectively to discern the population structure in this inbreeding species.     

Genetic diversity in wild and cultivated black raspberry (Rubus occidentalis L.) evaluated by simple sequence repeat markers

Breeding progress in black raspberry (Rubus occidentalis L.) has been limited by a lack of genetic diversity in elite germplasm. Black raspberry cultivars have been noted for showing very few phenotypic differences and seedlings from crosses between cultivars for a lack of segregation for important traits. Despite these challenges, little molecular work has been done to explore genetic diversity and relationships in wild and cultivated black raspberry germplasm. Microsatellite, or simple sequence repeat (SSR), markers are highly polymorphic codominant markers useful for studying genetic diversity, population genetics, genetic fingerprinting and other applications. We examined genetic diversity in 148 wild and cultivated black raspberry accessions using 21 polymorphic SSR markers. Black raspberry cultivars clustered tightly and showed higher than expected heterozygosity while that of wild accessions was low. Relationships between wild black raspberry accessions were poorly resolved and regional clusters were mostly absent from our analysis. Our results indicated that wild black raspberry germplasm is a relatively untapped resource available for future breeding.     

Genic SSRs for European and North American hop (Humulus lupulus L.)

Eight genic SSR loci were evaluated for genetic diversity assessment and genotype identification in Humulus lupulus L. from Europe and North America. Genetic diversity, as measured by three diversity indices, was significantly lower in European cultivars than in North American wild accessions. Neighbor Joining cluster analysis separated the hop genotypes into European and North American groups. These eight SSRs were useful in uniquely identifying each accession with the exception of two sets of European landraces and a pair of Japanese cultivars, ‘Shinshuwase’ and ‘Kirin II’. An accession from Manitoba grouped with the European (EU) cluster reflecting the group’s genetic similarity to older Manitoba germplasm used to develop ‘Brewer's Gold’ and the gene pool arising from this cultivar. Cultivars grouped closely with one of their immediate parents. ‘Perle’ grouped with its parent ‘Northern Brewer and ‘Willamette’ grouped with its parent ‘Fuggle H’. Wild American accessions were divided into two subgroups: a North Central group containing mostly H. lupulus var. lupuloides and a Southwestern group containing H. lupulus var. neomexicanus accessions. These eight SSRs will be valuable for genotype identification in European and wild American germplasm and may potentially prove useful for marker-assisted selection in hop. PCR products from four previously reported primer pairs that amplify the same intronic SSR regions as do the genic SSRs in this study were compared in eight common cultivars. Different primer pairs generated robust markers at the chs2 and chi loci. However, only the HLC-004B and HLC-006 primer pairs amplified successfully at the chs3 and chs4 loci. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessing genetic diversity of wild southeastern North American <Emphasis Type="Italic">Vaccinium</Emphasis> species using microsatellite markers

Wild species representatives from Northwestern, Central and Southern Florida, and neighboring U.S. states were collected in multiple United States Department of Agriculture (USDA) exploration expeditions and are being preserved at the USDA, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon. Germplasm from these southeastern regions of North America is particularly vulnerable to loss in the wild due to encroachment of human development in key habitats and biotic and abiotic stresses from climate change. Fourteen simple sequence repeats (SSRs), previously developed from the highbush blueberry (Vaccinium corymbosum) cultivar ‘Bluecrop’, were used to estimate genetic diversity and genetic differentiation of 67 diploid individuals from three species, including 19 V. elliottii, 12 V. fuscatum, and 35 V. darrowii accessions collected throughout the species’ ranges. Results from our analyses indicated that the samples from each species could be reliably resolved using genetic distance measures with ordination and neighbor joining approaches. In addition, we estimated admixture among these species by using Bayesian assignment tests, and were able to identify a mis-labeled accession of V. darrowii ‘Johnblue’, two mis-classified accessions (CVAC 735.001 and CVAC 1223.001), and four accessions of previously undescribed hybrid origin (CVAC 734.001, CVAC 1721.001, CVAC 1741.001, and Florida 4B CVAC 1790). Allele composition at the 14 SSRs confirmed that Florida 4B CVAC 1790, the donor of low chilling for the southern highbush blueberry, was the critical parent of US 74. Genetic diversity assessment and identification of these wild accessions are crucial for optimal germplasm management and expand opportunities to utilize natural variation in breeding programs.     

A microsatellite and morphological assessment of the Russian National cultivated potato collection

The germplasm collections of the Vavilov Institute of Plant Industry, Russia represent the first germplasm collection made for potatoes, now numbering 8,680 accessions. It has tremendous historical and practical importance and a rich history, having been used to document a polyploid series in the cultivated species, to formulate initial taxonomic hypotheses in potato, for studies of interspecific hybridization, and serving as the germplasm base for Russian breeding efforts. Despite its importance and size, there has never been a study of its molecular diversity, and there were many gaps in its passport data. The purpose of the present study is to obtain morphological, ploidy, and microsatellite (SSR) data needed to set up a useful subset of the collection of cultivated potatoes and closely related wild species, and to use this collection to study cultivated potato taxonomy and phylogeny. Through assessments of viability, passport data, and chromosome counts, we selected a subset of 238 cultivated and 54 wild accessions. A morphological and nuclear SSR study of these collections distinguished only three cultivated species: Solanum curtilobum, S. juzepczukii and S. tuberosum, not the many more cultivated potato species of prior taxonomic treatments. The SSR study supports the ideas of S. acaule as one of the parental species for S. curtilobum and S. juzepczukii. The morphological and SSR results are very similar to other recent studies of cultivated species, and show the need to reclassify the collection of cultivated potatoes by modern taxonomic criteria.     

Cross-transferability of SSR markers in <Emphasis Type="Italic">Osmanthus</Emphasis>

Developing a molecular tool kit for hybrid breeding of Osmanthus species and related genera is an important step in creating a systematic breeding program for this species. To date, molecular resources have been aimed solely at Osmanthus fragrans with little work to develop markers for other species and cultivars. The objectives of this study were to (1) determine cross-transferability of O. fragrans and Chionanthus retusus derived SSRs in diverse Osmanthus taxa, (2) quantify the influence of locus-specific factors on cross-transferability, and (3) determine the genetic relationships between accessions. We tested 70 SSR markers derived from O. fragrans and C. retusus in 24 accessions of Osmanthus. Sixty-seven markers showed transfer to at least one other Osmanthus species with an overall transfer rate of 84% of loci across taxa. Genotyping with 42 microsatellite markers yielded a total of 367 loci. Number of alleles per locus ranged from 2 to 17 with a mean of 8.7 ± 4.8. Mean observed and expected heterozygosities were 0.560 ± 0.225 and 0.688 ± 0.230, respectively. Percent of polymorphic loci ranged from 40% in Osmanthus delavayi to 100% in O. fragrans. Osmanthus fragrans had the highest mean number of alleles per locus (4.2) while O. delavayi had the lowest (1.1). A reduced suite of eight-markers can distinguish between accessions with non-exclusion probabilities of identity from 3.91E?⁰⁴ to 2.90E?⁰⁷. The SSR markers described herein will be immediately useful to characterize germplasm, identify hybrids, and aid in understanding the level of genetic diversity and relationships within the cultivated germplasm.     

Genetic diversity in populations of the medicinal plant <Emphasis Type="Italic">Leonurus cardiaca</Emphasis> L. revealed by inter-primer binding site (iPBS) markers

Motherwort (Leonurus cardiaca L.) is a medicinal plant indigenous to the Mediterranean regions in Europe and Asia. The objective of this study is to apply inter-primer binding site (iPBS) markers to assess the molecular variation and genetic relationships of 89 genotypes of motherwort to assist the genetic improvement of this species. The genotypes comprised 79 from Iran and 10 collected in Australia and 15 additional accessions of two related species (L. heterophyllus Sweet and L. sibiricus L.) collected in Australia, were also included. PCR of 7 iPBS primers (dominant markers) produced a total of 191 bands ranging from 180 to 4000 bp and the mean PIC for primers ranged from 0.2213 to 0.3206 with a mean value 0.2921. The mean expected heterozygosity (0.134), the mean unbiased expected heterozygosity (0.140) and Shannon’s information index (0.213) indicated a high level of inbreeding among the accessions tested. Ordination and cluster analysis showed that the genetic relationships among all accessions could be separated into three major groups—L. cardiaca, L. heterophyllus and L. sibiricus. However, among the 89 accessions of L. cardiaca, genotypes collected from the same geographic region tended to cluster together thus indicating greater genetic similarity. The Motherwort accessions originating in Iran are highly divergent and possess abundant genetic diversity and clearly provide a basis for selection and breeding. The iPBS PCR-based genome fingerprinting technology used in this study is low-cost and effective in differentiating accessions of motherwort and their related species.     

Molecular Characterization by AFLPs of Capsicum Germplasm from the Amazon Department in Colombia

Using AFLPs, 71 peppers (Capsicum) accessions from the species C. chinense Jacq., C. baccatum L., C. annuum L. and C. frutescens L. from indigenous communities of the Amazon Department (Colombia) were studied to assess the genetic diversity of the collections, and delineate species gene groups. Ten additional accessions were included as a reference species group. Three clusters were identified in the Amazonian accessions by Multiple Correspondence Analyses (MCA) and a dendrogram from the UPGMA analyses of Nei – Li genetic similarity. The clusters correspond to gene groups of the species Capsicum chinense (the majority of the accessions), C. baccatum and the complex annuum - frutescens. A fourth cluster corresponds to the reference accession C. pubescens. The MCA analyses accounted for 95% of the total variation. The total genetic variation was low (Ht 0.119) and a genetic diversity index (Gst) of 0.331 was obtained. This suggests a limited genetic diversity of the accessions and a close relatedness of the species. This study is the first molecular marker assessment of genetic diversity for peppers from the Colombian Amazon, and provides information of biodiversity that can be employed in the preservation and use of Capsicum germplasm.     

Assessment of molecular diversity and population structure of the Ethiopian sorghum [Sorghum bicolor (L.) Moench] germplasm collection maintained by the USDA–ARS National Plant Germplasm System using SSR markers

The molecular diversity and population structure present in the Ethiopian sorghum collection maintained at the USDA–ARS National Plant Germplasm System (NPGS) has not been studied. In addition, 83 % of the accessions in the Ethiopian collection lack passport information which has constrained their evaluation and utility. Therefore, 137 Ethiopian accessions from NPGS were randomly selected and characterized with 20 strategically selected simple sequence repeat markers. These markers indentified 289 alleles with average polymorphic information content of 0.78. The allele frequency distribution reflects that 62 % of the alleles were rare (<0.05), 17 % range from 0.05 to 0.10, and 22 % were higher than 0.10. Expected and observed heterozygosity were estimated at 0.78 and 0.23, respectively, demonstrating Ethiopia has high sorghum genetic diversity germplasm. Population structure analysis indentified two subpopulations of 77 and 41 accessions, respectively, while a third group was constituted by 19 accessions whose classifications were not defined (i.e. hybrids). Analysis of molecular variances determined variation within subpopulations as the major source of variation. Likewise, genetic differentiation between subpopulations was moderate (F_st = 0.10). These results indicated that a continuous exchange of genes between subpopulations of sorghum exists in Ethiopia. The absence of a well defined population structure positioned this germplasm as an important resource for the study and dissection of agricultural traits by association mapping. However, genetic redundancy analysis indicated the presence of highly related accessions, therefore, strategic selected accessions must be consider prior to phenotype evaluation of larger number of accessions. The Ethiopian collection is composed of highly genetically diverse germplasm, and the genetic information presented herein is valuable to ex situ and in situ conservation programs to promote the use of this germplasm for breeding programs.     

Genetic diversity,population structure and differentiation of rice species from Niger and their potential for rice genetic resources conservation and enhancement

Rice genetic resources conservation and evaluation is crucial to ensure germplasm sources for further crop breeding. We conducted a wide collection of Oryza species in Niger and characterize its diversity with microsatellites (or simple sequence repeats, SSR). The aims of this research were to get a better understanding of the extent of genetic diversity, its structure and partition within rice eco-geographical zones of Niger. There were 264 accessions found in farmers’ and other fields: 173 O. sativa (Asia’s rice), 65 O. glaberrima (Africa’s rice), 25 O. barthii, and 1 O. longistaminata (weedy perennial rice), which were genotyped with 18 SSR. A total of 178 alleles were detected, with a mean of 9.89 alleles per locus. The polymorphism information content was 0.65 and heterozygosity was estimated as 0.14. Two main well-differentiate genotypic groups, which correspond to Asian and African rice species, were identified. The SSR set divided the Asia’s rice group (solely indica) into irrigated and floating rice, with rainfed lowland rice in between. The African rice species group was composed of O. glaberrima, O. longistaminata and O. barthii accessions, but without any clear genetic differentiation among them likely due admixtures within the samples of O. barthii. Five accessions that could be natural interspecific hybrids were too admixed for assigning them to any of the two well-differentiated groups. The partitioning of the overall diversity showed that maximum variation was within genotypic groups and subgroups or cropping ecologies, rather than between eco-geographical zones. The eco-geographical distribution of the diversity suggests germplasm exchange in Niger. Next-steps for conserving rice and crop wild relatives in Niger could be taken using the findings of this research.     

Genetic diversity, conservation, and utilization of Theobroma cacao L.: genetic resources in the Dominican Republic

Cacao (Theobroma cacao L.) is a significant agricultural commodity in the Dominican Republic, which ranks 11th in the world for cacao exports. To estimate genetic diversity, determine genetic identity, and identify any labeling errors, 14 SSR markers were employed to fingerprint 955 trees among cacao germplasm accessions and local farmer selections (LFS). Comparisons of homonymous plants across plots revealed a significant misidentification rate estimated to be 40.9 % for germplasm accessions and 17.4 % for LFS. The 14 SSRs amplified a total of 117 alleles with a mean allelic richness of 8.36 alleles per locus and average polymorphism information content (PIC) value of 0.67 for the germplasm collection. Similar levels of variation were detected among the LFS where a total of 113 alleles were amplified with a mean of 8.07 alleles per locus and PIC of 0.57. The observed heterozygosity (H_obs) was 0.67 for the germplasm collection and 0.60 for LFS. Based on population structure analysis 43.9 % of the germplasm accessions and 72.1 % of the LFS are predominantly of the Amelonado ancestry. Among these Amelonado, 51.7 % for the germplasm collection and 50.6 % for LFS corresponded to Trinitario hybrid lineage. Criollo ancestry was found in 7.6 and 9.5 % of the germplasm accessions and LFS, respectively. The Contamana, Nacional, and Iquitos backgrounds were also observed in both populations, but the Curaray background was only detected in the germplasm accessions. No Purús or Guiana ancestry was found in either of the populations. Overall, significant genetic diversity, which could be exploited in the Dominican Republic breeding and selection programs, was identified among the germplasm accessions and LFS.