Inferring population structure and genetic diversity of broad range of wild diploid alfalfa (Medicago sativa L.) accessions using SSR markers

Diversity analyses in alfalfa have mainly evaluated genetic relationships of cultivated germplasm, with little known about variation in diploid germplasm in the M. sativa–falcata complex. A collection of 374 individual genotypes derived from 120 unimproved diploid accessions from the National Plant Germplasm System, including M. sativa subsp. caerulea, falcata, and hemicycla, were evaluated with 89 polymorphic SSR loci in order to estimate genetic diversity, infer the genetic bases of current morphology-based taxonomy, and determine population structure. Diploid alfalfa is highly variable. A model-based clustering analysis of the genomic data identified two clearly discrete subpopulations, corresponding to the morphologically defined subspecies falcata and caerulea, with evidence of the hybrid nature of the subspecies hemicycla based on genome composition. Two distinct subpopulations exist within each subsp. caerulea and subsp. falcata. The distinction of caerulea was based on geographical distribution. The two falcata groups were separated based on ecogeography. The results show that taxonomic relationships based on morphology are reflected in the genetic marker data with some exceptions, and that clear distinctions among subspecies are evident at the diploid level. This research provides a baseline from which to systematically evaluate variability in tetraploid alfalfa and serves as a starting point for exploring diploid alfalfa for genetic and breeding experiments.     

Population structure and combining ability of diverse Medicago sativa germplasms

Although unadapted germplasms have been used to improve disease and insect resistance in alfalfa, there has been little effort to use these for improving forage yield. We evaluated genetic diversity and combining ability among two unadapted germplasms (Medicago sativa ssp. sativa Peruvian and M. sativa ssp. falcata WISFAL) and three Northern U.S. adapted alfalfa cultivars. Population structure analyses indicated that the WISFAL and Peruvian germplasms were genetically distinct from the cultivars, although Peruvian was relatively closer to the cultivars. Peruvian and WISFAL germplasms were intermated to generate a novel hybrid population. This population was crossed to the three cultivars as testers, and the testcross progenies were evaluated for forage yield along with the hybrid population, the original germplasms (Peruvian, WISFAL and cultivars), testcrosses of Peruvian and WISFAL to the three cultivars and a three-way hybrid of the cultivars. The experiment was carried out in the field in Temuco, Chile and Arlington, Wisconsin, USA, and forage was harvested during two seasons. Results from these evaluations showed that hybrids between the Peruvian × WISFAL population and the cultivar testers yielded as much as the cultivar testers. Heterosis was observed between Peruvian and WISFAL, and between these germplasms and the cultivar testers, suggesting that each germplasm may contain different favorable alleles. If Peruvian and WISFAL populations contain alleles at different loci that complement cultivar testers, then combining and enriching these alleles in a single population could result in improved combining ability with alfalfa cultivars.     

Genetic diversity and population structure in the US Upland cotton (Gossypium hirsutum L.)

Key message

Genetic diversity and population structure in the US Upland cotton was established and core sets of allelic richness were identified for developing association mapping populations in cotton.

Abstract

Elite plant breeding programs could likely benefit from the unexploited standing genetic variation of obsolete cultivars without the yield drag typically associated with wild accessions. A set of 381 accessions comprising 378 Upland (Gossypium hirsutum L.) and 3 G. barbadense L. accessions of the United States cotton belt were genotyped using 120 genome-wide SSR markers to establish the genetic diversity and population structure in tetraploid cotton. These accessions represent more than 100 years of Upland cotton breeding in the United States. Genetic diversity analysis identified a total of 546 alleles across 141 marker loci. Twenty-two percent of the alleles in Upland accessions were unique, specific to a single accession. Population structure analysis revealed extensive admixture and identified five subgroups corresponding to Southeastern, Midsouth, Southwest, and Western zones of cotton growing areas in the United States, with the three accessions of G. barbadense forming a separate cluster. Phylogenetic analysis supported the subgroups identified by STRUCTURE. Average genetic distance between G. hirsutum accessions was 0.195 indicating low levels of genetic diversity in Upland cotton germplasm pool. The results from both population structure and phylogenetic analysis were in agreement with pedigree information, although there were a few exceptions. Further, core sets of different sizes representing different levels of allelic richness in Upland cotton were identified. Establishment of genetic diversity, population structure, and identification of core sets from this study could be useful for genetic and genomic analysis and systematic utilization of the standing genetic variation in Upland cotton.     

Germination salt resistance of alfalfa (Medicago sativa L.) germplasm in relation to subspecies and centers of diversity

Saline soils and water severely limit the productivity of crop and pasture lands in semiarid and arid environments. The breeding of salt resistant cultivars of some crops is a partial solution to this problem. To breed for increased salt resistance, scientists must characterize the potentials and limitations of germplasm resources. This study measured the salt resistance of 761 alfalfa (Medicago sativa L. Emend. Sensu Lato) plant introduction accessions to NaCl during germination and characterized the resistance by subspecies, country of origin, and center of diversity. Experiments indicated that germplasm from the arid Indian and African centers excelled in NaCl resistance during germination. Germplasm from the Falcata center was least resistant. M. sativa L. subsp. sativa was more than twice as resistant as M. sativa L. subsp. ambigua or subsp. falcata. Thus, more resistant germplasm potentially adapted to the warm desert regions is available than resistant germplasm better suited to alfalfa production in more temperate regions. Joint contribution of the USDA-Agricultural Research Service and the Utah Agricultural Experimental Station Journal Paper no. 3782. Joint contribution of the USDA-Agricultural Research Service and the Utah Agricultural Experimental Station Journal Paper no. 3782.     

Reducing the tetraploid non-nodulating alfalfa (Medicago sativa) MnNC-1008(NN) germ plasm to the diploid level

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn ₁ and nn ₂ )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F₁ plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F₂ population segregating the nn ₁ and nn ₂ genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F₁ plants.     

Genetic diversity and population structure of common bean (Phaseolus vulgaris) landraces from China revealed by a new set of EST-SSR markers

A new set of EST-SSR markers were developed and employed to analyze the genetic diversity and population structure of Phaseolus vulgaris in China. A total of 2452 microsatellites were identified in 2144 unigenes assembled from P. vulgaris ESTs, indicating that merely 6.9% of the 30,952 unigene sequences contained SSRs. Seventeen of 153 randomly designed EST-SSR primer pairs successfully amplified polymorphic products in 31 landraces from six major production provinces of China, with the mean number of alleles per locus of 2.700 and polymorphism information content of 0.378. The observed and expected heterozygosity ranged from 0.100 to 0.954 and 0.081 to 0.558, respectively. Using these markers, both an unrooted neighbor-joining tree and principal coordinates analysis showed that almost all of the landraces were separated according with their regional distribution. Moreover, population structure analysis revealed that all genotypes formed into three distinct clusters (k = 3), suggesting that geographic and climatic factors could provide diverse degrees of selection pressure. Accordingly, germplasm collection and cross breeding among different regions are suggested to accelerate the process of diverse germplasm creation and broaden germplasm resources of Chinese common bean.     

Estimating the genetic diversity and spatial structure of Bulgarian Castanea sativa populations by SSRs: implications for conservation

Sweet chestnut (Castanea sativa Mill.) is a multipurpose species of great ecological and economic importance in southwest Bulgaria. Bulgarian chestnut forests are severely degraded, however, due to the intensive exploitation and bad management that have occurred over the last 2000 years. Given the urgent need to define conservation strategies to preserve the biodiversity of Bulgarian chestnut, we estimated its genetic variability. A set of eight microsatellite primers were used to analyze the genetic diversity and structure of six C. sativa populations distributed throughout the range of species in Bulgaria. Results showed a generally high level of genetic diversity but little divergence among populations. A significant, positive, within-population inbreeding coefficient (Fis) was observed in four populations. A STRUCTURE analysis revealed three genetic clusters. Using a landscape approach, significant genetic barriers among populations were found by integrating genetics with geographical distance. We hypothesize that one population is a relict from a glacial refugium; the structure of the remaining populations is probably the result of a combination of natural events and human impacts. For the purposes of conservation planning, we have identified populations that are particularly rich in diversity and private alleles that are good candidates for preservation.     

Novel microsatellite loci reveal high genetic diversity yet low population structure for alfalfa leafcutting bees in North America

The alfalfa leafcutting bee, Megachile rotundata (ALCB) is an economically important pollinator necessary for seed production of the critical forage crop alfalfa, Medicago sativa. The pollinator was accidentally introduced to North America from Europe approximately 70 years ago, and it is primarily produced in Canada and shipped to the United States annually en masse for seed field pollination. We investigate how the large-scale commercial movement of this bee affects the genetic structure of populations in the North American seed growing system and compare the genetic diversity and structure of introduced North American bees with two native European populations. Using 16 newly developed microsatellite loci, we describe the North American population structure of this bee. ALCBs collected from alfalfa seed farms have a degree of genetic variability similar to one native European population, but lower than the second. Considering that the species was accidentally introduced into North America, we anticipated more signature of a founder effect. Despite the level of genetic variability, we found little, if any, genetic structuring across North America, other than that the North American populations were distinct from the European populations sampled. While we detected some sub-structure in North American populations using Bayesian methods, the structuring was without geographic pattern, and we propose it is the result of the intense human management and movement of these bees. The trade and movement of these bees by humans has created a nearly panmictic M. rotundata population across the continent, which has implications relevant to the preservation and conservation of other bee pollinators.     

Genetic Diversity and Population Structure in Aromatic and Quality Rice (Oryza sativa L.) Landraces from North-Eastern India

The North-eastern (NE) India, comprising of Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim and Tripura, possess diverse array of locally adapted non-Basmati aromatic germplasm. The germplasm collections from this region could serve as valuable resources in breeding for abiotic stress tolerance, grain yield and cooking/eating quality. To utilize such collections, however, breeders need information about the extent and distribution of genetic diversity present within collections. In this study, we report the result of population genetic analysis of 107 aromatic and quality rice accessions collected from different parts of NE India, as well as classified these accessions in the context of a set of structured global rice cultivars. A total of 322 alleles were amplified by 40 simple sequence repeat (SSR) markers with an average of 8.03 alleles per locus. Average gene diversity was 0.67. Population structure analysis revealed that NE Indian aromatic rice can be subdivided into three genetically distinct population clusters: P1, joha rice accessions from Assam, tai rices from Mizoram and those from Sikkim; P2, chakhao rice germplasm from Manipur; and P3, aromatic rice accessions from Nagaland. Pair-wise F_ST between three groups varied from 0.223 (P1 vs P2) to 0.453 (P2 vs P3). With reference to the global classification of rice cultivars, two major groups (Indica and Japonica) were identified in NE Indian germplasm. The aromatic accessions from Assam, Manipur and Sikkim were assigned to the Indica group, while the accessions from Nagaland exhibited close association with Japonica. The tai accessions of Mizoram along with few chakhao accessions collected from the hill districts of Manipur were identified as admixed. The results highlight the importance of regional genetic studies for understanding diversification of aromatic rice in India. The data also suggest that there is scope for exploiting the genetic diversity of aromatic and quality rice germplasm of NE India for rice improvement.     

Origin of Oryza sativa in China Inferred by Nucleotide Polymorphisms of Organelle DNA

China is rich of germplasm resources of common wild rice (Oryza rufipogon Griff.) and Asian cultivated rice (O. sativa L.) which consists of two subspecies, indica and japonica. Previous studies have shown that China is one of the domestication centers of O. sativa. However, the geographic origin and the domestication times of O. sativa in China are still under debate. To settle these disputes, six chloroplast loci and four mitochondrial loci were selected to examine the relationships between 50 accessions of Asian cultivated rice and 119 accessions of common wild rice from China based on DNA sequence analysis in the present study. The results indicated that Southern China is the genetic diversity center of O. rufipogon and it might be the primary domestication region of O. sativa. Molecular dating suggested that the two subspecies had diverged 0.1 million years ago, much earlier than the beginning of rice domestication. Genetic differentiations and phylogeography analyses indicated that indica was domesticated from tropical O. rufipogon while japonica was domesticated from O. rufipogon which located in higher latitude. These results provided molecular evidences for the hypotheses of (i) Southern China is the origin center of O. sativa in China and (ii) the two subspecies of O. sativa were domesticated multiple times.     

Chromosomal and molecular rearrangements in somatic hybrids between tetraploid Medicago sativa and diploid Medicago falcata

The aim of this study was to produce somatic hybrids between tetraploid (2n=4x=32) M. sativa and diploid (2n=2x=16) M. ?falcata and analyse their genomic structure. Protoplasts from genotypes selected for regeneration ability from the cultivar Rangelander of M. sativa and Wisfal-1 of M. falcata were electrofused. Seven somatic hybrid calli were produced and one of them regenerated plants. The hybrid nature of these plants and their genetic composition were assessed with morphological, cytological, and molecular analyses. The resulting plants were hyper-aneuploid (2n=33) and contained one extra long chromosome, indicating that a translocation had taken place. The presence of both types of parental sequences in the RAPDs analysis confirmed the true hybrid nature of the plants. Rearrangements within the parental genomes and the presence of somaclonal variation among hybrid plants were observed through an RFLP analysis of the nucleolar organizing region (NOR). The possible causes for the gross genomic alterations, and the suitability of this method for transferring useful agronomic traits from wild species to cultivated alfalfa, are discussed.     

Development of Microsatellite Markers and Analysis of Genetic Diversity and Population Structure of Colletotrichum gloeosporioides from Ethiopia

Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus, Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (F_ST = 0.049) as evidenced by high level of gene flow estimate (N_m = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.     

Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa

Cultivated alfalfa (Medicago sativa) is an autotetraploid. However, all three existing alfalfa genetic maps resulted from crosses of diploid alfalfa. The current study was undertaken to evaluate the use of Simple Sequence Repeat (SSR) DNA markers for mapping in diploid and tetraploid alfalfa. Ten SSR markers were incorporated into an existing F₂ diploid alfalfa RFLP map and also mapped in an F₂ tetraploid population. The tetraploid population had two to four alleles in each of the loci examined. The segregation of these alleles in the tetraploid mapping population generally was clear and easy to interpret. Because of the complexity of tetrasomic linkage analysis and a lack of computer software to accommodate it, linkage relationships at the tetraploid level were determined using a single-dose allele (SDA) analysis, where the presence or absence of each allele was scored independently of the other alleles at the same locus. The SDA diploid map was also constructed to compare mapping using SDA to the standard co-dominant method. Linkage groups were generally conserved among the tetraploid and the two diploid linkage maps, except for segments where severe segregation distortion was present. Segregation distortion, which was present in both tetraploid and diploid populations, probably resulted from inbreeding depression. The ease of analysis together with the abundance of SSR loci in the alfalfa genome indicated that SSR markers should be a useful tool for mapping tetraploid alfalfa. Received: 10 September 1999 / Accepted: 11 November 1999     

Landscape genetics structure of European sweet chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill): indications for conservation priorities

Sweet chestnut is a tree of great economic (fruit and wood production), ecological, and cultural importance in Europe. A large-scale landscape genetic analysis of natural populations of sweet chestnut across Europe is applied to (1) evaluate the geographic patterns of genetic diversity, (2) identify spatial coincidences between genetic discontinuities and geographic barriers, and (3) propose certain chestnut populations as reservoirs of genetic diversity for conservation and breeding programs. Six polymorphic microsatellite markers were used for genotyping 1608 wild trees sampled in 73 European sites. The Geostatistical IDW technique (ArcGIS 9.3) was used to produce maps of genetic diversity parameters (He, Ar, PAr) and a synthetic map of the population membership (Q value) to the different gene pools. Genetic barriers were investigated using BARRIER 2.2 software and their locations were overlaid on a Digital Elevation Model (GTOPO30). The DIVA-GIS software was used to propose priority areas for conservation. High values of genetic diversity (He) and allelic richness (Ar) were observed in the central area of C. sativa’s European distribution range. The highest values of private allelic richness (PAr) were found in the eastern area. Three main gene pools and a significant genetic barrier separating the eastern from the central and western populations were identified. Areas with high priority for genetic conservation were indicated in Georgia, eastern Turkey, and Italy. Our results increase knowledge of the biogeographic history of C. sativa in Europe, indicate the geographic location of different gene pools, and identify potential priority reservoirs of genetic diversity.     

Preliminary analysis of the genetic diversity and population structure of mortiño (Vaccinium floribundum Kunth)

Mortiño (Vaccinium floribundum Kunth) is a deciduous perennial shrub endemic to the high Andes of South America. Despite a rich ethnobotanic history among indigenous communities, mortiño remains a wild species vulnerable to extinction from the ongoing fragmentation of its natural habitat. This study assessed the degree of genetic diversity and population structure of Ecuadorian V. floribundum as a preliminary step towards the establishment of effective conservation and sustainable-agriculture strategies. Mortiño individuals (126 in total) sampled from 3 regions in the northern highlands of Ecuador were characterized using 11 heterologous SSR markers originally developed for Vaccinium corymbosum. Expected heterozygosity (He = 0.49) revealed a moderate degree of genetic diversity for Ecuadorian mortiño, and pairwise F statistics between sampling regions (0.019≤ Fst ≤0.041) demonstrated low-to-moderate population differentiation. Population structure analysis clustered mortiño germplasm into 3 groups, each representing the 3 distinct regions from where samples were collected. The geographic patterning of genetic diversity for V. floribundum could be explained by an isolation-by-distance model, where physical barriers along the Andean highlands reduce genetic exchange between distanced populations. To confirm the latter, this study should extend to a wider sampling range, covering other regions along the Andean alley where the species is found.     

Development of simple sequence repeat markers and diversity analysis in alfalfa (Medicago sativa L.)

Efficient and robust molecular markers are essential for molecular breeding in plant. Compared to dominant and bi-allelic markers, multiple alleles of simple sequence repeat (SSR) markers are particularly informative and superior in genetic linkage map and QTL mapping in autotetraploid species like alfalfa. The objective of this study was to enrich SSR markers directly from alfalfa expressed sequence tags (ESTs). A total of 12,371 alfalfa ESTs were retrieved from the National Center for Biotechnology Information. Total 774 SSR-containing ESTs were identified from 716 ESTs. On average, one SSR was found per 7.7 kb of EST sequences. Tri-nucleotide repeats (48.8 %) was the most abundant motif type, followed by di—(26.1 %), tetra—(11.5 %), penta—(9.7 %), and hexanucleotide (3.9 %). One hundred EST–SSR primer pairs were successfully designed and 29 exhibited polymorphism among 28 alfalfa accessions. The allele number per marker ranged from two to 21 with an average of 6.8. The PIC values ranged from 0.195 to 0.896 with an average of 0.608, indicating a high level of polymorphism of the EST–SSR markers. Based on the 29 EST–SSR markers, assessment of genetic diversity was conducted and found that Medicago sativa ssp. sativa was clearly different from the other subspecies. The high transferability of those EST–SSR markers was also found for relative species.     

SSR-based population structure, molecular diversity and linkage disequilibrium analysis of a collection of flax (Linum usitatissimum L.) varying for mucilage seed-coat content

Flax seed mucilage (SM) presents specific biological activities useful for the food and pharmaceutical industries. Understanding the population structure, genetic diversity and linkage disequilibrium (LD) of germplasm varying for mucilage content is pivotal for the identification of genes and quantitative trait loci underlying mucilage variation by association mapping (AM). In this study, 150 microsatellite loci were used to assess the population structure, genetic diversity and LD of a set of 60 flax cultivars/accessions capturing the breadth of SM variation in flax germplasm. STRUCTURE analysis and similarity-based methods revealed the presence of three populations reflecting mainly their geographic origins (South Asia, South America and North America), and the impact of germplasm exchange within and between North American flax breeding programs. Analysis of molecular variance showed that 78.32% of the genetic variation resided within populations and 21.68% among populations. The phi-statistic (??_st) value of 0.22 confirmed the presence of a strong population structure. A total of 408 alleles were detected, with the South American population capturing the highest overall diversity. However, the genetic diversity was narrow, as indicated by the small number of alleles per locus (2.72) and gene diversity (mean?=?0.34). LD was significant between 3.9% (r ²) and 36.2% (D??) of the loci pairs (FDR?<?0.05). The mean r ² and D?? were 0.26 and 0.53, respectively. The results suggest that the collection could be useful in AM studies aimed at the discovery of genes/alleles involved in SM; however a greater diversity may be required to improve the AM resolution.     

High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applications in rice (Oryza sativa L.), we designed seven GoldenGate VeraCode oligo pool assay (OPA) sets for the Illumina BeadXpress Reader. Validated markers from existing 1536 Illumina SNPs and 44?K Affymetrix SNP chips developed at Cornell University were used to select subsets of informative SNPs for different germplasm groups with even distribution across the genome. A 96-plex OPA was developed for quality control purposes and for assigning a sample into one of the five O. sativa population subgroups. Six 384-plex OPAs were designed for genetic diversity analysis, DNA fingerprinting, and to have evenly-spaced polymorphic markers for quantitative trait locus (QTL) mapping and background selection for crosses between different germplasm pools in rice: Indica/Indica, Indica/Japonica, Japonica/Japonica, Indica/O. rufipogon, and Japonica/O. rufipogon. After testing on a diverse set of rice varieties, two of the SNP sets were re-designed by replacing poor-performing SNPs. Pilot studies were successfully performed for diversity analysis, QTL mapping, marker-assisted backcrossing, and developing specialized genetic stocks, demonstrating that 384-plex SNP genotyping on the BeadXpress platform is a robust and efficient method for marker genotyping in rice.