Population structure and genetic diversity analysis of Indian and exotic rice (Oryza sativa L.) accessions using SSR markers

In order to understand the population structure and genetic diversity among a set of 82 rice genotypes collected from different parts of the Asian countries including India were characterized using 39 microsatellite loci. The Population structure analysis suggested that the optimum number of subpopulations was four (K = 4) among the rice genotypes, whereas phylogenetic analysis grouped them into three populations. The results obtained from phylogenetic and STRUCTURE analysis proved to be very powerful for the differentiation of rice genotypes based on their place of origin. The genetic diversity analysis using 39 SSR loci yielded 183 scorable alleles, out of which 182 alleles were observed to be polymorphic with an average of 4.8 alleles per locus. The Polymorphism Information Content (PIC) values for all the polymorphic primers across 82 rice genotypes varied from 0.02 to 0.77, with an average of 0.50. Gene diversity (He) was found to be in the range of 0.02 (RM484) to 0.80 (OSR13) with an average value of 0.55, while heterozygosity (Ho) was observed with an average of 0.07, ranging from 0.01 (RM334) to 0.31 (RM316). The present study resulted in identification of seven highly polymorphic SSR loci viz., OSR13, RM152, RM144, RM536, RM489, RM259 and RM271 based on the parameters like PIC value (≥0.70), gene diversity (≥0.71), and polymorphic alleles (≥6). These seven polymorphic primers can effectively be used in further molecular breeding programs and QTL mapping studies of rice since they exhibited very high polymorphism over other loci. SSR analysis resulted in a more definitive separation of clustering of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions.     

Molecular genetic diversity and population structure in Lycium accessions using SSR markers

This study was conducted to assess the genetic diversity and population structure of 139 Lycium chinense accessions using 18 simple sequence repeat (SSR) markers. In total, 108 alleles were detected. The number of alleles per marker locus ranged from two to 17, with an average of six. The gene diversity and polymorphism information content value averaged 0.3792 and 0.3296, with ranges of 0.0793 to 0.8023 and 0.0775 to 0.7734, respectively. The average heterozygosity was 0.4394. The model-based structure analysis revealed the presence of three subpopulations, which was consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 15.3%, in contrast to 84.7% for the within-population component. The overall F_ST value was 0.1178, indicating a moderate differentiation among groups. The results could be used for future L. chinense allele mining, association mapping, gene cloning, germplasm conservation, and designing effective breeding programs.     

Assessment of genetic diversity and population structure in pomegranate (Punica granatum L.) using hypervariable SSR markers

Physiology and Molecular Biology of Plants - The present study investigates the genetic diversity and population structure among 42 diverse pomegranate genotypes using a set of twenty one class I...     

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.     

Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.)

Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70–80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.     

Liming of alfalfa (Medicago sativa L.)

Summary A growth-chamber experiment was conducted to study the effect of liming upon growth of alfalfa. The beneficial effects observed were related to changes in soil properties brought about by lime application. Reductions of aluminum and manganese toxicities were the major factors responsible for the increased yields and the decreased growth period required to reach harvest stage. Significant correlations between plant growth parameters and various measures of extractable aluminum were found.     

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     

Genetic diversity among alfalfa (Medicago sativa) cultivars coming from a breeding program, using SSR markers

Alfalfa (Medicago sativa) is an autotetraploid, allogamous and heterozygous species whose cultivars are synthetic populations. The breeders apply selection pressure for some agronomic traits within a breeding pool to increase the frequency of favorable individuals. The objective of this study was to investigate the differentiation level among seven cultivars originating from one breeding program, and between these cultivars and the breeding pool, with eight SSR markers. These highly polymorphic and codominant markers, together with recent population genetic statistics extended to autotetraploids, offer tools to analyse genetic diversity in alfalfa. The number of alleles per locus varied between 3 and 24. All loci were at a panmictic equilibrium in the cultivars, except one, probably because of null alleles. With seven SSR loci, each cultivar was at panmictic equilibrium. The mean gene diversity was high, ranging from 0.665 to 0.717 in the cultivars. The parameter F _ST indicated a low but significant diversity among cultivars. Among 21 pairs of cultivars, 15 were significantly different. The breeding pool also had a high diversity, and was significantly different from each cultivar except the most recent one. Considering the characteristics of the breeding program and the mode of cultivar elaboration, we found that they were unable to generate a large variety differentiation. Estimation of population genetics parameters at SSR loci can be applied for assessing the differences between cultivars or populations, either for variety distinction or the management of genetic resources.     

Crystal structure of vestitone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids are commonly found in leguminous plants, where they play important roles in plant defense and have significant health benefits for animals and humans. Vestitone reductase catalyzes a stereospecific NADPH-dependent reduction of (3R)-vestitone in the biosynthesis of the antimicrobial isoflavonoid phytoalexin medicarpin. The crystal structure of alfalfa (Medicago sativa L.) vestitone reductase has been determined at 1.4 A resolution. The structure contains a classic Rossmann fold domain in the N terminus and a small C-terminal domain. Sequence and structural analysis showed that vestitone reductase is a member of the short-chain dehydrogenase/reductase (SDR) superfamily despite the low levels of sequence identity, and the prominent structural differences from other SDR enzymes with known structures. The putative binding sites for the co-factor NADPH and the substrate (3R)-vestitone were defined and located in a large cleft formed between the N and C-terminal domains of enzyme. Potential key residues for enzyme activity were also identified, including the catalytic triad Ser129-Tyr164-Lys168. A molecular docking study showed that (3R)-vestitone, but not the (3S) isomer, forms favored interactions with the co-factor and catalytic triad, thus providing an explanation for the enzyme's strict substrate stereo-specificity.     

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.     

Population structure and genetic diversity in a rice core collection (Oryza sativa L.) investigated with SSR markers

The assessment of genetic diversity and population structure of a core collection would benefit to make use of these germplasm as well as applying them in association mapping. The objective of this study were to (1) examine the population structure of a rice core collection; (2) investigate the genetic diversity within and among subgroups of the rice core collection; (3) identify the extent of linkage disequilibrium (LD) of the rice core collection. A rice core collection consisting of 150 varieties which was established from 2260 varieties of Ting's collection of rice germplasm were genotyped with 274 SSR markers and used in this study. Two distinct subgroups (i.e. SG 1 and SG 2) were detected within the entire population by different statistical methods, which is in accordance with the differentiation of indica and japonica rice. MCLUST analysis might be an alternative method to STRUCTURE for population structure analysis. A percentage of 26% of the total markers could detect the population structure as the whole SSR marker set did with similar precision. Gene diversity and MRD between the two subspecies varied considerably across the genome, which might be used to identify candidate genes for the traits under domestication and artificial selection of indica and japonica rice. The percentage of SSR loci pairs in significant (P<0.05) LD is 46.8% in the entire population and the ratio of linked to unlinked loci pairs in LD is 1.06. Across the entire population as well as the subgroups and sub-subgroups, LD decays with genetic distance, indicating that linkage is one main cause of LD. The results of this study would provide valuable information for association mapping using the rice core collection in future.     

Induced androgenesis in alfalfa (Medicago sativa L.)

Summary Anthers of 10 alfalfa (Medicago sativa L.) lines were used as initial material for the production of androgenic haploids. More than 30 variants of nutrient media were tested. Twenty five different treatments with low temperatures and gamma rays were tried in order to find optimal conditions for callus induction and organogenesis.The genotype, stage of microspore development, phytohormonal composition of the nutrient media and pretreatment with physical agents, alone or in combination, affected the efficiency of organogenesis and regeneration in anther cultures of alfalfa.Plants exhibited a high degree of variability in their chromosome number. Haploids, dihaploids and mixoploids were obtained.Cytological studies of in vitro pollen development revealed the origin of the regenerants from microspores.Abbreviations BAP 6-Benzylaminopurine - 2-ip 6-(,-dimethylallylamino)Purine - IAA Indolylacetic Acid - NAA Naphthaleneacetic Acid - 2,4-D Dichlorophenoxyacetic Acid - CMS Cytoplasmic Male Sterility     

Assessment of population structure,genetic diversity and relationship of Mediterranean olive accessions using SSR markers and computational tools

Biotechnology Letters - Olive tree is an emblematic crop of the Mediterranean region, mainly renowned for its fruit oil, although the species provides several industrial purposes. The Mediterranean...     

A note on the measurement of genetic diversity within genebank accessions of lettuce (Lactuca sativa L.) using AFLP markers

This paper discusses a statistical approach for measuring genetic diversity within genebank accessions of a self-fertilising species. This approach is applied to lettuce (Lactuca sativa L.), using AFLP marker data on a set of 1,390 accessions, representing six different lettuce types. Knowledge of the within-accession genetic diversity is important for decisions about the way accessions have to be maintained by genebanks. It is argued that if the within-accession diversity is small, as can be expected for a self-fertilising species like L. sativa, the best approach is to sample as many accessions as possible with only two plants per accession and estimate the within-accession diversity by the proportion of accessions of which the individuals are different.     

Fine root demography in alfalfa (Medicago sativa L.)

In perennial forages like alfalfa (Medicago sativa L.), repeated herbage removal may alter root production and mortality which, in turn, could affect deposition of fixed N in soil. Our objective was to determine the extent and patterns of fine-diameter root production and loss during the year of alfalfa stand establishment. The experiment was conducted on a loamy sand soil (Udorthentic Haploboroll) in Minnesota, USA, using horizontally installed minirhizotrons placed directly under the seeded rows at 10, 20, and 40 cm depths in four replicate blocks. We seeded four alfalfa germplasms that differed in N₂ fixation capacity and root system architecture: Agate alfalfa, a winter hardy commercially-available cultivar; Ineffective Agate, which is a non-N₂-fixing near isoline of Agate; a new germplasm that has few fibrous roots and strong tap-rooted traits; and a new germplasm that has many fibrous roots and a strongly branched root system architecture. Video images collected biweekly throughout the initial growing season were processed using C-MAP-ROOTS software.More than one-half of all fine roots in the upper 20 cm were produced during the first 7 weeks of growth. Root production was similar among germplasms, except that the highly fibrous, branch-rooted germplasm produced 29% more fine roots at 20 cm than other germplasms. In all germplasms, about 7% of the fine roots at each depth developed into secondarily thickened roots. By the end of the first growing season, greatest fine root mortality had occurred in the uppermost depth (48%), and least occurred at 40 cm (36%). Survival of contemporaneous root cohorts was not related to soil depth in a simple fashion, although all survivorship curves could be described using only five rates of exponential decline. There was a significant reduction in fine root mortality before the first herbage harvest, followed by a pronounced loss (average 22%) of fine roots at the 10- and 20-cm depths in the 2-week period following herbage removal. Median life spans of these early-season cohorts ranged from 58 to 131 days, based on fitted exponential equations. At all depths, fine roots produced in the 4 weeks before harvest (early- to mid-August) tended to have shorter median life spans than early-season cohorts. Similar patterns of fine root mortality did not occur at the second harvest. Germplasms differed in the pattern, but not the ultimate extent, of fine root mortality. Fine root turnover during the first year of alfalfa establishment in this experiment released an estimated 830 kg C ha^–1 and 60 kg N ha^–1, with no differences due to N₂ fixation capacity or root system architecture.     

Hairy root transformation in alfalfa (Medicago sativa L.)

Summary The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.Research work was partially supported by Progetto Strategico Agrobiotecnologia C.N.R., Italy     

Assessment of genetic diversity in broomcorn millet （Panicum miliaceum L.） using SSR markers

The genetic diversity of 118 accessions of broomcom millet （Panicum miliaceum L.）, collected from various ecological areas, was analyzed. Using 46 SSR （Simple Sequence Repeat） polymorphic markers from rice, wheat, oat and barley, a total of 226 alleles were found, which exhibited moderate level of diversity. The number of alleles per primer ranged from two to nine, with an average of 4.91. The range of polymorphism information content （PIC） was 0.2844）.980 （average, 0.793）. The expected heterozygosity （He） varied from 0.346 to 0.989, with an average of 0.834. The average coefficient of the genetic similarity of SSR markers among the 118 accessions was 0.609, and it ranged from 0.461 to 0.851. The UPGMA （Unweight Pair Group Method with Arithmetic Mean） clustering analysis at the genetic similarity value of 0.609 grouped the 118 accessions into five groups. Mantel test meant that geographical origin and genetic distance presented positive correlation. The clustering results were consistent with known information on ecological growing areas. The genetic similarity coefficient of the accessions in the Loess Plateau ecotype was significantly lower than those in the other ecotypes. It indicates that the highest level of genetic diversity occurred in the Loess Plateau, which is probably the original site of Panicum miliaceum.     

Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Medicago sativa L.) cells and study the physiological changes occurring in cells during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (fall-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiated regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-treatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid N₂ (EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of the cv. 5262 cells after cryopreservation was two- to three-fold greater than that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2°C) improved viability after cryopreservation. The addition of 7.6 µM ABA to the B5 medium enhanced viability in ED but did not improve cell cryopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold-acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased concentration of cell protein and an altered protein composition. Suspension cultures were re-initiated from both ED and EDN treatments by transferring beads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures, whereas EDN cells needed four or five subcultures to resume rapid growth.     

Purification of beta-amylase from alfalfa (Medicago sativa L.) seeds

An amylase from alfalfa (Medicago sativa L. c.v. Moapa) seeds was purified by column chromatography and gel filtration, followed by chromatofocusing on Mono P HR 5/20. The last step was effective for separation of the alfalfa amylase to a homogeneous state. The purified amylase was identified as beta-amylase from the fact that only beta-maltose was formed by the enzymatic degradation of soluble starch. The molecular weight and specific activity of the beta-amylase (E1%(280 nm) = 18.3) were determined to be 61,000 and 1,077 A.U./mg, respectively. The beta-amylase activity was inhibited by the modification of sulfhydryl groups with p-chloromercuribenzoic acid. The optimum pH and isoelectric point of alfalfa beta-amylase were 7.0 and 4.8, respectively, which were different from other plant beta-amylases.     

Evaluation of the genetic diversity and population structure of sesame (Sesamum indicum L.) using microsatellite markers

Sixteen polymorphic microsatellite (SSR) markers, developed from an SSR-enriched genomic DNA library of sesame (Sesamum indicum L.), were used to assess genetic diversity, phylogenetic relationships, and population structure among 150 sesame accessions collected from 22 countries. A total of 121 alleles were detected among the sesame accessions. The number of detected alleles varied from 2 to 18, with an average of 7.6 alleles per locus. Polymorphism information content values ranged from 0.03 to 0.79, with an average of 0.42. These values indicated an excess of heterozygous individuals at 16 loci and an excess of homozygous individuals at three loci. Of these, 32 genotype-specific alleles were identified at 11 of 16 polymorphic SSR markers. Cluster analyses were performed by accession and population, revealing a complex accession distribution pattern with mean genetic similarity coefficient of 0.45 by accession and 0.52 by population. The wide variation in genetic similarity among the accessions revealed by SSRs reflected a high level of polymorphism at the DNA level. Model-based structure analysis revealed the presence of three groups that were basically consistent with the clustering results based on genetic distance. These findings may be used to augment the sesame germplasm and to increase the effectiveness of sesame breeding.