Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers

Cucurbita pepo is a highly polymorphic species. The cultivars can be grouped into eight morphotypes in two subspecies, ssp. pepo and ssp. ovifera. A collection of 69 accessions representative of the morphotypes and some unclassified types was used for analysing the morphological and molecular diversity of this species. This collection includes commercial cultivars and Spanish landraces, which represent the great diversification of types that have arisen in Europe after this species arrived from America. For the molecular variability studies, two PCR-based systems were employed, AFLP and SRAP, which preferentially amplify ORFs. Principal coordinates analysis and cluster analysis using the UPGMA method clearly separate the accessions into the two subspecies through the use of both markers. However, the gene diversity and the genetic identity values among morphotypes and subspecies varied between the two marker systems. The information given by SRAP markers was more concordant to the morphological variability and to the evolutionary history of the morphotypes than that of AFLP markers. In ssp. ovifera, the accessions of the different morphotypes were basically grouped according to the fruit colour. This may indicate different times of development and also the extent of breeding in the accessions used. This study has allowed identification of new types that can be employed for the development of new cultivars. The landraces of the spp. ovifera, used as ornamental in Europe, have proved to be of great interest for preserving the diversity of C. pepo.     

Assessing the genetic diversity of Panicum coloratum var. makarikariense using agro‐morphological traits and microsatellite‐based markers

Panicum coloratum var. makarikariense is a perennial C₄ grass native to South Africa with relatively good forage production under limited‐resource conditions. Genetic characterisation and breeding efforts have been scant, thus limiting its use in cattle raising systems. The goal of the present study was to assess the genetic diversity of a collection of P. coloratum var. makarikariense using agro‐morphological traits and molecular markers, in comparison with one accession of var. coloratum and one population of Panicum bergii. Agro‐morphological variability between and within accessions of var. makarikariense in a common garden setting was observed, showing that there is still opportunity for selection. Some accessions performed better than the commercialised material in relation to potential forage production. A total of 117 ISSR bands and 48 SSR alleles allowed the detection of genetic variability between and within accessions. The presence of accession‐specific bands suggested distinctness and limited gene flow. The genetic variability encountered in the commercialised material suggested that it is a stabilised population which has not undergone a strong selection process. Low correlation between agro‐morphologic and molecular variability was observed indicating that both approaches provide complementary information. Both morphological and molecular markers reveal genetic differentiation between varieties and species. This study provides a set of new SSR markers available for diversity assessment and valuable information that can be applied directly in collection management for breeding and conservation programmes.     

Genetic Diversity and Population Structure of Tetraploid Wheats (Triticum turgidum L.) Estimated by SSR,DArT and Pedigree Data

Levels of genetic diversity and population genetic structure of a collection of 230 accessions of seven tetraploid Triticum turgidum L. subspecies were investigated using six morphological, nine seed storage protein loci, 26 SSRs and 970 DArT markers. The genetic diversity of the morphological traits and seed storage proteins was always lower in the durum wheat compared to the wild and domesticated emmer. Using Bayesian clustering (K = 2), both of the sets of molecular markers distinguished the durum wheat cultivars from the other tetraploid subspecies, and two distinct subgroups were detected within the durum wheat subspecies, which is in agreement with their origin and year of release. The genetic diversity of morphological traits and seed storage proteins was always lower in the improved durum cultivars registered after 1990, than in the intermediate and older ones. This marked effect on diversity was not observed for molecular markers, where there was only a weak reduction. At K >2, the SSR markers showed a greater degree of resolution than for DArT, with their identification of a greater number of groups within each subspecies. Analysis of DArT marker differentiation between the wheat subspecies indicated outlier loci that are potentially linked to genes controlling some important agronomic traits. Among the 211 loci identified under selection, 109 markers were recently mapped, and some of these markers were clustered into specific regions on chromosome arms 2BL, 3BS and 4AL, where several genes/quantitative trait loci (QTLs) are involved in the domestication of tetraploid wheats, such as the tenacious glumes (Tg) and brittle rachis (Br) characteristics. On the basis of these results, it can be assumed that the population structure of the tetraploid wheat collection partially reflects the evolutionary history of Triticum turgidum L. subspecies and the genetic potential of landraces and wild accessions for the detection of unexplored alleles.     

Molecular characterization of an international cacao collection using microsatellite markers

Plant germplasm collections invariably contain varying levels of genetic redundancy, which hinders the efficient conservation and utilization of plant germplasm. Reduction of genetic redundancies is an essential step to improve the accuracy and efficiency of genebank management. The present study targeted the assessment of genetic redundancy and genetic structure in an international cacao (Theobroma cacao L.) collection maintained in Costa Rica. A total of 688 cacao accessions maintained in this collection were genotyped with 15 simple sequence repeat (SSR) loci, using a capillary electrophoresis genotyping system. The SSR markers provided a high resolution among the accessions. Thirty-six synonymously labeled sets, involving 135 accessions were identified based on the matching of multilocus SSR profiles. After the elimination of synonymous sets, the level of redundancy caused by closely related accessions in the collection was assessed using a simulated sampling scheme that compared allelic diversity in different sample sizes. The result of the simulation suggested that a random sample of 113 accessions could capture 90% of the total allelic diversity in this collection. Principal Coordinate Analysis revealed that the Trinitario hybrids from Costa Rica shared a high similarity among groups as well as among individual accessions. The analysis of the genetic structure illustrated that the within-country/within-region difference accounted for 84.6% of the total molecular variation whereas the among-country/among-region difference accounted for 15.4%. The Brazilian germplasm contributed most to this collection in terms of total alleles and private alleles. The intercountry/interregion relationship by cluster analysis largely agreed with the geographical origin of each germplasm group and supported the hypothesis that the Upper Amazon region is the center of diversity for cacao. The results of the present study indicated that the CATIE International Cacao Collection contains a high level of genetic redundancy. It should be possible to rationalize this collection by reducing redundancy and ensuring optimal representation of the genetic diversity from distinct germplasm groups. The results also demonstrated that SSR markers, together with the statistical tools for individual identification and redundancy assessment, are technically practical and sufficiently informative to assist the management of a tropical plant germplasm collection.     

小麦地方品种繁殖更新过程中的遗传多样性变化分析

采用分别保存于长期库及中期库的3个小麦地方品种的6份材料,进行了9项农艺性状及35个与农艺性状相关的微卫星标记检测,每份材料选取30个单株进行遗传多样性与遗传结构分析。结果表明:(1)更新前,3个小麦地方品种均为遗传异质性群体,在SSR位点上的异质度分别为57.14%、48.57%和5.71%。(2)在农艺性状表现上,只有温泉小麦3在株高和穗粒数上更新后比更新前显著增加,其他材料无显著差异。(3)在SSR位点多态性表现上,3个品种在更新后均发生了遗传多样性变化,8个与粒重、产量、生育期性状相关位点存在等位位点丢失现象,其中2个与粒重、生育期相关位点频率变化显著。(4)综合农艺性状调查与SSR分子标记检测结果发现,3个品种更新前后在多样性指数上无显著差异,遗传分化系数Gst分别为0.0269、0.0324和0.0380,即更新前后遗传差异分别为2.69%、3.24%和3.80%。上述结果建议,经繁殖更新的小麦种质资源能够比较完好地保持其遗传多样性和遗传结构。对于遗传异质性小麦地方品种在繁殖更新后存在遗传多样性丢失的危险,为了保证更新前后的遗传完整性,建议在繁殖更新过程中每个品种至少应保持300个单株的群体。     

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.     

Molecular genetic diversity of the Turkish national hazelnut collection and selection of a core set

European hazelnut (Corylus avellana L.) is an economically and nutritionally important nut crop with wild and cultivated populations found throughout Europe and in parts of Asia. This study examined the molecular genetic diversity and population structure of 402 genotypes including 143 wild individuals, 239 landraces, and 20 cultivars from the Turkish national hazelnut collection using simple sequence repeat (SSR) markers. A total of 30 SSR markers yielded 407 polymorphic fragments. Diversity analysis of the Turkish hazelnut genotypes indicated that they fell into three subpopulations according to ad hoc statistics and neighbor-joining algorithm. Although all cultivars clustered together, they overlapped with the wild accessions and landraces. Thus, the dendrogram, principal coordinate, and population structure analyses suggest that they share the same gene pool. A total of 78 accessions were selected as a core set to encompass the molecular genetic and morphological diversity present in the national collection. This core set should have priority in preservation efforts and in trait characterization.     

SSR-based genetic diversity and structure of garlic accessions from Brazil

Garlic is a spice and a medicinal plant; hence, there is an increasing interest in ‘developing’ new varieties with different culinary properties or with high content of nutraceutical compounds. Phenotypic traits and dominant molecular markers are predominantly used to evaluate the genetic diversity of garlic clones. However, 24 SSR markers (codominant) specific for garlic are available in the literature, fostering germplasm researches. In this study, we genotyped 130 garlic accessions from Brazil and abroad using 17 polymorphic SSR markers to assess the genetic diversity and structure. This is the first attempt to evaluate a large set of accessions maintained by Brazilian institutions. A high level of redundancy was detected in the collection (50 % of the accessions represented eight haplotypes). However, non-redundant accessions presented high genetic diversity. We detected on average five alleles per locus, Shannon index of 1.2, H_O of 0.5, and H_E of 0.6. A core collection was set with 17 accessions, covering 100 % of the alleles with minimum redundancy. Overall F_ST and D values indicate a strong genetic structure within accessions. Two major groups identified by both model-based (Bayesian approach) and hierarchical clustering (UPGMA dendrogram) techniques were coherent with the classification of accessions according to maturity time (growth cycle): early-late and midseason accessions. Assessing genetic diversity and structure of garlic collections is the first step towards an efficient management and conservation of accessions in genebanks, as well as to advance future genetic studies and improvement of garlic worldwide.     

Comparison of anonymous and targeted molecular markers for the estimation of genetic diversity in <Emphasis Type="Italic">ex situ</Emphasis> conserved <Emphasis Type="Italic">Lactuca</Emphasis>

The anonymous marker systems microsatellites (simple sequence repeats), amplified fragment length polymorphisms and sequence-specific amplified polymorphisms were compared with the targeted marker systems sequence-related amplified polymorphisms, target region amplification polymorphisms and nucleotide binding site profiling for their ability to describe the genetic diversity in a selected set of 80 Lactuca accessions. The accessions were also described morphologically, and all characterisation methods were evaluated against the genetic diversity assessed by a panel of three crop experts. The morphological data showed a low level of association with the molecular data, and did not display a consistently better relationship with the experts’ assessments in comparison with the molecular data. In general, the diversity described by the targeted molecular markers did not differ markedly from that of the anonymous markers, resulting in only slight differences in performance when related to the expert-based assessments. It was argued that markers targeted to specific gene sequences may still behave as anonymous markers and that the type of marker system used is irrelevant when at low taxonomic levels a clear genetic structure is absent due to intensive breeding activities.     

Genetic diversity and population structure detection in sponge gourd ( Luffa cylindrica ) using ISSR,SCoT and morphological markers

Investigation of genetic diversity is essential for the selection of parents for crop breeding and conservation of genetic resources. To estimate the genetic variability and population structure in the midst of 45 accessions of sponge gourd brought together from different geographical areas of India, morphological traits and two molecular markers, ISSR and SCoT markers were compared. Principal components analysis of 20 morphological traits showed 72.70% variability and significant positive correlations between fruit traits. All three marker techniques clustered all accessions into two groups with few outgroups. High level of polymorphism was observed among ISSR (74.6%) and SCoT (71.5%) primers. The Bayesian model revealed the hidden grouping and showed admixture type of population. The diversity pattern is influenced by genetic marker used, as different molecular markers have different polymorphism evaluation efficiency. This study can be helpful in amplifying the genetic base and selection of specific traits for breeding. Thus, ISSR and SCoT markers are potential marker for identification in sponge gourd and provide valuable data on its genetic correlation and structure.     

Design of a Brassica rapa core collection for association mapping studies

A Brassica rapa collection of 239 accessions, based on two core collections representing different morphotypes from different geographical origins, is presented and its use for association mapping is illustrated for flowering time. We analyzed phenotypic variation of leaf and seed pod traits, plant architecture, and flowering time using data collected from three field experiments and evaluated the genetic diversity with a set of SSR markers. The Wageningen University and Research Centre (WUR) and the Vavilov Research Institute of Plant Industry (VIR) core collections had similar representations of most morphotypes, as illustrated by the phenotypic and genetic variation within these groups. The analysis of population structure revealed five subgroups in the collection, whereas previous studies of the WUR core collection indicated four subgroups; the fifth group identified consisted mainly of oil accessions from the VIR core collection, winter oils from Pakistan, and a number of other types. A very small group of summer oils is described, that is not related to other oil accessions. A candidate gene approach was chosen for association mapping of flowering time with a BrFLC1 biallelic CAPS marker and a BrFLC2 multiallelic SSR marker. The two markers were significantly associated with flowering time, but their effects were confined to certain morphotypes and (or) alleles. Based on these results, we discuss the optimal design for an association mapping population and the need to fix the heterogeneous accessions to facilitate phenotyping and genotyping.     

The patterns of population differentiation in a <Emphasis Type="Italic">Brassica rapa</Emphasis> core collection

With the recent advances in high throughput profiling techniques the amount of genetic and phenotypic data available has increased dramatically. Although many genetic diversity studies combine morphological and genetic data, metabolite profiling has yet to be integrated into these studies. For our study we selected 168 accessions representing the different morphotypes and geographic origins of Brassica rapa. Metabolite profiling was performed on all plants of this collection in the youngest expanded leaves, 5 weeks after transplanting and the same material was used for molecular marker profiling. During the same season a year later, 26 morphological characteristics were measured on plants that had been vernalized in the seedling stage. The number of groups and composition following a hierarchical clustering with molecular markers was highly correlated to the groups based on morphological traits (r = 0.420) and metabolic profiles (r = 0.476). To reveal the admixture levels in B. rapa, comparison with the results of the programme STRUCTURE was needed to obtain information on population substructure. To analyze 5546 metabolite (LC–MS) signals the groups identified with STRUCTURE were used for random forests classification. When comparing the random forests and STRUCTURE membership probabilities 86% of the accessions were allocated into the same subgroup. Our findings indicate that if extensive phenotypic data (metabolites) are available, classification based on this type of data is very comparable to genetic classification. These multivariate types of data and methodological approaches are valuable for the selection of accessions to study the genetics of selected traits and for genetic improvement programs, and additionally provide information on the evolution of the different morphotypes in B. rapa.     

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.     

SSR and morphological trait based population structure analysis of 130 diverse flax (Linum usitatissimum L.) accessions

A total of 130 flax accessions of diverse morphotypes and worldwide origin were assessed for genetic diversity and population structure using 11 morphological traits and microsatellite markers (15 gSSRs and 7 EST–SSRs). Analysis performed after classifying these accessions on the basis of plant height, branching pattern, seed size, Indian/foreign origin into six categories called sub-populations viz. fibre type exotic, fibre type indigenous, intermediate type exotic, intermediate type indigenous, linseed type exotic and linseed type indigenous. The study assessed different diversity indices, AMOVA, population structure and included a principal coordinate analysis based on different marker systems. The highest diversity was exhibited by gSSR markers (SI = 0.46; He = 0.31; P = 85.11). AMOVA based on all markers explained significant difference among fibre type, intermediate type and linseed type populations of flax. In terms of variation explained by different markers, EST-SSR markers (12%) better differentiated flax populations compared to morphological (9%) and gSSR (6%) markers at P = 0.01. The maximum Nei's unbiased genetic distance (D = 0.11) was observed between fibre type and linseed type exotic sub-populations based on EST-SSR markers. The combined structure analysis by using all markers grouped Indian fibre type accessions (63.4%) in a separate cluster along with the Indian intermediate type (48.7%), whereas Indian accessions (82.16%) of linseed type constituted an independent cluster. These findings were supported by the results of the principal coordinate analysis. Morphological markers employed in the study found complementary with microsatellite based markers in deciphering genetic diversity and population structure of the flax germplasm.     

Evaluation of Genetic Diversity and Development of a Core Collection of Wild Rice (Oryza rufipogon Griff.) Populations in China

Common wild rice (Oryza rufipogon Griff.), the progenitor of Asian cultivated rice (O. sativa L.), is endangered due to habitat loss. The objectives of this research were to evaluate the genetic diversity of wild rice species in isolated populations and to develop a core collection of representative genotypes for ex situ conservation. We collected 885 wild rice accessions from eight geographically distinct regions and transplanted these accessions in a protected conservation garden over a period of almost two decades. We evaluated these accessions for 13 morphological or phenological traits and genotyped them for 36 DNA markers evenly distributed on the 12 chromosomes. The coefficient of variation of quantitative traits was 0.56 and ranged from 0.37 to 1.06. SSR markers detected 206 different alleles with an average of 6 alleles per locus. The mean polymorphism information content (PIC) was 0.64 in all populations, indicating that the marker loci have a high level of polymorphism and genetic diversity in all populations. Phylogenetic analyses based on morphological and molecular data revealed remarkable differences in the genetic diversity of common wild rice populations. The results showed that the Zengcheng, Gaozhou, and Suixi populations possess higher levels of genetic diversity, whereas the Huilai and Boluo populations have lower levels of genetic diversity than do the other populations. Based on their genetic distance, 130 accessions were selected as a core collection that retained over 90% of the alleles at the 36 marker loci. This genetically diverse core collection will be a useful resource for genomic studies of rice and for initiatives aimed at developing rice with improved agronomic traits.     

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.     

Morphology,Carbohydrate Composition and Vernalization Response in a Genetically Diverse Collection of Asian and European Turnips (Brassica rapa subsp. rapa)

Brassica rapa displays enormous morphological diversity, with leafy vegetables, turnips and oil crops. Turnips (Brassica rapa subsp. rapa) represent one of the morphotypes, which form tubers and can be used to study the genetics underlying storage organ formation. In the present study we investigated several characteristics of an extensive turnip collection comprising 56 accessions from both Asia (mainly Japanese origin) and Europe. Population structure was calculated using data from 280 evenly distributed SNP markers over 56 turnip accessions. We studied the anatomy of turnip tubers and measured carbohydrate composition of the mature turnip tubers of a subset of the collection. The variation in 16 leaf traits, 12 tuber traits and flowering time was evaluated in five independent experiments for the entire collection. The effect of vernalization on flowering and tuber formation was also investigated. SNP marker profiling basically divided the turnip accessions into two subpopulations, with admixture, generally corresponding with geographical origin (Europe or Asia). The enlarged turnip tuber consists of both hypocotyl and root tissue, but the proportion of the two tissues differs between accessions. The ratio of sucrose to fructose and glucose differed among accessions, while generally starch content was low. The evaluated traits segregated in both subpopulations, with leaf shape, tuber colour and number of shoots per tuber explaining most variation between the two subpopulations. Vernalization resulted in reduced flowering time and smaller tubers for the Asian turnips whereas the European turnips were less affected by vernalization.     

The use of molecular markers for germplasm management in a French olive collection

With more than 100 accessions, the CBNMP olive collection includes a major part of the French germplasm. We used molecular markers to characterise all accessions and to study genetic relationships between cultivars. Firstly, 497 olive trees were genotyped using 32 RAPD markers. We identified 114 RAPD profiles and detected several cases of mislabelling, synonymy and homonymy. Secondly, for each RAPD profile, one tree was analysed using mtDNA RFLPs to determine the cytoplasmic lineage of each cultivar and using five nuclear SSR loci. French germplasm displayed ME1, MOM and MCK mitotypes with ME1 prevailing (84%). Based on SSR markers, we revealed a slight differentiation between French cultivars growing in the West and the East side of the Rh?ne Valley. This study allowed us to construct a molecular data-base for the reference collection and to analyse genetic diversity for further prospecting, and for introducing new olive accessions.