Simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop

Cassava (Manihot esculenta) is an allogamous, vegetatively propagated, Neotropical crop that is also widely grown in tropical Africa and Southeast Asia. To elucidate genetic diversity and differentiation in the crop's primary and secondary centers of diversity, and the forces shaping them, SSR marker variation was assessed at 67 loci in 283 accessions of cassava landraces from Africa (Tanzania and Nigeria) and the Neotropics (Brazil, Colombia, Peru, Venezuela, Guatemala, Mexico and Argentina). Average gene diversity (i.e., genetic diversity) was high in all countries, with an average heterozygosity of 0.5358 ± 0.1184. Although the highest was found in Brazilian and Colombian accessions, genetic diversity in Neotropical and African materials is comparable. Despite the low level of differentiation [F_st(theta) = 0.091 ± 0.005] found among country samples, sufficient genetic distance (1-proportion of shared alleles) existed between individual genotypes to separate African from Neotropical accessions and to reveal a more pronounced substructure in the African landraces. Forces shaping differences in allele frequency at SSR loci and possibly counterbalancing successive founder effects involve probably spontaneous recombination, as assessed by parent-offspring relationships, and farmer-selection for adaptation.Communicated by H.C. Becker     

Variation of isozyme patterns among Arachis species

The genus Arachis contains a large number of species and undescribed taxa with patterns of genetic variation that are little understood. The objectives of this investigation were to estimate genetic diversity among species of Arachis by utilizing electrophoretic techniques and to establish the potential for use of isozymes as markers for germplasm introgression. One-hundred-and-thirteen accessions representing six of the seven sections of the genus were analyzed for isozyme variation of 17 enzymes. Section Rhizomatosae species were not included because they produce very few seeds. Seeds were macerated and the crude extract was used for starch-gel electrophoretic analyses. Although the cultivated species has few polymorphic isozymes, the diploid species are highly variable and two-to-six bands were observed for each isozyme among accessions. Because of the large number of isozyme differences between A. hypogaea and A. batizocoi (the presumed donor of the B genome), this species can no longer be considered as a progenitor of the cultivated peanut. Seed-to-seed polymorphisms within many accessions were also observed which indicate that germplasm should be maintained as bulk seed lots, representative of many individuals, or as lines from individual plants from original field collections. The area of greatest interspecific genetic diversity was in Mato Grosso, Brazil; however, the probability of finding unique alleles from those observed in A. hypogaea was greatest in north, north-central, south and southeast Brazil. The large number of polymorphic loci should be useful as genetic markers for interspecific hybridization studies.     

Genetic diversity of traditional South American Landraces of Cassava (Manihot esculenta Crantz): an analysis using microsatellites

The extent and structure of the genetic variability of traditional varieties of cassava (Manihot esculenta Crantz) have been little documented, despite considerable evidence for this crop? great varietal diversity in traditional agroecosystems. We used microsatellite markers to assess the genetic structure of traditional landraces of sweet and bitter cassava collected from five South American sites. As reference, we used a sample of 38 accessions from a world collection of cultivated cassava. For a total of 10 loci examined, we found 15 alleles that were not represented in this sample. Ten of these had been previously detected in wild Manihot species. The geographical structure of genetic variability was weak, but the genetic differentiation between bitter and sweet landraces was significant, suggesting that each form had evolved separately after domestication. Our results showed that traditional landraces form an important source of genetic diversity and merit more attention from managers of crop genetic resources.     

Microsatellite Polymorphisms in Cassava Landraces from the Cerrado Biome, Mato Grosso do Sul, Brazil

Using nine microsatellite loci, we investigated genetic structure and diversity in 83 Brazilian cassava accessions, including several landraces, in the Cerrado biome in Mato Grosso do Sul, Brazil. All nine loci were polymorphic, averaging 6.00 alleles per locus. Treating each of seven municipalities as a cassava group or population, they averaged 3.5 alleles per locus, with 97% polymorphic loci, high values for observed heterozygosity (0.32) and gene diversity (0.56). Total genetic variability was high (0.668), and most of this genetic variability was concentrated within municipalities (0.577). Cluster and structure analyses divided accessions into two major clusters or populations (K = 2). Also, a significant genetic versus geographic correlation was found (r = 0.4567; P < 0.0260). Migratory routes in the Cerrado are considered main contributors to the region’s high cassava diversity and spatial genetic structure, amplifying interactions between traditional farmers and the evolutionary dynamics of this crop.     

Restriction fragment variation in the nuclear and chloroplast genomes of cultivated and wild Sorghum bicolor

Summary Fifty-six accessions of cultivated and wild sorghum were surveyed for genetic diversity using 50 low-copy-number nuclear DNA sequence probes to detect restriction fragment length polymorphisms (RFLPs). These probes revealed greater genetic diversity in wild sorghum than in cultivated sorghum, including a larger number of alleles per locus and a greater portion of polymorphic loci in wild sorghum. In comparison to previously published isozyme analyses of the same accessions, RFLP analysis reveals a greater number of alleles per locus. Furthermore, many RFLP alleles have frequencies between 0.25–0.75, while the vast majority of isozyme alleles are either rare (< 0.25) or near fixation (> 0.75). Correlations between genetic and geographic distances among the accessions were stronger when calculated with RFLP than with isozyme data. Systematic relationships revealed by nuclear and chloroplast restriction site analysis indicate that cultivated sorghum is derived from the wild ssp. arundinaceum. The portion of the wild gene pool most genetically similar to the cultivars is from central-northeastern Africa. Previous published data also suggested that this is most likely the principal area of domestication of sorghum. Introgression between wild and cultivated sorghum was inferred from disconcordant relationships shown by nuclear and chloroplast DNA markers. Introgression apparently occurs infrequently enough that the crop and its wild relatives maintain distinct genetic constitutions.     

Genetic Diversity and Core Collection Evaluations in Common Wheat Germplasm from the Northwestern Spring Wheat Region in China

Fluorescence microsatellite markers were employed to reveal genetic diversity of 340 wheat accessions consisting of 229 landraces and 111 modern varieties from the Northwest Spring Wheat Region in China. The 340 accessions were chosen as candidate core collections for wheat germplasm in this region. A core collection representing the genetic diversity of these accessions was identified based on a cluster dendrogram of 78 SSR loci. A total of 967 alleles were detected with a mean of 13.6 alleles (5–32) per locus. Mean PIC was 0.64, ranged from 0.05 to 0.91. All loci were distributed relatively evenly in the A, B and D wheat genomes. Mean genetic richness of A, B and D genomes for both landraces and modern varieties was B > A > D. However, mean genetic diversity indices of landraces changed to B > D > A. As a whole, genetic diversity of the landraces was considerably higher than that of the modern varieties. The big difference of genetic diversity indices in the three genomes suggested that breeding has exerted greater selection pressure in the D than the A or B genomes in this region. Changes of allelic proportions represented in the proposed core collection at different sampling scales suggested that the sampling percentage of the core collection in the Northwest Spring Wheat Region should be greater than 4% of the base collection to ensure that more than 70% of the variation is represented by the core collection. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Optimal sampling strategy and core collection size of Andean tetraploid potato based on isozyme data - a simulation study

Selection of an appropriate sampling strategy is an important prerequisite to establish core collections of appropriate size in order to adequately represent the genetic spectrum and maximally capture the genetic diversity in available crop collections. We developed a simulation approach to identify an optimal sampling strategy and core-collection size, using isozyme data from a CIP germplasm collection on an Andean tetraploid potato. Five sampling strategies, constant (C), proportional (P), logarithmic (L), square-root (S) and random (R), were tested on isozyme data from 9,396 Andean tetraploid potato accessions characterized for nine isozyme loci having a total of 38 alleles. The 9,396 accessions, though comprising 2,379 morphologically distinct accessions, were found to represent 1,910 genetically distinct groups of accessions for the nine isozyme loci using a sort-and-duplicate-search algorithm. From each group, one accession was randomly selected to form a genetically refined entire collection (GREC) of size 1,910. The GREC was used to test the five sampling strategies. To assess the behavior of the results in repeated sampling, k = 1,500 and 5,000 independent random samples (without replacement) of admissible sizes n = 50(50)1,000 for each strategy were drawn from GREC. Allele frequencies (AF) for the 38 alleles and locus heterozygosity (LH) for the nine loci were estimated for each sample. The goodness of fit of samples AF and LH with those from GREC was tested using the L² test. A core collection of size n = 600, selected using either the P or the R sampling strategy, was found adequately to represent the GREC for both AF and LH. As similar results were obtained at k = 1,500 and 5,000, it seems adequate to draw 1,500 independent random samples of different sizes to test the behavior of different sampling strategies in order to identify an appropriate sampling approach, as well as to determine an optimal core collection size.     

Comparison of different methods to construct a core germplasm collection in woody perennial species with simple sequence repeat markers. A case study in cherimoya (Annona cherimola, Annonaceae), an underutilised subtropical fruit tree species

Although molecular markers are becoming the tool of choice to develop core collections in plants, the examples of their use in woody perennial species are very scarce. In this work, we used simple sequence repeat (SSR) marker data to develop a core collection in an underutilised subtropical fruit tree species, cherimoya ( Annona cherimola , Annonaceae), from an initial collection of 279 genotypes from different countries. We compared six alternative allocation methods to construct the core collection, four not based upon the similarity dendrogram [random sampling, maximisation strategy (M strategy) and simulated annealing algorithm maximising both genetic diversity and number of SSR alleles] and two based on dendrogram data (logarithmic strategy and stepwise clustering). The diversity maintained in each subset was compared with that present in the entire collection. The results obtained indicate that the use of SSRs together with the M strategy is the most efficient method to develop a core collection in cherimoya. In the best subset, with 40 accessions, all the SSR alleles present in the whole collection were recovered and no significant differences in frequency distribution of alleles for any of the loci studied or in variability parameters ( H _O, H _E) were recorded between the core and the whole collection.     

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.     

Mining EST-Derived SSR Markers to Assess Genetic Diversity in Cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is a very important staple and industrial crop in tropical and subtropical regions of the world. The paucity of markers is a serious limitation in marker-assisted breeding. A total of 35,992 expressed sequence tags (ESTs) from cassava, which were clustered in 13,173 unigenes, were used in this study. A total of 1,889 microsatellites were identified, with an average density of one simple sequence repeats (SSRs) every 4.40 kb. Of the 1,058 designed EST-SSRs from cultivars SC06, TMS60444, and W14, 431 were polymorphic. Then, 31 randomly selected EST-SSRs from the 431 polymorphic EST-SSRs were used to evaluate the genetic diversity of 76 cassava accessions. A total of 93 alleles were identified, and the number detected for each EST-SSR ranged from one to four. Based on the 93 alleles, the 76 cassava accessions could be classified into six groups, and the genetic similarity coefficient ranged from 0.55 to 0.94. This study demonstrated the potential of EST-derived SSRs in cassava. The resources developed in this study enriched the available molecular markers for cassava.     

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.     

Characterization of genetic diversity in core collection accessions of wild barley,Hordeum vulgare ssp. spontaneum

Genetic variability in the 143 core accessions of wild barley, Hordeum vulgare ssp. spontaneum, was assessed by allozyme analysis. A total of 34 alleles were detected at ten isozyme loci. All loci were polymorphic except Pgd-1, which was monomorphic. Est-2 and Est-4 were the most diverse loci, with genetic diversity values of 0.747 and 0.686, respectively. The comparison of the results with those of previous studies indicates that all alleles occurring in cultivated and wild barley are observed in this set of the wild Barley Core Collection. Only one allele (Pgd-1 Tj) was absent. It is noteworthy that one new allele at the Ndh-2 locus and another new allele at Aco-2 locus were first detected in the present study. Nine of the 34 alleles were rare and detected only in one to four accessions. The genetic similarities among the 143 accessions ranged from 0.18 to 1.00. Data analysis based on clustering and principal coordinate analysis showed that a high level of genetic variability exists in this set of core accessions, and indicated that some duplication probably exists in this set core based on the present study.     

Developing core collections to optimize the management and the exploitation of diversity of the coffee Coffea canephora

The management of diversity for conservation and breeding is of great importance for all plant species and is particularly true in perennial species, such as the coffee Coffea canephora. This species exhibits a large genetic and phenotypic diversity with six different diversity groups. Large field collections are available in the Ivory Coast, Uganda and other Asian, American and African countries but are very expensive and time consuming to establish and maintain in large areas. We propose to improve coffee germplasm management through the construction of genetic core collections derived from a set of 565 accessions that are characterized with 13 microsatellite markers. Core collections of 12, 24 and 48 accessions were defined using two methods aimed to maximize the allelic diversity (Maximization strategy) or genetic distance (Maximum-Length Sub-Tree method). A composite core collection of 77 accessions is proposed for both objectives of an optimal management of diversity and breeding. This core collection presents a gene diversity value of 0.8 and exhibits the totality of the major alleles (i.e., 184) that are present in the initial set. The seven proposed core collections constitute a valuable tool for diversity management and a foundation for breeding programs. The use of these collections for collection management in research centers and breeding perspectives for coffee improvement are discussed.     

Characterization and multiplexing of EST-SSR primers in Cynodon (Poaceae) species1

? Premise of the study: Cynodon species are multiple-use grasses that display varying levels of adaptation to biotic and abiotic stress. Previously identified EST-SSR primers were characterized and multiplexed to assess the level of genetic diversity present within a collection of almost 1200 Cynodon accessions from across Australia. ? Methods and Results: Two multiplex reactions were developed comprising a total of 16 EST-SSR markers. All SSR markers amplified across different Cynodon species and different levels of ploidy. The number of alleles ranged from one to eight per locus and the total number of alleles for the germplasm collection was 79. ? Conclusions: The 16 markers show sufficient variation for the characterization of Cynodon core collections and analysis of population genetic diversity in Cynodon grasses.     

A worldwide bread wheat core collection arrayed in a 384-well plate

Bread wheat (Triticum aestivum), one of the world’s major crops, is genetically very diverse. In order to select a representative sample of the worldwide wheat diversity, 3,942 accessions originating from 73 countries were analysed with a set of 38 genomic simple sequence repeat (SSR) markers. The number of alleles at each locus ranged from 7 to 45 with an average of 23.9 alleles per locus. The 908 alleles detected were used together with passport data to select increasingly large sub-samples that maximised both the number of observed alleles at SSR loci and the number of geographical origins. A final core of 372 accessions (372CC) was selected with this M strategy. All the different geographical areas and more than 98% of the allelic diversity at the 38 polymorphic loci were represented in this core. The method used to build the core was validated, by using a second set of independent markers [44 expressed sequence tag (EST)-SSR markers] on a larger sample of 744 accessions: 96.74% of the alleles observed at these loci had already been captured in the 372CC. So maximizing the diversity with a first set of markers also maximised the diversity at a second independent set of locus. To relate the genetic structure of wheat germplasm to its geographical origins, the two sets of markers were used to compute a dissimilarity matrix between geographical groups. Current worldwide wheat diversity is clearly divided according to wheat’s European and Asian origins, whereas the diversity within each geographical group might be the result of the combined effects of adaptation of an initial germplasm to different environmental conditions and specific breeding practices. Seeds from each accession of the 372CC were multiplied and are now available to the scientific community. The genomic DNA of the 372CC, which can be entirely contained in a 384-deep-well storage plate, will be a useful tool for future studies of wheat genetic diversity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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

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

粤东橄榄资源核心种质取样方案的研究

为研究基于分子标记数据构建橄榄(Canarium album L.)核心种质的取样方案,以粤东地区64份橄榄种质材料的ISSR分析结果为基础,分别利用SM和Nei&Li遗传距离,采用UPGMA聚类法进行多次聚类随机取样,比较了不同分组情况下P、S、L和G等取样策略对核心种质构建的影响。结果表明,通过比较不同样品群的多态性位点数、多态性位点百分率、观测等位基因数、有效等位基因数、Nei’s遗传多样性指数和Shannon’s信息指数等参数,最终选择根据Nei&Li法计算遗传距离,G策略取样得到的16个样品作为核心种质。该核心种质保留了初始种质25%的样品,多态性位点和多态性位点百分率保留率分别为92.93%和98.31%,观测等位基因数、有效等位基因数、Nei’s遗传多样性指数、Shannon’s信息指数的保留率分别为9.26%、102.56%、107.39%和106.29%。因此,按该方案进行取样的核心种质可以较好地代表原有种质库的遗传多样性。     

Genetic diversity analysis of common beans based on molecular markers

A core collection of the common bean (Phaseolus vulgaris L.), representing genetic diversity in the entire Mexican holding, is kept at the INIFAP (Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico) Germplasm Bank. After evaluation, the genetic structure of this collection (200 accessions) was compared with that of landraces from the states of Oaxaca, Chiapas and Veracruz (10 genotypes from each), as well as a further 10 cultivars, by means of four amplified fragment length polymorphisms (AFLP) +3/+3 primer combinations and seven simple sequence repeats (SSR) loci, in order to define genetic diversity, variability and mutual relationships. Data underwent cluster (UPGMA) and molecular variance (AMOVA) analyses. AFLP analysis produced 530 bands (88.5% polymorphic) while SSR primers amplified 174 alleles, all polymorphic (8.2 alleles per locus). AFLP indicated that the highest genetic diversity was to be found in ten commercial-seed classes from two major groups of accessions from Central Mexico and Chiapas, which seems to be an important center of diversity in the south. A third group included genotypes from Nueva Granada, Mesoamerica, Jalisco and Durango races. Here, SSR analysis indicated a reduced number of shared haplotypes among accessions, whereas the highest genetic components of AMOVA variation were found within accessions. Genetic diversity observed in the common-bean core collection represents an important sample of the total Phaseolus genetic variability at the main Germplasm Bank of INIFAP. Molecular marker strategies could contribute to a better understanding of the genetic structure of the core collection as well as to its improvement and validation.     

Genetic diversity and association mapping in a collection of selected Chinese soybean accessions based on SSR marker analysis

For broadening the narrow genetic base of modern soybean cultivars, 159 accessions were selected from the Chinese soybean collection which contained at least one of seven important agronomic traits: resistance to soybean cyst nematode (SCN) or soybean mosaic virus (SMV), tolerance to salt, cold, or drought, high seed oil content or high protein content. Genetic diversity evaluation using 55 microsatellite loci distributed across the genome indicated that a large amount of genetic diversity (0.806) and allelic variation (781) were conserved in this selected set, which captured 65.6% of the alleles present in Chinese soybean collection (1,863 accessions). On average, 9.4 rare alleles (frequency <5%) per locus were present, which were highly informative. Using model-based Bayesian clustering in STRUCTURE we distinguished four main clusters and a set of accessions with admixed ancestry. The four clusters reflected different geographic regions of origin of the accessions. Since the clusters were also clearly different with respect to the seven agronomic traits, the inferred population structure was introduced when association analysis was conducted. A total of 21 SSR markers on 16 chromosomes were identified as significantly (P < 0.01) associated with high oil content (6), high protein content (1), drought tolerance (5), SCN resistance (6) and SMV resistance (3). Twelve of these markers were located in or near previously identified quantitative trait loci (QTL). The results for both genetic relationship and trait-related markers will be useful for effective conservation and utilization of soybean germplasm.     

辣椒种质遗传多样性的RAPD和ISSR及其表型数据分析

用RAPDI、SSR分子标记及28个表型性状数据对辣椒属5个栽培种的13份材料进行了分析,结果表明:23条RAPD引物共扩增出209条带,平均每个引物扩增出9.09条,多态性位点比率为83.73%;16条ISSR引物共扩增出94条带,平均每个引物扩增出5.88条,多态性位点比率为79.79%.与RAPD相比,ISSR标记检测到的有效等位基因数(Ne)及Shannon多样性指数(I)、遗传离散度(Ht)和遗传分化系数(Gst)等遗传多样性参数都较大,多态性位点比例在亲缘关系较近的一年生辣椒(Capsicum annuum)种内较高,说明ISSR有更高的多态性检测效率,并且适合亲缘关系较近的种群间遗传多样性分析.基于RAPDI、SSR的聚类与基于表型数据的聚类之间存在极显著正相关,且都能将C.annuum与其它栽培种区分开来.