Evaluation of genetic diversity in a Camelina sativa (L.) Crantz collection using microsatellite markers and biochemical traits

A genomic DNA library enriched with GA/TC repeats from Camelina sativa variety Calena has been analysed. After sequencing of about 200 randomly selected clones, approximately 60 % of them showed to contain simple or compound microsatellites with a high number of repeats. Among all microsatellite markers analysed 15 primer pairs amplified polymorphic fragments. Forty C. sativa accessions of different origin were genotyped with 15 microsatellite markers that generated 134 alleles with an average of 8.93 alleles per locus. The observed heterozygosity (Ho) among the accessions ranged from 0.0 to 0.15 with an average of 0.0370, whereas the average of expected heterozygosity (He) among accessions was 0.2769. The analysis of the average total heterozygosity (H_T = 0.651), the intrapopulation genetic diversity (H_S = 0.260), the interpopulation genetic diversity (D_ST = 0.391) and the coefficient of genetic differentiation among populations (G_ST = 0.574) demonstrated that 57.4 % of the genetic diversity is among the accessions, while 42.6 % resides within them. Phylogenetic tree of the 40 C. sativa accessions was constructed based on Nei’s genetic distance. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram shows, except for CAM108 and CAM170, a clear discrimination among C. sativa accessions grouping them in five subgroups. ANOVA analysis indicates significant differences in some biochemical and agronomic parameters among the C. sativa accessions grouped according to Nei’s genetic distance. The result of the Tukey HSD test demonstrated that the A4 subgroup showed a significant higher TWS and linoleic acid (LA) content, while the subgroup A1 showed a significant higher linolenic and lower LA content compared to the remaining groups.     

Genetic diversity analysis in Phaseolus vulgaris L. using morphological traits

Common bean (Phaseolus vulgaris) is an important export crop in Kyrgyzstan since the end of the twentieth century. Genetic diversity analysis of common bean populations is useful for breeding programs, as it helps to select genetic material to be used for further crossing. Twenty-seven common bean accessions were analyzed using 13 qualitative morphological traits. In some cases, obtained morphological data were combined with previously published results based on microsatellite markers. The similarity matrices generated from the molecular and morphological data were significantly correlated (r = 0.49, P < 0.01). Cluster analyses based on Dice’s similarity coefficient were constructed based on morphological data and the combined data set of morphology and microsatellite, and both grouped the 27 accessions according to their origin: 15 belonged to the Andean and 12 to the Mesoamerican gene pool. On average, the Andean accessions were less diverse than the Mesoamerican accessions. The average diversity based on the Shannon diversity index for the 13 qualitative morphological traits was 0.05. Overall, this study revealed that qualitative morphological markers are efficient in assigning modern cultivars to their gene pools of origin.     

Water yam (Dioscorea alata L.) diversity pattern in Brazil: an analysis with SSR and morphological markers

Substantial progress was made in the last decade in understanding the diversity in Dioscorea species. However, most of the studies so far concentrated in the ‘yam belt in Africa’. We present a genetic diversity and structure analysis among commercial and local varieties of water yam (Dioscorea alata L.) in Brazil using microsatellite and morphological markers. Twelve microsatellite primers were used to generate DNA profiles of 72 local varieties and 17 commercial accessions of water yam collected in four different regions in Brazil. Also, four morphological traits were evaluated on individual plants under field conditions. The morphological characterization showed considerable diversity. High polymorphism was found with 100 % polymorphism observed for 11 primers and a discriminating power value of 0.92, on average. We did not observe a strong population structure among sampling regions, while most of the genetic diversity was concentrated within regions (95.9 %). Analysis of the relationship between accessions did not clearly separate the local and commercial genotypes. However, the molecular analyses of D. alata showed high intraspecific diversity in local accessions from different regions in Brazil, with the highest Shannon index value (H′ = 0.41) for the Southeast region. These results confirmed an admixture of accessions in all sampling regions, consistent with the lack of a significant correlation between geographic and genetic distances, suggesting that farmers exchanged water yam materials extensively. The genetic diversity can be explained by the result of a continuous exchange of accessions by farmers throughout Brazil.     

Variations in morphological and agronomic traits among Jerusalem artichoke (Helianthus tuberosus L.) accessions

Jerusalem artichoke is a diversely-utilized crop. Selection for high yield, inulin content and other economically important traits are useful for improving this crop. The objectives of the present study were to evaluate genetic variability for qualitative and quantitative traits among Jerusalem artichoke accessions and to identify different groups of accessions using morphological and agronomic traits. Seventy-nine accessions were evaluated in a randomized complete block design with two replications in the late rainy season 2008, the early rainy season 2009 and the late rainy season 2009 at Khon Kaen University agronomy farm, Thailand. Morphological and agronomic characteristics were evaluated for genetic variations. High variations were found among Jerusalem artichoke accessions for qualitative and quantitative characters, and selection for these characters is possible. High variations were observed for tuber width, number of tubers/plant, biomass, fresh tuber yield and tuber size. Correlation coefficient between fresh tuber yield and tuber size was positive and significant (0.58, P ≤ 0.01). Improvement of tuber size is a means to improve yield and tuber quality. Based on morphological and agronomic characteristics, Jerusalem artichoke accessions were clustered into four distinct groups (R² = 0.88). These groups may be used as parental material to generate progenies for further improvement of this crop. This information will enable breeders to make informed decisions about possible heterotic groups for their breeding programs and germplasm conservation.     

Genetic diversity and relationships of ancient Chinese fir (<Emphasis Type="Italic">Cunninghamia lanceolata</Emphasis>) genotypes revealed by sequence-related amplified polymorphism markers

Some more than a century old Chinese fir genotypes with high biomass that are now rare in China were historically called “the king of Chinese fir”. The genetic diversity and relationships of those ancient Chinese fir genotypes were investigated using morphological analysis and sequence-related amplified polymorphism markers. The morphological analysis indicated that the tree volume parameter had the maximum variable coefficient value (70.2 %). In total, 18 selected primers generated 154 bands, and 150 bands were polymorphic (97.4 %). Higher values of genetic diversity parameters (h = 0.3593, I = 0.5283) were maintained at the species level, indicating that the ancient Chinese fir in China has retained a relatively high level of genetic diversity. AMOVA indicated that 73.16 % of the variation resided within provenances. The UPGMA dendrogram and genetic structure analysis identified 3 major clusters and grouped the genotypes in agreement with their geographic origins. A Mantel test revealed a significant correlation between genetic distances and geographic distances among the genotypes (r = 0.4275, p < 0.01). Overall, we recommend a combination of conservation measures including a germplasm repository and the implementation of in situ conservation.     

Genetic diversity in farmer grown spring barley material from Kyrgyzstan

Barley (Hordeum vulgare L.) is one of the economically important cereal crops grown in the highlands of the Kyrgyz Republic. In marginal agricultural areas of the country barley is considered as irreplaceable and very suitable fodder crop for livestock. In this study the genetic diversity of currently grown farmers’ spring barley material from Naryn and Issik-Kul provinces is described. In order to capture maximum diversity present in farmer field’s different morphological types were collected. The 22 spring barley accessions collected in 2008 were described morphologically and analysed using fourteen simple sequences repeat (SSR) markers. Most material was two-rowed barley, 78 %, whereas six-rowed barley was only 22 %. The spike colours present were light yellow, dark yellow, brown and violet. The material varied also for such characters as length of spike and number of kernels per spike. For the SSR loci 129 alleles were detected with an average of 9.3 alleles per locus and a genetic diversity with an average of h = 0.721 per locus. Based on analysis of molecular variance the 71.3 % of genetic diversity accounted for within accession variation, whereas 27.4 % for among accession variation. The variation between provinces was not significantly different. A group of two-rowed barley was identified that was more closely related to six-rowed barley than to the rest of the two-rowed barley material. Two-rowed material currently grown in Kyrgyzstan is more diverse than six-rowed barley (h = 0.65, A = 8.8 vs. h = 0.59, A = 3.1). From this study we could conclude that the materials nowadays grown by farmers are mixtures. Since there are no geographic differentiation between these mixtures suggests that materials is frequently inter-exchanged between farmers and are most probably not landrace material. The question concerning the origin of these mixtures remains unsolved and further investigation will be carried out to identify sources of variation in farmers’ field.     

Morphological,SSR and ploidy analysis of water yam (<Emphasis Type="Italic">Dioscorea alata</Emphasis> L.) accessions for utilization of aerial tubers as planting materials

The high cost and supply shortage of seed yam propagules for planting are major constraints in yam production. In the water yam (Dioscorea alata L.), aerial tubers have potential as alternative sources of planting material. In this study, we investigated morphological, molecular and ploidy variation across multiple aerial tuber producing accessions of Dioscorea alata. Initial screening of over 800 accessions from the International Institute of Tropical Agriculture germplasm collection for aerial tuber production identified a subset (15 %) of accessions, which produced aerial tubers. The aerial tuber producing accessions (along with 18 non-aerial tuber accessions) were further characterized for phenotypic and ploidy variation. In addition, using SSR markers we characterized the genetic diversity amongst all of the aerial tuber producing accessions, along with six non-aerial tuber producing accessions. Multiple Correspondence Analysis (MCA) using morphological data grouped the accessions according to their aerial tuber production. The aerial tuber production characteristics of accessions were associated with phenotypic variables and ploidy levels. The MCA analysis revealed three main groups consisting of; Group 1) all non aerial tuber producing accessions (n = 15), hastate leaf shape, less or no anthocyanin pigmentation and diploid (2n = 2x = 40), Group 2) group with some aerial tuber producing accessions, different extent of anthocyanin pigmentation, sagittate leaf shape, mainly diploid (n = 44) and three triploid (2n = 3x = 60) and 3) a group where all individuals bear aerial tuber, cordate leaf shape, intermediate anthocyanin pigmentation and majority (n = 74) tetraploid (2n = 4x = 80) and three triploid individuals. Aerial tuber production may be subject to a genome dosage effect as an increase in aerial tuber production was associated with increased ploidy level. For instance, tetraploid plants produce more aerial tubers per sprout than either triploids or diploids. Principal coordinate analysis based on SSR markers using Jaccard’s coefficient also revealed distinct groups associated with the pattern of aerial tuber formation, leaf shape and anthocyanin pigmentation. Overall our study indicates the usefulness of combining SSR markers, ploidy level and phenotypic data for identification and classification of Dioscorea alata accessions according to their extent of aerial tuber production.     

Spatially structured genetic diversity of the Amerindian yam (Dioscorea trifida L.) assessed by SSR and ISSR markers in Southern Brazil

Dioscorea trifida L. (Dioscoreaceae) is among the economically most important cultivated Amerindian yam species, whose origin and domestication are still unresolved issues. In order to estimate the genetic diversity maintained by traditional farmers in Brazil, 53 accessions of D. trifida from 11 municipalities in the states of São Paulo, Santa Catarina, Mato Grosso and Amazonas were characterized on the basis of eight Simple Sequence Repeats (SSR) and 16 Inter Simple Sequence Repeats (ISSR) markers. The level of polymorphism among the accessions was high, 95 % for SSR and 75.8 % for ISSR. The SSR marker showed higher discrimination power among accessions compared to ISSR, with D parameter values of 0.79 and 0.44, respectively. Although SSR and ISSR markers led to dendrograms with different topologies, both separated the accessions into three main groups: I—Ubatuba-SP; II—Iguape-SP and Santa Catarina; and III—Mato Grosso. The accessions from Amazonas State were classified in group II with SSR and in a separate group with ISSR. Bayesian and principal coordinate analyzes conducted with both molecular markers corroborated the classification into three main groups. Higher variation was found within groups in the AMOVA analysis for both markers (66.5 and 60.6 % for ISSR and SSR, respectively), and higher Shannon diversity index was found for group II with SSR. Significant but low correlations were found between genetic and geographic distances (r = 0.08; p = 0.0007 for SSR and r = 0.16; p = 0.0002 for ISSR). Therefore, results from both markers showed a slight spatially structured genetic diversity in D. trifida accessions maintained by small traditional farmers in Brazil.     

Genetic diversity and structure of natural and agronomic switchgrass (Panicum virgatum L.) populations

Panicum virgatum L. (switchgrass) is an obligate outcrossing C₄ perennial prairie grass currently being pursued for the production of lignocellulosic ethanol. Commercial production of switchgrass for bioenergy has increased substantially in the United States. Understanding the degree of native genetic diversity within and among switchgrass populations will facilitate effective germplasm improvement, conservation, and management programs. In this study, the genetic diversity and differentiation among natural and agronomic switchgrass populations were analyzed at the molecular level by using random amplified polymorphic (RAPD) DNA markers. The mean genetic diversity among populations ranged from 0.051 ± 0.136 to 0.243 ± 0.214 and the mean genetic similarity among all the switchgrass populations was 0.775. The clustering pattern of switchgrass populations grouped the individuals based on their sites of origin, with agronomic cultivars predominantly separated into distinct clusters. The grouping of individuals within and across the populations was corroborated by principal component analysis. These results are consistent with previous reports for switchgrass accessions. RAPD DNA markers were suitable for quickly estimating the genetic diversity of native and agronomic switchgrass populations, and suggest that introgression of agronomic genes into natural switchgrass populations and subsequent changes in genetic structure may be detectable.     

Genetic diversity,population structure and linkage disequilibrium in Nordic spring barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L. subsp. <Emphasis Type="Italic">vulgare</Emphasis>)

Genetic diversity, population structure and genome-wide linkage disequilibrium (LD) was estimated in Nordic spring barley (Hordeum vulgare L. subsp. vulgare) by genotyping 180 breeding lines with 48 SSR markers and 7842 high-confidence SNPs using the Illumina Infinium 9K assay. In total 6208 SNPs were polymorphic and selected for further statistical analysis. A Mantel test revealed a strong positive correlation with a Pearson’s correlation coefficient (r) of 0.86, between the estimates of genetic distances based on SSR and SNP data. Population structure analysis identified two groups with a clear ancestry and one group with an admixed ancestry. The groups were primarily separated based on row-type and geographical origin. Average LD for the whole population decayed below a critical level of r² = 0.20 within a range of 0–4 cM. To avoid confounding effects of the strong population structure, LD decay for the different groups was analysed separately and ranged from 0 to 12 cM. A slower LD decay was found within the two-rowed lines compared to the six-rowed lines and the two-rowed lines originating from the northern part, which could be the result of strong selection for malting quality and yield in the southern part. No large difference in genetic diversity was observed between population sub-groups, but differences at certain chromosomal regions were evident.     

Diversity of Nordic Landrace Potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) Revealed by AFLPs and Morphological Characters

The management of genebank collections of cultivated potato is costly due to the need for in vitro maintenance and virus eradication. Therefore, it is important to set up conservation strategies, which prevent duplicates entering the collections. In this study, 32 Nordic potato landraces were studied for 57 morphological traits and analysed for amplified fragment length polymorphism (AFLP). Most landraces could be distinguished based on the morphological characters, except five accessions. Using five primer combinations to generate 114 reproducible AFLPs, of which 63 (55%) were polymorphic, the five morphologically indistinguishable accessions were placed into two groups with identical AFLP patterns, suggesting that some of the accessions were redundant for long-term preservation. The AFLP data showed that the Nordic collection of potato landraces is composed of genetically different clones, and morphological analysis revealed a wide range of variability. This variability seems to be distributed randomly over the Nordic region since the cluster analysis based on AFLPs and morphological traits revealed no grouping based on the country of origin. Principal component analysis suggests that fewer morphological traits than used in this study will be sufficient to discriminate between different genotypes of cultivated potato (Solanum tuberosum L.). Future possibilities for rationalising potato collections are discussed.     

RAPD-based assessment of genetic relationships among and within American ginseng (Panax quinquefolius L.) populations and their implications for a future conservation strategy

American ginseng (Panax quinquefolius) is a native North American medicinal plant that is becoming increasingly vulnerable despite government harvest restrictions. To better understand the genetic diversity and gene flow of American ginseng, we studied RAPD variation in cultivated and wild populations. Classical and Bayesian analogues of genetic diversity statistics were estimated in seven wild and two cultivated populations. The wild populations were more highly structured (G _stβ  = 0.41) than the cultivated populations (G _stβ  = 0.24). The genetic diversity within populations ranged from H _eβ  = 0.05 to 0.38. Based on genetic pairwise distances, six of the wild populations clustered with the locally-derived cultivated population, while one wild population was more similar to the non-local cultivated population than the local populations. This wild population was highly diverse (P = 1.0; U = 1.0) suggesting that it was supplemented from exotic seed. A set of eight RAPD markers was identified that differentiated plants of local and non-local origin. As a conservation strategy, we recommend that regional gene banks be established based on molecular and geographic diversity to preserve the locally adapted germplasm. These regional gene banks would serve as a conservation tool and also provide a source of genes for genetic improvement of cultivated ginseng.     

Inferring geographic origin of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L. subsp. <Emphasis Type="Italic">vulgare</Emphasis>) accessions using molecular markers

The USDA Agricultural Research Service (ARS) National Small Grains Collection (NSGC) has 207 landrace barleys obtained from a nursery grown in the Ukraine in 1930 by N.I. Vavilov, many of which have multiple resistance (MR) to disease similar to accessions from Ethiopia. The original collection locations of the accessions from the Vavilov nursery are unknown. The purpose of this study was to determine, using molecular markers, if the MR accessions of unknown origin from the Vavilov nursery are genetically related to MR accessions from Ethiopia. The genetic relatedness among susceptible accessions of unknown origin and among a selection of landrace accessions from the NSGC barley core subset was also assessed. Simple sequence repeat (SSR) and Diversity Array Technology PL (DArT) marker data were used to generate similarity matrices with Dice’s similarity coefficients. Cluster analysis from these results showed that the unknown-origin MR accessions grouped with accessions from Ethiopia. Susceptible accessions of unknown origin were genetically similar to accessions from western Asia, especially near the Caucasus, and from both southern and northern Europe. Based on similarity of marker profiles, some accessions from the core collection are likely duplicates. Future work will seek to identify the probable origin of the remainder of the unknown origin accessions and to more thoroughly characterize the genetic diversity within NSGC barley core subset.     

Analysis of genetic diversity of Ruthenia Medic (Medicago ruthenica (L.) Trautv.) in Inner Mongolia using ISSR and SSR markers

Ruthenia Medic is tolerant to drought, cold, high salinity, resistance to trampling and high quality features. Inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR) molecular markers were employed for the first time to access the genetic diversity and relationships of 30 wild Ruthenia Medic accessions obtained from Inner Mongolia in the present study. A total of 94 bands were amplified by ten ISSR primers, of which 83 (88.5 %) were polymorphic, and 57 polymorphic bands (80.4 %) were observed in 69 bands amplified by ten SSR primers. Shannon’s information index (I = 0.487), and average expected heterozygosis (He = 0.329) generated by ISSR primer were higher than that of SSR analysis (I = 0.372, He = 0.231). The study indicated that ISSR were more effective than SSR markers for assessing the degree of genetic variation of Ruthenia Medic. UPGMA cluster analysis revealed inconsistencies in the clustering patterns, as the Mantel’s test between the dendrograms for ISSR and SSR data indicated a poor fit for the ISSR and SSR data types (r = 0.0970). Whereas the pattern of clustering of the genotypes remained relatively the same in ISSR and combined data of ISSR and SSR. The results of principal components analysis also supports their UPGMA clustering. These results have an important implication for Ruthenia Medic germplasm characterization, improvement, and conservation.     

Morphological variation among shea tree (Vitellaria paradoxa subsp. nilotica) ‘ethnovarieties’ in Uganda

Vitellaria paradoxa C. Gaertn. (shea butter tree) is an indigenous African tree species that is widely distributed in the dry areas of northern and eastern Uganda. The species is widely known for its oil which is used in cooking, cosmetics and traditional medicine. Local folk classification recognises the presence of different ethno-varieties on the basis of fruit and nut characters. In the present study, 176 trees representing 44 ethno-varieties from three farming systems of Uganda were assessed to determine the patterns of morphological variation and establish the congruence between morphological variation and folk classification. The results show high variation in pulp weight (CV = 35.9 %), stem diameter (CV = 28.48 %), fruit weight (CV = 27.81 %) and canopy diameter (CV = 26.69 %). There was a strong positive correlation between pulp and fruit weight (r = 0.963, p < 0.001), leaf length and leaf width (r = 0.652, p < 0.001) and between petiole length and leaf length (r = 0.788, p < 0.001). There was no underlying quantitative morphological structuring among the 44 ethno-varieties. Hierarchical cluster analysis using quantitative morphometric data produced three groups without clear aggregation based on ethnographic or geographic separation. However, a combination with qualitative traits as perceived by farmers provided good congruence with folk classification. Quantitative morphological data alone does not resolve any discrete forms of V. paradoxa that are related to folk classification. There is need to utilise biochemical and molecular markers to unravel the underlying variation for use in selection and improvement of shea butter tree ethno-varieties.     

Target region amplification polymorphism (TRAP) for assessing genetic diversity and marker-trait associations in chickpea (Cicer arietinum L.) germplasm

The effective utilization of crop diversity held in genebanks depends on our knowledge of useful traits and available markers associated with the target traits. Target region amplification polymorphism (TRAP) was used to evaluate the genetic diversity and underlying relationships among 263 accessions of chickpea landraces maintained by the USDA-ARS Western Regional Plant Introduction Station in Pullman, WA, USA. Two-hundred sixty-two TRAP markers were amplified by eight primer combinations. Altogether, 110 (42 %) markers were polymorphic, the other 152 (58 %) displayed no variation. These polymorphic markers revealed important differences among the accessions, with an estimated, mean pair-wise genetic distance of 25.82 %, ranging from 2.8 to 50.0 %. Genetic distance analysis divided the accessions into two major groups, with 113 and 150 accessions each, and substantial association between molecular diversity and geographic origin was evident. Bayesian analysis of population structure revealed two groups (K = 2) with evidence for six sub-groups. Additionally, the population structure of a subset of 110 lines was determined (K = 3) for testing marker-trait associations (MTAs). Phenotypic traits included the concentrations of protein and nine mineral elements in the seeds. Two MTAs were significant (p < 0.01) for concentrations of Ca and K, and three MTAs were significant for Cu and Ni concentrations. The results indicate that this population is useful for genome-wide association studies on other economic traits given the level of genetic diversity uncovered and the marker-trait associations in seed minerals discovered.