Chloroplast microsatellite variations in tetraploid alfalfa

Four chloroplast simple sequence repeat (cpSSR) loci were identified in alfalfa (lucerne), Medicago sativa L. The occurrence of allelic variation at these loci was evaluated in 100 plants from 10 populations of tetra‐ploid alfalfa, belonging to four Italian ecotypes and to an Italian (‘Lodi’) and an Egyptian (‘Iside’) variety. Twenty‐four different alleles were identified, four of which were exclusive to the ‘Iside’ variety. The genetic relationship among plants and among populations was analysed by computing an analysis of molecular variance and an unweighted pair group means analysis clustering. This analysis allowed clear separation of the ‘Iside’ variety from Italian germplasm and the recognition of close relationships within the Italian populations. The data presented suggest that cpSSR analysis of tetraploid alfalfa could be used for germplasm polymorphism analysis.     

Use of RAPD and microsatellite (SSR) variation to assess genetic relationships among populations of tetraploid alfalfa, Medicago sativa

The level of random amplified polymorphic DNA (RAPD) and microsatellite variation present in four ecotypes and two varieties of alfalfa (lucerne) from Italian and Egyptian germplasm sources was evaluated. A sample of 100 plants from 10 populations was analysed by means of 41 RAPD markers and 37 simple sequence repeat (SSR) markers. Both molecular approaches revealed a high degree of genetic diversity within each of the cultivated populations and enabled each of the plants considered to be uniquely fingerprinted. The genetic relationships among plants and populations were analysed by computing AMOVA (analysis of molecular variance) and F_ST analyses. RAPDs were able to separate the Italian populations from the Egyptian variety. SSRs allowed strong separation of the four Italian alfalfa ecotypes. It was concluded that RAPD and microsatellites could be useful and powerful tools for assessing genetic variation and genetic relationships in tetraploid alfalfa.     

Genetic diversity of feral alfalfa (<Emphasis Type="Italic">Medicago</Emphasis> <Emphasis Type="Italic">sativa</Emphasis> L.) populations occurring in Manitoba,Canada and comparison with alfalfa cultivars: an analysis using SSR markers and phenotypic traits

Feral populations of cultivated crops may act as reservoirs for novel traits and aid in trait movement across the landscape. Knowledge on the genetic diversity of feral populations may provide new insights into their origin and evolution and may help in the design of efficient novel trait confinement protocols. In this study, the genetic diversity of 12 feral alfalfa (Medicago sativa) populations originating from southern Manitoba (Canada) and 10 alfalfa cultivars and a M. falcata germplasm were investigated using eight SSR markers (i.e., microsatellites) and 14 phenotypic traits. We found that the genetic diversity observed in feral populations was similar to the diversity detected among the 10 cultivars. Analysis of molecular variance revealed that there was great genetic variation within (99.8%) rather than between different feral populations. Cluster analysis (unweighted pair-group method using arithmetic average) revealed no differentiation between feral populations and cultivars for neutral loci. High levels of population differentiation for phenotypic traits (and not for neutral markers) suggest the occurrence of heterogeneous selection for adaptive traits. The phenotypic traits we studied did not distinctly separate feral populations from cultivars but there was evidence of natural selection in feral populations for traits including winter survivability, rhizome production, and prostrate growth habit. Our results suggest that feral alfalfa populations need to be considered in the risk assessment of alfalfa containing novel genetically modified (GM) traits. Further, feral alfalfa populations may be regarded as a source of new germplasm for plant improvement.     

Genetic diversity and population structure of four Iranian alfalfa populations revealed by sequence-related amplified polymorphism (SRAP) markers

In the present study, genetic diversity of 48 individual plants from four Iranian cultivated populations of alfalfa (Medicago sativa L.) was evaluated using sequence-related amplified polymorphism (SRAP) markers. Fourteen SRAP primer combinations produced 193 fragments of which 95 were polymorphic. The number of polymorphic fragments detected per primer combination ranged from 3 to 10 bands with an average of 6.78 bands. Average polymorphic information content (PIC) was 0.343 for all primer combinations. Although the AMOVA (analysis of molecular variance) results showed a significant difference in the genetic diversity among the populations (P < 0.0001), the genetic variation mainly caused by the variation of intra population accounted for 93.17% of the total genetic variation. Unweighted pair-group method arithmetic average (UPGMA) analysis of the marker data clearly separated the populations of subtropical (Yazdi) and semi-cold (Hamadani and Nikshahri) as well as Kodi, an improved population. It can be concluded that SRAP markers are useful for studying diversity and relationships among and within alfalfa populations.     

Genetic diversity of garlic (Allium sativum L.) germplasm from China by fluorescent‐based AFLP,SSR and InDel markers

Garlic (Allium sativum L.), an asexually propagated crop, is an important vegetable and medicinal plant. China is the biggest garlic producer in the world; however, the genetic background of garlic from China is not well understood. In this study, population structure and clustering analysis of garlic germplasm was performed using amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) and insertion–deletion (InDel) markers. Among 212 accessions of garlic, genetic diversity analysis identified 546 alleles amplified by AFLP, SSR and InDel primers, and 492 of these were polymorphic. All accessions were divided into five groups by structure analysis and neighbor‐joining clustering. Most traits, including allicin content, were only slightly affected by population structure, which indicated that this germplasm can be used as populations for association mapping. The results provide a molecular basis for understanding the genetic diversity of the garlic germplasm preserved in China.     

Morphological differences among Raphanus raphanistrum populations and their relationship to related crops

Wild radish (Raphanus raphanistrum) has developed introgressed populations after hybridization with its cultivated counterpart (R. sativus) in California. Hybridization with various Brassica and Sinapis species is also possible. To determine if hybridization is responsible of the genetic diversity of European populations, six wild radish populations with distinct morphological traits were sampled from geographically distant regions in Europe. Plants were cultivated in an oilseed rape field and in insect‐proof cages. Silique and flower morphology, growth, and reproductive traits were measured. The wild radish populations could be discriminated by the morphological traits, but not related to geographic regions. In particular, populations of one region showed wide variability in terms of silique shape and growth behaviour, and small‐sized flowers. Although the origin of morphological diversity in wild radish is unclear, i.e. native or due to gene flow from the cultivated radish or other Brassicaceae, significant morphological divergence was found that could have relevant effects on plant ecology and adaptation.     

Molecular marker analysis of genetic variation characterizing a grasspea (Lathyrus sativus) collection from central Italy

Levels of genetic similarity characterizing 20 grasspea (Lathyrus sativus L.) populations collected in central Italy (17 populations in the Marche region and three populations in the Abruzzo region) were analysed with amplified fragment length polymorphism (AFLP) molecular markers. Two main clusters were found: one included large‐seeded populations from farms that were not market‐oriented (named Household populations) and the second, small‐seeded populations, cultivated in market‐oriented farms (named Commercial populations). Relationships among populations collected in different regions were found, although one population of the Abruzzo region was placed between the two main clusters, suggesting a possible further genetic differentiation within this grasspea germplasm collection. Principal component analysis based on AFLP marker frequency was effective in identifying polymorphic markers showing high discriminating ability between clusters H and C. In particular, seven markers showing high positive and three markers with low negative PC1 scores showed an almost cluster‐specific distribution. These results will be useful for enhancing Italian grasspea germplasm use in plant‐breeding programmes and for extending grasspea cultivation within the sustainable agricultural systems of central Italy.     

Habitat and management affect genetic structure of Festuca pratensis but not Lolium multiflorum ecotype populations

Genetic diversity present in permanent grassland may be valuable for broadening gene pools in breeding programmes and for conservation of genetic resources. However, little is known about the amount of genetic diversity present at specific habitats and about site‐related factors affecting it. To identify valuable habitats, genetic diversity of 12 ecotype populations and four reference cultivars of both Festuca pratensis Huds. and Lolium multiflorum Lam. was analysed using Simple sequence repeat markers (SSR). Analysis of molecular variance revealed a larger within population variation for L. multiflorum (97.1%) than for F. pratensis (92.6%). F. pratensis ecotype populations were clearly separated from cultivars and formed three distinct subclusters according to the geographic regions they were sampled from. Differences between L. multiflorum ecotype populations and cultivars were small and no grouping of populations was observed. Thus, only F. pratensis ecotype populations were structured and habitat as well as management had a slight influence on genetic structure. This information may allow the design of individual strategies for targeted utilization of genetic resources in plant breeding programmes.     

Challenges and opportunities for conservation of forest genetic resources

Increased use of forest resources and a shrinking forestland base threaten the sustainability of forest genetic resources and highlight the importance of conservation and sustainable management of these resources. As forest trees are normally the keystone species of forest ecosystems, their continued existence is essential for many floral and faunal associations of these ecosystems. Major concepts, challenges and opportunities for conservation of forest genetic resources are briefly discussed in this paper. The major challenges include population decline and population structure changes due to forest removal and conversion of forest land to other uses,forest fragmentation, forestry practices, climate change, disease conditions,introduced pests, atmospheric pollution, and introgressive hybridization. Developing scientifically sound conservation strategies, maintaining minimum viable population sizes, and deployment of genetically engineered organisms represent other important challenges in conservation. The usefulness of various biochemical and molecular genetic markers, adaptive traits, and genetic diversity measures for developing conservation strategies for in situ and ex situ genetic resource conservation are also discussed. Major opportunities for conservation of forest genetic resources include: use of molecular genetic markers and adaptive traits for developing conservation strategies; in situ conservation through natural reserves,protected areas, and sustainable forest management practices; ex situ conservation through germplasm banks, common garden archives, seed banks, DNA banks, and tissue culture and cryopreservation; incorporation of disease, pest, and stress tolerance traits through genetic transformation;plantation forestry; and ecological restoration of rare or declining tree species and populations. Forest genetic resource conservation and resource use should be considered complementary rather than contradictory to each other. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity in cornsalad (Valerianella locusta) and related species as determined by AFLP markers

Fifteen amplified fragment length polymorphism (AFLP) EcoRI/MseI‐based primer combinations with five selective bases (Eco RI‐ANN, MseI‐CN) were used to estimate genetic diversity among 45 line varieties of cultivated cornsalad and 19 genebank accessions classified into nine different species related to cornsalad. Polymorphic fragments were scored for calculation of Jaccard's coefficient of genetic similarity (GS). The average GS estimate in elite germplasm (GS = 0.90) was substantially higher than in exotic germplasm (GS = 0.47). UPGMA‐cluster analysis revealed genetic relationships among recently bred varieties, old varieties and genebank accessions. Analysis of molecular variance indicated almost threefold variability within sets compared with between sets due to a high level of polymorphism among wild species. Sources for increasing genetic diversity in elite germplasm of cornsalad were suggested and a duplicate among the genebank accessions was detected. AFLPs could be considered a powerful tool for genetic diversity estimation in cornsalad germplasm and are recommended for systematic fingerprinting of remaining cornsalad species.     

Genetic Diversity of Farmers’Preferred Sorghum Accessions and Improved Lines from ICRISAT Reveal a Disconnect Between Innovation and Technology Transfer

The genetic diversity of 65 accessions of sorghum [Sorghum bicolor (L.) Moench] collected from various farmers and germplasm lines from ICRISAT-Kenya were analyzed. Simple sequence repeats (SSR) markers were used in order to determine the extent and distribution of its genetic diversity. Twenty-nine (29) SSRs markers were polymorphic and a total of 192 alleles were detected which showed diversity. The number of alleles per primer ranged from 2 to 17, with an average of 6.62. The range of polymorphism information content (PIC) ranged from 0.03 to 0.86, with total average of 0.82. According to the results analyzed, estimates of the mean allelic pattern across the two populations was generated; expected heterozygosity (He; 0.45, 0.54), average observed alleles (Na; 3.40, 6.20), number of private allele (0.23, 3.03), and Shannon information index (I; 0.85, 1.13) for farmer and ICRISAT-Kenya germplasm, respectively. The expected heterozygosity (He) varied from 0 to 0.26 with an average of 0.05. The Neighbor-joining phenogram based on Nei’s genetic distance grouped the 65 accessions into three main groups. The analysis of molecular variance (AMOVA) revealed that 99% of the total genetic variation was within accessions in a population whereas the genetic variation among populations in accessions accounted for 1% of the total genetic variation. Genetic diversity in ICRISAT sorghum material compared to the farmer’s collection suggested little infiltration of improved germplasm to the farmers.     

Choosing rice germplasm for evaluation

Summary Using the evaluation database on the world collection of rice, Oryza sativa, conserved at the International Rice Research Institute, different sampling strategies for choosing germplasm were compared. Random, stratified, sequential and analysed sets of germplasm were chosen and the frequency of finding resistance to different rice pests, the brown planthopper, green leafhopper and whitebacked planthopper, and diseases, bacterial blight and blast were compared. The frequency of the geographically restricted javanica race of rice was also compared in the different germplasm sets. The results indicate that where no prior information is available to choose germplasm for evaluation, for the same sample number, germplasm representing broad genetic diversity are preferable to other sampling strategies.     

Inter and intra-population variability of <Emphasis Type="Italic">Jatropha curcas</Emphasis> (L.) characterized by RAPD and ISSR markers and development of population-specific SCAR markers

Jatropha curcas (Euphorbiaceae) is an oil-bearing species with multiple uses and considerable potential as a bioenergy crop. The present investigation has been undertaken to assess the extent of genetic diversity in a representative set of 42 accessions of J. curcas encompassing different crop growing regions in India along with a non-toxic genotype from Mexico as a prelude for utilization of promising and genetically divergent materials in the breeding programmes. Molecular polymorphism was 42.0% with 400 RAPD primers and 33.5% with 100 ISSR primers between accessions indicating modest levels of genetic variation in the Indian germplasm. The within-population variation based on RAPD polymorphism was 64.0% and was on par with the inter-population variation. Polymorphic ISSR markers have been identified that could differentiate the Indian accessions from the Mexican genotype and two of them were converted to SCAR markers. The SCAR primer pair ISPJ1 amplified a 543 bp fragment in all the Indian populations, while ISPJ2 with a specific amplicon of 1,096 bp was specific to the Mexican genotype. Population-specific bands have been identified for the accession from Kerala (2 RAPD markers), Neemuch-1 from Rajasthan (1 each of RAPD and ISSR markers) and the non-toxic genotype from Mexico (17 RAPD and 4 ISSR markers), which serve as diagnostic markers in genotyping. The study indicates an immediate need for widening the genetic base of J. curcas germplasm through introduction of accessions with broader geographical background.     

Population genetic structure and genetic diversity of <Emphasis Type="Italic">Jatropha curcas</Emphasis> germplasm as investigated by 5′-anchored simple sequence repeat primers

Jatropha (Jatropha curcas L.) is widely distributed in the tropics with a promise for use as an oil crop for biodiesel. Understanding population genetic structure and diversity of J. curcas is important for germplasm collection and planning for breeding programs. Twelve 5′-anchored simple sequence repeat primers were selected and used for evaluation of population genetic structure and genetic diversity of J. curcas accessions collected from different regions within Thailand and introduced from other countries. A moderate level of genetic variation was found in all accessions studied. Cluster analysis did not reflect geographical distance in general as revealed by the Mantel test. An analysis of molecular variance showed that the majority (83.3%) of genetic diversity was within populations, indicating a high level of overlap and little population subdivision with respect to geographic area. An absence of subpopulation structure among neighboring regions may be caused by exchanging germplasm with vegetative propagation. This study provides a basis for breeders on selection of parental material to maximize genetic polymorphism in J. curcas breeding programs.     

Allozyme variation and conservation genetics of common wild rice (Oryza rufipogon Griff.) in Yunnan, China

In order to reveal levels and distribution of genetic variation within Oryza rufipogon Griff. of Yunnan, China, where one of the centers of genetic diversity for Asian cultivated rice O. sativa L. is located, allozyme variation encoded by 22 loci was electrophoretically analyzed in 149 individuals of all three existing populations as well as five from other regions (Guangxi, Hainan and Jiangxi provinces) of China. As compared to the level of genetic diversity (the mean A = 1.2, P = 24.1%, Ho = 0.045 and He = 0.079) for the populations from other regions, a rather low genetic diversity (the mean A = 1.1, P = 7.6%, Ho = 0.007 and He = 0.011) was found in Yunnan, which may originate from marginal nature of these populations, recent reduction of populations and consequent drift. The result suggests that the current center of genetic diversity for O. rufipogon fail to agree with that for cultivated rice in China. The genetic differentiation for all the eight populations(F_ST = 0.254) was slightly lower than that for three populations from Yunnan (F_ST = 0.302), indicating a fairly high genetic differentiation in the region. Finally, a conservation plan for sampling/preserving fewer populations but more individuals from each population for the species was given, and an appropriate strategy for conserving the three surviving populations from Yunnan was proposed. This revised version was published online in August 2006 with corrections to the Cover Date.     

AFLP fingerprinting in Medicago spp.: Its development and application in linkage mapping

Cultivated alfalfa (Medicago sativa L., 2n= 4x= 32) is one of the most important forage crops in temperate climates. The genus Medicago includes diploid species that are a valuable source of wild germplasm for studying the reproductive system of alfalfa and its abnormalities. A linkage map of an apomeiotic mutant of Medicago falcata (L.) Arcang. (2n= 2x= 16) that spanned 368.6 cM and included 29 amplified fragment length polymorphism (AFLP), 35 random amplified polymorphic DNA (RAPD) and three restriction fragment length polymorphism (RFLP) loci was constructed using a one-way pseudo-testcross mapping strategy. The success of such a strategy depends on the presence of sufficiently high levels of heterozygosity in the individual plant which is being mapped and on the informativeness of the marker system that is used. In general: (1) highly informative and reproducible RAPD and AFLP fingerprints were generated and several genome-specific primers selected; (2) of 67 marker loci mapped, 51 were arranged in 11 main linkage groups and eight additional couples of linked marker loci were detected; (3) mapping of an F₁ population theoretically allowed a better estimation of linkage distances since it avoided segregation distortion (x² analyses revealed segregation distortion in only 5.2% of marker loci); (4) the high frequency of unlinked marker loci obtained suggests that, in this alfalfa genotype, DNA markers are distributed throughout the genome. This type of genetic map should find application and prove useful in marker-assisted selection and map-based breeding programmes in meiotic mutants of alfalfa for which there is a lack of suitable genetic markers.     

Characterizing the molecular and morphophysiological diversity of Italian red clover

Red clover (Trifolium pratense L.) is the third major forage species in Europe, but there is limited information on the biodiversity and the genetic structure of landraces and natural populations which evolved in this region. The objective of this study was producing such information for Italian germplasm on the ground of molecular and morphophysiological diversity. The study included 16 Italian natural populations from a wide range of environments, four landraces representing the four traditional commercial ecotypes, and two varieties. Eight morphophysiological traits were assessed in a replicated trial under field conditions, whereas two AFLPs primer combinations with 140 polymorphic markers were recorded on a random sample of 13 genotypes per population. Ordination and classification results based on morphophysiological data clearly kept track of the type of germplasm (i.e. landrace or natural population) and its geographic origin, unlike results based on molecular markers. Euclidean distances among populations based on morphophysiological traits were not correlated with Nei’s genetic distances based on molecular markers according to Mantel’s test. Geographical distances among landrace or natural population material was correlated with distances based on morphophysiological traits but not with those based on molecular markers. The average within-population variation estimated via molecular markers was about 2.6-fold higher than that among populations, preventing an acceptable discrimination among most populations. On average, natural populations tended to have within-population variation similar to varieties and somewhat lower than landraces. Our results have implications for collection, conservation, exploitation and registration in a sui generis system of red clover genetic resources.     

Genetic variation and heritability of forage yield in Mediterranean tall fescue

The genetic control of tall fescue forage yield has been poorly investigated. Full‐sib families from diallel crosses of Mediterranean germplasm were evaluated for forage yield over 34 months in a Mediterranean environment with severe drought stress (diallel 1, with 20 parents) and over 16 months under irrigation in a heated greenhouse simulating the Mediterranean temperature pattern (diallel 2, with 15 parents). Genetic parameters were estimated for fresh biomass in diallel 1 and dry‐matter yield in diallel 2. Additive genetic variance was always larger than non‐additive (dominance) variance. Narrow‐sense heritability was fairly high (h² = 0.61) in diallel 1 and moderate (h² = 0.45) in diallel 2. Predicted yield gains from one selection cycle were larger in the former diallel (23.9%) than in the latter (10.5%), suggesting that gains can be enhanced by selection under severe drought stress and over a time span sufficient to allow the variation in persistence to fully emerge. General combining ability effects of eight parents that were common to both diallel crosses were highly correlated (r = 0.94) across the contrasting evaluation environments. The extent and consistency of additive genetic effects across environments suggest that rapid improvement of forage yield is possible.     

Collection and evaluation of grasspea (Lathyrus sativus L.) germplasm of central Italy

Characterization of 16 grasspea (Lathyrus sativus L.) accessions collected in central Italy (Marche region) was performed for morphological and agronomic traits and for 3‐(‐N‐oxalyl)‐L‐2,3 diaminoprop‐ionic acid (ODAP) content. Field trials, carried out in two locations, revealed high among‐population genetic variation. In particular, an evident differentiation between commercial populations, cultivated by market‐oriented farms, and household populations (not market‐oriented) was found. Genotype × environment interaction was negligible. Medium‐high values of ODAP content were observed following colorimetric and capillary electrophoresis analyses. A high positive correlation between the two methods was found (r = 0.83**), but the colorimetric values showed, on average, significant 14% lower ODAP values. This research represents a pre‐breeding activity aimed at developing a breeding programme for the preservation and utilization of Italian grasspea germplasm within sustainable or organic agricultural systems.     

Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms

Fifteen alfalfa populations consisting of six public cultivars and nine historically recognized sources of alfalfa germplasm in North American cultivars were examined using sequence related amplified polymorphisms (SRAPs). Three bulk DNA samples from each population were evaluated with fourteen different SRAP primer pairs. This resulted in 249 different amplicons, of which over 90% were polymorphic. A dendrogram from the analysis suggests that the public cultivars are quite diverse from all the historical sources of germplasm. The highest mean genetic similarity among the nine original sources of Medicago germplasm was 0.85 between PI 536535 (Peruvian) and 536536 (Indian), while the lowest (0.47) was between PI 560333 (M. falcata) and 536539 (African). The highest mean genetic similarity among the nine original sources of Medicago germplasm and the public alfalfa cultivars was 0.78 between PI 536532 (Ladak) and Vernal, while the lowest (0.59) was between PI 536539 (African) and Oneida. Relationships based on SRAP analysis appear to generally concur with expected relationships based on fall dormancy. This report demonstrates that SRAPs are a promising marker system for detecting polymorphisms in alfalfa.