Allozyme diversity in wild Phaseolus vulgaris: further evidence for two major centers of genetic diversity

Summary Allozyme analysis was performed on 83 wild Phaseolus vulgaris accessions, representing a wide geographical distribution from Mesoamerica to Argentina, to determine levels of genetic diversity and geographic patterns of variability at nine polymorphic isozyme loci. The collection can be divided into two major groups, one consisting of accessions from Mexico, Central America, Colombia and Peru, and the other consisting of accessions from Peru and Argentina. One accession from northern Peru is distinct from the two major groups, and may delineate a transition zone between the two divergent groups. The level of genetic diversity within wild P. vulgaris (Ht=0.132) is comparable with those found in other Phaseolus species. There was no significant within-accession gene diversity (Hs=0.006); however, there is a moderate level of genetic diversity (Dst=0.126) between accessions. Our results are consistent with previous studies on the genetic diversity of wild P. vulgaris using phaseolin, the major seed storage protein of beans.     

Bidirectional backcrosses between wild and cultivated lettuce identify loci involved in nonhost resistance to downy mildew

Key message

The nonhost resistance of wild lettuce to lettuce downy mildew seems explained by four components of a putative set of epistatic genes.

Abstract

The commonplace observation that plants are immune to most potential pathogens is known as nonhost resistance (NHR). The genetic basis of NHR is poorly understood. Inheritance studies of NHR require crosses of nonhost species with a host, but these crosses are usually unsuccessful. The plant-pathosystem of lettuce and downy mildew, Bremia lactucae, provides a rare opportunity to study the inheritance of NHR, because the nonhost wild lettuce species Lactuca saligna is sufficiently cross-compatible with the cultivated host Lactuca sativa. Our previous studies on NHR in one L. saligna accession led to the hypothesis that multi-locus epistatic interactions might explain NHR. Here, we studied NHR at the species level in nine accessions. Besides the commonly used approach of studying a target trait from a wild donor species in a cultivar genetic background, we also explored the opposite, complementary approach of cultivar introgression in a wild species background. This bidirectional approach encompassed (1) nonhost into host introgression: identification of L. saligna derived chromosome regions that were overrepresented in highly resistant BC1 plants (F1?×?L. sativa), (2) host into nonhost introgression: identification of L. sativa derived chromosome regions that were overrepresented in BC1 inbred lines (F1?×?L. saligna) with relatively high infection levels. We demonstrated that NHR is based on resistance factors from L. saligna and the genetic dose for NHR differs between accessions. NHR seemed explained by combinations of epistatic genes on three or four chromosome segments, of which one chromosome segment was validated by the host into nonhost approach.

     

LSGermOPA,a custom OPA of 384 EST-derived SNPs for high-throughput lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) germplasm fingerprinting

To deploy a high-throughput genotyping platform in germplasm management, we designed and tested a custom OPA (Oligo Pool All), LSGermOPA, for assessing the genetic diversity and population structure of the USDA cultivated lettuce (Lactuca sativa L.) germplasm collection using Illumina’s GoldenGate assay. This OPA contains 384 EST (expressed sequence tag)-derived SNP (single nucleotide polymorphism) markers selected from a large set of SNP markers experimentally validated and mapped by the Compositae Genome Project. Used for genotyping were DNA samples prepared from bulked leaves of five randomly-selected seedlings from each of 380 lettuce accessions. High-quality genotype data were obtained from 354 of the 384 SNPs. The reproducibility of automatic genotype calls was 99.8% as calculated from the four pairs of duplicated DNA samples in the assay. An unexpectedly high percentage of heterozygous genotypes at the polymorphic loci for most accessions indicated a high level of heterogeneity within accessions. Only 148 homogenous accessions, collectively comprising all five horticultural types, were used in subsequent analyses to demonstrate the usefulness of LSGermOPA. The results of phylogenetic relationship, population structure and genetic differentiation analyses were consistent with previous reports using other marker systems. This suggests that LSGermOPA is capable of revealing sufficient levels of polymorphism among lettuce cultivars and is appropriate for rapid assessment of genetic diversity and population structure in the lettuce germplasm collection. Challenges and strategies for effective genotyping and managing lettuce germplasm are discussed.     

Loss of genetic diversity from heterogeneous self-pollinating genebank accessions

Genebank seed accessions of predominantly self-pollinating species may be stored either as bulked (mixed) seed lines or as pure line cultivars. If seed lines are bulked in storage then when considered over several regeneration cycles, loss of genetic diversity within heterogeneous self pollinating genebank accessions is shown to be severe. This within-accession loss of diversity represents opportunities foregone through the random loss of individual genotypes. Amongst working collections, the utility and repeatability of genebank accessions is paramount in the justification of the germ plasm resource. Therefore, the only practical solution to the management of predominantly self-pollinating species is to preserve individual accessions as pure lines.     

Molecular diversity at the major cluster of disease resistance genes in cultivated and wild Lactuca spp.

Diversity was analyzed in wild and cultivated Lactuca germplasm using molecular markers derived from resistance genes of the NBS-LRR type. Three molecular markers, one microsatellite marker and two SCAR markers that amplified LRR-encoding regions, were developed from sequences of resistance gene homologs at the main resistance gene cluster in lettuce. Variation for these markers were assessed in germplasm including accessions of cultivated lettuce, Lactuca sativa L. and three wild Lactuca spp., L. serriola L., L. saligna and L. virosa L. Diversity was also studied within and between natural populations of L. serriola from Israel and California; the former is close to the center of diversity for Lactuca spp. while the latter is an area of more recent colonization. Large numbers of haplotypes were detected indicating the presence of numerous resistance genes in wild species. The diversity in haplotypes provided evidence for gene duplication and unequal crossing-over during the evolution of this cluster of resistance genes. However, there was no evidence for duplications and deletions within the LRR-encoding regions studied. The three markers were highly correlated with resistance phenotypes in L. sativa. They were able to discriminate between accessions that had previously been shown to be resistant to all known isolates of Bremia lactucae. Therefore, these markers will be highly informative for the establishment of core collections and marker-aided selection. A hierarchical analysis of the population structure of L. serriola showed that countries, as well as locations, were significantly differentiated. These differences may reflect local founder effects and/or divergent selection. Received: 7 March 1999 / Accepted: 25 March 1999     

Selenium accumulation in lettuce germplasm

Selenium (Se) is an essential micronutrient for animals and humans. Increasing Se content in food crops offers an effective approach to reduce the widespread selenium deficiency problem in many parts of the world. In this study, we evaluated 30 diverse accessions of lettuce (Lactuca sativa L.) for their capacity to accumulate Se and their responses to different forms of Se in terms of plant growth, nutritional characteristics, and gene expression. Lettuce accessions responded differently to selenate and selenite treatment, and selenate is superior to selenite in inducing total Se accumulation. At least over twofold change in total Se levels between cultivars with high and low Se content was found. Synergistic relationship between Se and sulfur accumulation was observed in nearly all accessions at the selenate dosage applied. The change in shoot biomass varied between lettuce accessions and the forms of Se used. The growth-stimulated effect by selenate and the growth-inhibited effect by selenite were found to be correlated with the alteration of antioxidant enzyme activities. The different ability of lettuce accessions to accumulate Se following selenate treatment appeared to be associated with an altered expression of genes involved in Se/S uptake and assimilation. Our results provide important information for the effects of different forms of Se on plant growth and metabolism. They will also be of help in selecting and developing better cultivars for Se biofortification in lettuce.     

Assessment of genetic diversity within and between pearl millet landraces

A minimum core subset of pearl millet [Pennisetum glaucum (L.) R. Br.], which comprised 504 landrace accessions, was recently established from the global pearl millet germplasm collection of ICRISAT. The accessions for this core were selected by a random proportional sampling strategy following stratification of the entire landrace collection (about 16,000 accessions) according to their geographic origin and morpho-agronomic traits. In this study RFLP probes were used to quantify the genetic diversity within and between landrace accessions of this minimum core using a subset comprising ten accessions of Indian origin. Twenty five plants per accession were assayed with EcoRI, EcoRV, HindIII and DraI restriction enzymes, and 16 highly polymorphic RFLP probes, nine associated with a quantitative trait loci (QTLs) for downy mildew resistance, and five associated with a QTL for drought tolerance. A total of 51 alleles were detected using 16 different probe-enzyme combinations. The partitioning of variance components based on the analysis of molecular variance (AMOVA) for diversity analysis revealed high within-accession variability (30.9%), but the variability between accessions was significantly higher (69.1%) than that within the accessions. A dendrogram based on the dissimilarity matrix obtained using Ward's algorithm further delineated the 250 plants into ten major clusters, each comprised of plants from a single accession (with the exception of two single plants). A similar result was found in an earlier study using morpho-agronomic traits and geographic origin. This study demonstrated the utility of RFLP markers in detecting polymorphism and estimating genetic diversity in a highly cross-pollinated species such as pearl millet. When less-tedious marker systems are available, this method could be further extended to assess the genetic diversity between and within the remaining accessions in the pearl millet core subset.     

An insight into the genetic polymorphism among European populations of Lactuca serriola assessed by AFLP

Prickly lettuce (Lactuca serriola) is world-wide distributed and very variable species generally considered as a progenitor of the cultivated lettuce (Lactuca sativa). Altogether, 50 populations of L. serriola were characterized by means of amplified fragment length polymorphism (AFLP) and by isozyme analysis. Relationships among individuals and populations were examined by applying the unweighted pair-group method with the arithmetic averages (UPGMA) clustering algorithm, principal coordinate analysis (PCA) and the Nei's gene diversity index. The studied set of populations split into three main groups based on the AFLP polymorphism analysis. The first group contained L. sativa (control). The second group comprised two L. serriola accessions; one of them was identified as L. serriola f. integrifolia and the other as a mixture of two L. serriola forms. The largest and the most diverse third group contained the remaining L. serriola accessions. The population clustering corresponded approximately to their geographical distribution in Europe. At least five distinct geographic groups were recognised: 1) Northern European; 2) Slovenian; 3) very heterogeneous Central and Western European (mostly north of the Alps); 4) Mediterranean; 5) prevalence of L. serriola f. integrifolia, mostly comprising accessions from the United Kingdom and the Netherlands. This study showed that accessions originating in various eco-geographical conditions of Europe differ significantly in their genetic and protein polymorphism, as well as in morphology. Some European L. serriola populations (e.g. from Scandinavia and United Kingdom/British Isles/) seems to be isolated and homogeneous; in contrast, populations occurring in Central Europe are very diverse and genetically overlapping.     

Crossing experiments of lettuce cultivars and species (Lactuca sect.Lactuca,Compositae)

The degree of relationships withinLactuca sativa and three wild relativesL. serriola, L. saligna, andL. virosa was studied by observing the performance, vigour and fertility of the F 1 hybrids obtained from crosses made in and between the four species. The crosses ofL. saligna ×L. virosa and the reciprocal crosses produced no hybrids.L. saligna andL. virosa are the least related of the four species.L. sativa ×L. serriola and the reciprocal crosses were successful and produced fertile hybrids These two species are genetically very closely related.L. saligna is known to produce, as a female parent, hybrids withL. sativa andL. serriola. Now the reciprocal cross was successful for the first time, so the unability to obtain hybrids in the past was based on the choice of accessions and not caused by unilateral incompatibility.L. virosa ×L. sativa and the reciprocal combination produced hybrids. The combinationL. serriola ×L. virosa produced hybrids with very limited fertility. In contrast to earlier reports (sterile hybrids) one combination of the reciprocal cross too produced hybrids with very limited fertility.—Some of theL. saligna ×L. sativa (and reciprocal) hybrids were found to look strikingly likeL. serriola. This adds evidence for the descent ofL. serriola andL. sativa:L. saligna also made part of the ancestral complex of the cultivated lettuce.     

Efficient QTL detection for nonhost resistance in wild lettuce: backcross inbred lines versus F2 population

In plants, several population types [F₂, recombinant inbred lines, backcross inbred lines (BILs), etc.] are used for quantitative trait locus (QTL) analyses. However, dissection of the trait of interest and subsequent confirmation by introgression of QTLs for breeding purposes has not been as successful as that predicted from theoretical calculations. More practical knowledge of different QTL mapping approaches is needed. In this recent study, we describe the detection and mapping of quantitative resistances to downy mildew in a set of 29 BILs of cultivated lettuce (L. sativa) containing genome segments introgressed from wild lettuce (L. saligna). Introgression regions that are associated with quantitative resistance are considered to harbor a QTL. Furthermore, we compare this with results from an already existing F₂ population derived from the same parents. We identified six QTLs in our BIL approach compared to only three in the F₂ approach, while there were two QTLs in common. We performed a simulation study based on our actual data to help us interpret them. This revealed that two newly detected QTLs in the BILs had gone unnoticed in the F₂, due to a combination of recessiveness of the trait and skewed segregation, causing a deficit of the wild species alleles. This study clearly illustrates the added value of extended genetic studies on two different population types (BILs and F₂) to dissect complex genetic traits.     

Orchardgrass (<Emphasis Type="Italic">Dactylis glomerata</Emphasis> L.) EST and SSR marker development,annotation, and transferability

Orchardgrass, or cocksfoot [Dactylis glomerata (L.)], has been naturalized on nearly every continent and is a commonly used species for forage and hay production. All major cultivated varieties of orchardgrass are autotetraploid, and few tools or information are available for functional and comparative genetic analyses and improvement of the species. To improve the genetic resources for orchardgrass, we have developed an EST library and SSR markers from salt, drought, and cold stressed tissues. The ESTs were bi-directionally sequenced from clones and combined into 17,373 unigenes. Unigenes were annotated based on putative orthology to genes from rice, Triticeae grasses, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Of 1,162 SSR markers developed, approximately 80% showed amplification products across a set of orchardgrass germplasm, and 40% across related Festuca and Lolium species. When orchardgrass subspecies were genotyped using 33 SSR markers their within-accession similarity values ranged from 0.44 to 0.71, with Mediterranean accessions having a higher similarity. The total number of genotyped bands was greater for tetraploid accessions compared to diploid accessions. Clustering analysis indicated grouping of Mediterranean subspecies and central Asian subspecies, while the D. glomerata ssp. aschersoniana was closest related to three cultivated varieties.     

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.     

A Bayesian analysis of gene flow from crops to their wild relatives: cultivated (Lactuca sativa L.) and prickly lettuce (L. serriola L.) and the recent expansion of L. serriola in Europe

Interspecific gene flow can lead to the formation of hybrid populations that have a competitive advantage over the parental populations, even for hybrids from a cross between crops and wild relatives. Wild prickly lettuce (Lactuca serriola) has recently expanded in Europe and hybridization with the related crop species (cultivated lettuce, L. sativa) has been hypothesized as one of the mechanisms behind this expansion. In a basically selfing species, such as lettuce, assessing hybridization in natural populations may not be straightforward. Therefore, we analysed a uniquely large data set of plants genotyped with SSR (simple sequence repeat) markers with two programs for Bayesian population genetic analysis, STRUCTURE and NewHybrids. The data set comprised 7738 plants, including a complete genebank collection, which provided a wide coverage of cultivated germplasm and a fair coverage of wild accessions, and a set of wild populations recently sampled across Europe. STRUCTURE analysis inferred the occurrence of hybrids at a level of 7% across Europe. NewHybrids indicated these hybrids to be advanced selfed generations of a hybridization event or of one backcross after such an event, which is according to expectations for a basically selfing species. These advanced selfed generations could not be detected effectively with crop‐specific alleles. In the northern part of Europe, where the expansion of L. serriola took place, the fewest putative hybrids were found. Therefore, we conclude that other mechanisms than crop/wild gene flow, such as an increase in disturbed habitats and/or climate warming, are more likely explanations for this expansion.     

Development and application of a 6.5 million feature affymetrix genechip® for massively parallel discovery of single position polymorphisms in lettuce (Lactuca spp.)

ABSTRACT: BACKGROUND: High-resolution genetic maps are needed in many crops to help characterize the genetic diversity that determines agriculturally important traits. Hybridization to microarrays to detect single feature polymorphisms is a powerful technique for marker discovery and genotyping because of its highly parallel nature. However, microarrays designed for gene expression analysis rarely provide sufficient gene coverage for optimal detection of nucleotide polymorphisms, which limits utility in species with low rates of polymorphism such as lettuce (Lactuca sativa). RESULTS: We developed a 6.5 million feature Affymetrix GeneChip? for efficient polymorphism discovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on the microarray were designed from 26,809 unigenes from cultivated lettuce and an additional 8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis). Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand; providing an average of 187 probes covering approximately 600 bp for each of over 35,000 unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developed protocols for hybridization of genomic DNA to the GeneChip? and refined custom algorithms that utilized coverage from multiple, high quality probes to detect single position polymorphisms in 2 bp sliding windows across each unigene. This allowed us to detect greater than 18,000 polymorphisms between the parental lines of our core mapping population, as well as numerous polymorphisms between cultivated lettuce and wild species in the lettuce genepool. Using marker data from our diversity panel comprised of 52 accessions from the five species listed above, we were able to separate accessions by species using both phylogenetic and principal component analyses. Additionally, we estimated the diversity between different types of cultivated lettuce and distinguished morphological types. CONCLUSION: By hybridizing genomic DNA to a custom oligonucleotide array designed for maximum gene coverage, we were able to identify polymorphisms using two approaches for pair-wise.     

An Association Between High Peroxidase Activity in Lettuce (Lactuca sativa) and Field Resistance to Downy Mildew (Bremia lactucae)

The association between variation for pre-infection peroxidase activity and levels of field resistance-susceptibility to downy mildew (Bremia lactucae) was investigated in lettuce (Lactuca sativa) cultivars, accessions of L. serriola (prickly lettuce), segregating F₂ populations and selected F₃ families from a cross between field resistant and susceptible lettuce cultivars. A trend was apparent in this series of experiments indicating that one component of field resistance could be related to a high level of peroxidase activity prior to infection. The data suggest that in breeding programmes there could be merit in imposing primary selection for high peroxidase activity prior to field selection for resistance.     

High efficiency and reliability of inter-simple sequence repeats (ISSR) markers for evaluation of genetic diversity in Brazilian cultivated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. accessions

Jatropha curcas L. is found in all tropical regions and has garnered lot of attention for its potential as a source of biodiesel. As J. curcas is a plant that is still in the process of being domesticated, interest in improving its agronomic traits has increased in an attempt to select more productive varieties, aiming at sustainable utilization of this plant for biodiesel production. Therefore, the study of genetic diversity in different accessions of J. curcas in Brazil constitutes a necessary first step in genetic programs designed to improve this species. In this study we have used ISSR markers to assess the genetic variability of 332 accessions from eight states in Brazil that produce J. curcas seeds for commercialization. Seven ISSR primers amplified a total of 21,253 bands, of which 19,472 bands (91%) showed polymorphism. Among the polymorphic bands 275 rare bands were identified (present in fewer than 15% of the accessions). Polymorphic information content (PIC), marker index (MI) and resolving power (RP) averaged 0.26, 17.86 and 19.87 per primer, respectively, showing the high efficiency and reliability of the markers used. ISSR markers analyses as number of polymorphic loci, genetic diversity and accession relationships through UPGMA-phenogram and MDS showed that Brazilian accessions are closely related but have a higher level of genetic diversity than accessions from other countries, and the accessions from Natal (RN) are the most diverse, having high value as a source of genetic diversity for breeding programs of J. curcas in the world.     

Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in cultivated lentils requires reliable information on genetic diversity in the germplasm. The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lens was evaluated for several geographically dispersed accessions/cultivars of four diploid Lens species. This study was carried out to assess whether RAPD data can provide additional evidence about the origin of the cultivated lentil and to measure genetic variability in lentil germplasm. Three cultivars of Lens culinaris ssp. culinaris, including one microsperma, and two macrosperma types, and four wild species (L. culinaris ssp. orientalis, L. odemensis and L. nigricans) were evaluated for genetic variability using a set of 1 11-mer and 14 random 10-mer primers. One hundred and fifty-eight reproducible and scorable DNA bands were observed from these primers. Genetic distances between each of the accessions were calculated from simple matching coefficients. Split decomposition analysis of the RAPD data allowed construction of an unrooted tree. This study revealed that (1) the level of intraspecific genetic variation in cultivated lentils is narrower than that in some wild species. (2) L. culinaris ssp. orientalis is the most likely candidate as a progenitor of the cultivated species, (3) L. nigricans accession W6 3222 (unknown) and L. c. ssp. orientalis W6 3244 (Turkey) can be reclassified as species of L. odemensis and (4) transmission of genetic material in Lens interspecific hybrids is genotypically specific, as identified by the RAPD markers in our study.     

Comparison of three genetic similarity coefficients based on dominant markers from predominantly self-pollinating species

Three genetic similarity coefficients were estimated and compared for their usefulness: simple matching (S _SM), Jaccard’s (S _J) and Dice’s (S _D), all based on dominant markers data from individuals representing predominantly self-pollinating species. AFLP markers were used to analyze 139 Phaseolus vulgaris L. (common bean) and 67 Lactuca saligna L. (least lettuce) accessions, and RAPD markers were used to analyze 110 Triticum dicoccoides Koern. (wild emmer wheat) accessions. Similar discriminating structure and power based on the three genetic similarity coefficients was found for each of the three species. This discriminating power was high for both P. vulgaris and L. saligna but moderate for T. dicoccoides. With closely related individuals, as in our study, the absence of a band in two individuals should be due to an identical cause inherited from the same ancestor. Accordingly we propose the use of S _SM, which alone out of the three examined coefficients involved shared absence of DNA bands, as contributing to genetic similarity. When RAPDs are employed, inferences about population structure and nucleotide divergence should be made with prudence as the nature of genetic variation uncovered by RAPDs is often unclear.