Genetic diversity of peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome

Background  

The genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Genetic relationships among seven sections of genus <Emphasis Type="Italic">Arachis</Emphasis>studied by using SSR markers

Background  

The genus Arachis, originated in South America, is divided into nine taxonomical sections comprising of 80 species. Most of the Arachis species are diploids (2n = 2x = 20) and the tetraploid species (2n = 2x = 40) are found in sections Arachis, Extranervosae and Rhizomatosae. Diploid species have great potential to be used as resistance sources for agronomic traits like pests and diseases, drought related traits and different life cycle spans. Understanding of genetic relationships among wild species and between wild and cultivated species will be useful for enhanced utilization of wild species in improving cultivated germplasm. The present study was undertaken to evaluate genetic relationships among species (96 accessions) belonging to seven sections of Arachis by using simple sequence repeat (SSR) markers developed from Arachis hypogaea genomic library and gene sequences from related genera of Arachis.     

Abundant Microsatellite Diversity and Oil Content in Wild Arachis Species

The peanut (Arachis hypogaea) is an important oil crop. Breeding for high oil content is becoming increasingly important. Wild Arachis species have been reported to harbor genes for many valuable traits that may enable the improvement of cultivated Arachis hypogaea, such as resistance to pests and disease. However, only limited information is available on variation in oil content. In the present study, a collection of 72 wild Arachis accessions representing 19 species and 3 cultivated peanut accessions were genotyped using 136 genome-wide SSR markers and phenotyped for oil content over three growing seasons. The wild Arachis accessions showed abundant diversity across the 19 species. A. duranensis exhibited the highest diversity, with a Shannon-Weaver diversity index of 0.35. A total of 129 unique alleles were detected in the species studied. A. rigonii exhibited the largest number of unique alleles (75), indicating that this species is highly differentiated. AMOVA and genetic distance analyses confirmed the genetic differentiation between the wild Arachis species. The majority of SSR alleles were detected exclusively in the wild species and not in A. hypogaea, indicating that directional selection or the hitchhiking effect has played an important role in the domestication of the cultivated peanut. The 75 accessions were grouped into three clusters based on population structure and phylogenic analysis, consistent with their taxonomic sections, species and genome types. A. villosa and A. batizocoi were grouped with A. hypogaea, suggesting the close relationship between these two diploid wild species and the cultivated peanut. Considerable phenotypic variation in oil content was observed among different sections and species. Nine alleles were identified as associated with oil content based on association analysis, of these, three alleles were associated with higher oil content but were absent in the cultivated peanut. The results demonstrated that there is great potential to increase the oil content in A. hypogaea by using the wild Arachis germplasm.     

Genetic relationships between peanut and wild species ofArachis sect.Arachis (Fabaceae): Evidence from RAPDs

Twenty-six accessions of wildArachis species and domesticated peanuts,A. hypogaea, introduced from South America were analyzed for random amplified polymorphic DNA (RAPD). The objective of the study was to investigate inter- and intraspecific variation and affinities among species of sect.Arachis which have been proposed as possible progenitors for the domesticated peanut. Ten primers resolved 132 DNA bands which were useful for separating species and accessions. The most variation was observed among accessions ofA. cardenasii andA. glandulifera whereas the least amount of variation was observed inA. hypogaea andA. monticola. The two tetraploid species could not be separated by using RAPDs.Arachis duranensis was most closely related to the domesticated peanut and is believed to be the donor of the A genome. The data indicated thatA. batizocoi, a species previously hypothesized to contribute the B genome toA. hypogaea, was not involved in its evolution. The investigation showed that RAPDs can be used to analyze both inter- and intraspecific variation in peanut species. Southern hybridization of RAPD probes to blots containing RAPD of theArachis species provided information on genomic relationships and revealed the repetitive nature of the amplified DNA.     

Characterization and transferability of microsatellite markers of the cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis>)

Background  

The genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species.     

CYTOLOGICAL AND INTERFERTILITY RELATIONSHIPS OF ARACHIS SECTION ARACHIS

Twenty-nine recently introduced diploid (2n = 2x = 20) accessions of section Arachis plus an A. correntina (Burk) Krap. et Greg. nom. nud. control were hybridized to the diploid A-genome species A. duranensis Krap. et Greg. nom. nud. (ace. 7988), the diploid B-genome species A. batizocoi Krap. et Greg. (acc. 9484), and with two subspecies of the A-B genome (2n = 4x = 40) A. hypogaea cultivars NC 4 and Argentine. Most attempted crosses were successful and the resulting plants were vigorous. However, A. batizocoi × accession 30008 hybrids died as seedlings and A. batizocoi × accession 30017 produced only dwarf plants. The 710 diploid F₁s from A. batizocoi were generally sterile, while those from A. duranensis had fertility ranges from 5% to 84%. Meiotic chromosome relationships in diploid crosses were cytologically evaluated in 185 plants plus tester accessions. Most taxa in section Arachis have an A genome, only A. batizocoi accessions have a B genome, a D genome is represented by accessions 30091 and 30099, and two other genomic groups, represented by accessions 30011 and 30033, may be present in the section. Most cytological differentiation was found among species originally collected in southern and eastern Bolivia. On the other hand, species collected at the extremes of the distribution of section Arachis species (northern Argentina to north-central Brazil) were cytologically very similar. Evidence is presented for speciation in Arachis being associated with both genetic differentiation and with translocated chromosomes. All taxa in the section except the D-genome species are believed to be cross-compatible with A. hypogaea, so germplasm introgression from most Arachis species should be possible.     

Comparative Embryo Sac Morphology at Anthesis of Cultivated and Wild Species of Arachis

Introgression of germplasm from wild to cultivated species ofArachis is severely impeded because abortion processes oftenoccur as a prepeg-, peg-(gynophore), or postpeg-elongation event.A comparative study of embryo sac morphology at anthesis wasundertaken to determine if observable differences were presentthat could possibly explain abortion prior to peg tip swellingfollowing soil penetration. Two wild Arachis species (A. duranensisand A. stenosperma) plus A. hypogaea cultivars NC 6 and Argentinewere studied. Differences in starch grain concentration andcytoplasmic stranding organization were observed between A.hypogaea cultivars and the wild Arachis species. These differencesprobably have a significant impact on energy availability atsyngamy and the subsequent early cell division of the embryo.An improper energy balance could contribute to the onset ofabortion in interspecific hybrids. Modification in egg apparatusorganization among all species was also observed which may accountfor low percentages of seed recovery resulting from interspecifichybridization attempts. Embryo sac morphology, interspecific hybridization, Arachis hypogaea L., Arachis species, light microscopy, scanning electron microscopy, embryo abortion, fertilization incompatibility, peanuts, groundnut     

Isozyme variation in germplasm accessions of the wild oat Avena sterilis L.

Summary Optimal exploitation of crop genetic resources requires a knowledge of the range and structure of the variation present in the gene pool of interest. Avena sterilis L., the cultivated oat progenitor, contains a store of genetic diversity that is readily accessible to the oat breeder. The objectives of the present paper were: (1) to evaluate isozyme polymorphisms in a sample of A. sterilis accessions from the U.S. National Small Grains Collection, (2) to analyze the distribution of isozyme diversity across the geographic range of the accessions, (3) to classify the accessions into groups based on isozyme variation, and (4) to suggest strategies for efficient sampling of this germplasm collection. One thousand and five accessions from 23 countries and 679 collection sites were screened for variation using 23 enzyme systems. Due to limited information about the genetic relationship among individual members of families of isozymes in hexaploid oat species, data were recorded solely for band presence. The frequencies of bands in accessions from the various countries were used to calculate the probability of genotypic identity (I_x.y), the probability of a unique genotype (U_x.y), and an adjusted polymorphic index (H_x). Accessions from Turkey and Lebanon had the largest polymorphic index values, Turkish and Moroccan accessions displayed the greatest numbers of bands. Accessions from Iran, Turkey, Iraq, and Lebanon had the largest mean probabilities of containing unique genotypes. Based on isozyme data, Turkey appeared to represent the center of diversity in this germplasm collection. Band frequencies calculated among countries were used in a principal component analysis. Accessions from Israel and Morocco clustered together; accessions from Iran, Iraq, Turkey, and Ethiopia formed another group; and Algerian accessions formed an outlying group. Several isozyme bands had a regional distribution. These results suggested that choosing accessions from countries based on their groupings in the principal component analysis should secure a greater range of diversity than sampling from the collection at random. Cluster analyses based on Jaccard's distances calculated for all pairwise combinations of the 1005 accessions revealed six broad genetic groups of accessions. Groups 1 and 6 contained accessions from many countries and encompassed half of all accessions. Groups 2 and 4 were heavily populated by accessions from Israel and Morocco. Groups 3 and 5 were composed almost exclusively of accessions from Iran, Iraq, and Turkey. By selecting representative accessions from these six groups, oat breeders could most effectively sample the range of genetic variation in this A. sterilis collection.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Genetic diversity and relationships detected by isozyme and RAPD analysis of crop and wild species of Amaranthus

 Genetic diversity and relationships of 23 cultivated and wild Amaranthus species were examined using both isozyme and RAPD markers. A total of 30 loci encoding 15 enzymes were resolved, and all were polymorphic at the interspecific level. High levels of inter-accessional genetic diversity were found within species, but genetic uniformity was observed within most accessions. In the cultivated grain amaranths (A. caudatus, A. cruentus, and A. hypochondriacus), the mean value of H_T was 0.094, H_S was 0.003, and G_ST was 0.977 at the species level. The corresponding values in their putative wild progenitors (A. hybridus, A. powellii, and A. quitensis) were 0.135, 0.004, and 0.963, respectively. More than 600 RAPD fragments were generated with 27 arbitrary 10-base primers. On average, 39.9% of the RAPD fragments were polymorphic among accessions within each crop species; a similar level of polymorphism (42.8%) was present in the putative progenitors, but much higher levels of polymorphism were found in vegetable (51%) and other wild species (69.5%). The evolutionary relationships between grain amaranths and their putative ancestors were investigated, and both the RAPD and isozyme data sets supported a monophyletic origin of grain amaranths, with A. hybridus as the common ancestor. A complementary approach using information from both isozymes and RAPDs was shown to generate more accurate estimates of genetic diversity, and of relationships within and among crop species and their wild relatives, than either data set alone. Received: 13 March 1997/Accepted: 6 May 1997     

Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of rDNA loci and heterochromatin detection: a new proposal for genome arrangement

Arachis hypogaea is an allotetraploid species with low genetic variability. Its closest relatives, all of the genus Arachis, are important sources of alleles for peanut breeding. However, a better understanding of the genome constitution of the species and of the relationships among taxa is needed for the effective use of the secondary gene pool of Arachis. In the present work, we focused on all 11 non-A genome (or B genome sensu lato) species of Arachis recognized so far. Detailed karyotypes were developed by heterochromatin detection and mapping of the 5S and the 18S–25S rRNA using FISH. On the basis of outstanding differences observed in the karyotype structures, we propose segregating the non-A genome taxa into three genomes: B sensu stricto (s.s.), F and K. The B genome s.s. is deprived of centromeric heterochromatin and is homologous to one of the A. hypogaea complements. The other two genomes have centromeric bands on most of the chromosomes, but differ in the amount and distribution of heterochromatin. This organization is supported by previously published data on molecular markers, cross compatibility assays and bivalent formation at meiosis in interspecific hybrids. The geographic structure of the karyotype variability observed also reflects that each genome group may constitute lineages that have evolved through independent evolutionary pathways. In the present study, we confirmed that Arachis ipaensis was the most probable B genome donor for A. hypogaea, and we identified a group of other closely related species. The data provided here will facilitate the identification of the most suitable species for the development of prebreeding materials for further improvement of cultivated peanut.     

Evaluation of Lespedeza germplasm genetic diversity and its phylogenetic relationship with the genus Kummerowia

The genetic diversity of the genus Lespedeza is not well known and the phylogenetic relationship of Lespedeza with the genus Kummerowia is unclear. We report the first study in which polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from Medicago, cowpea and soybean were used to assess the genetic diversity of the USDA Lespedeza germplasm collection and clarify its phylogenetic relationship with the genus Kummerowia. Phylogenetic analysis partitioned 44 Lespedeza accessions into three main groups some of which were species-specific and eight subgroups. This data set revealed some misidentified accessions, and indicated that the two species in the genus Kummerowia are closely related to the genus Lespedeza. Morphological reexamination was used to correct the misidentified accessions within the genus Lespedeza. Our results demonstrated that phylogenetic analysis with morphological reexamination provides a more complete approach to classify accessions in plant germplasm collection and conservation.     

Genetic diversity among wild and cultivated populations of Hevea brasiliensis assessed by nuclear RFLP analysis

Restriction fragment length polymorphism was assessed in wild and cultivated populations of Hevea brasiliensis using random probes from an Hevea nuclear library. One-hundred-and-sixty-four individuals were surveyed, and the results discussed in the light of previous work performed on isozyme variation. Both studies show that germplasm collections have led to an effective enrichment of the genetic resources available for Hevea breeding, and that cultivated clones have conserved a relatively high level of polymorphism, despite their narrow genetic base and their high level of inbreeding. An equivalent level of polymorphism is revealed by random nuclear probes and isozymes. However, the genetic structuring of the diversity appears more striking using RFLP markers. Wild accessions can be divided into three genetic groups according to their geographical origin. The present results are an essential guide to the incorporation of wild material in breeding schemes.     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

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.     

Development of an RFLP linkage map in diploid peanut species

An RFLP linkage map of peanut has been developed for use in genetic studies and breeding programs aimed at improving the cultivated species (Arachis hypogaea L.). An F₂ population derived from the interspecific hybridization of two related diploid species in the sectionArachis (A. stenosperma ×A. cardenasii) was used to construct the map. Both random genomic and cDNA clones were used to develop the framework of the map. In addition, three cDNA clones representing genes coding for enzymes involved in the lipid biosynthesis pathway have been mapped in peanut. Of the 100 genomic and 300 cDNA clones evaluated, 15 and 190, respectively, revealed polymorphisms among the parents of our mapping population. Unfortunately, a large number of these produced complex banding patterns that could not be mapped. Of the 132 markers analyzed for segregation, 117 are distributed among 11 linkage groups, while 15 have not yet been associated with any other marker. A total map distance of approximately 1063 cM has been covered to-date.     

Restriction fragment length polymorphism evaluation of six peanut species within the Avachis section

Summary Restriction fragment length polymorphisms (RFLP) were assessed among accessions within six peanut species of the Arachis section: tetraploid cultivated species, A. hypogaea; tetraploid wild species, A. monticola; and four diploid wild species, A. batizocoi,A. cardenasii, A. duranensis and A. glandulifera. While the two tetraploid species did not show polymorphism with 16 PstI-generated random genomic probes, two of seven seed cDNA probes detected polymorphisms. The RFLP variation detected by two seed cDNA probes appeared to be related to structural changes occurring within tetraploid species. The botanical var. fastigiata (Valencia market type) of A. hypogaea subspecies fastigiata was shown to be the most variable. Arachis monticola was found to be more closely related to A. hypogaea subspecies hypogaea than to subspecies fastigiata. Diploid species A. cardenasii, A. duranensis, and A. glandulifera showed considerable intraspecific genetic diversity, but A. batizocoi showed little polymorphism. The genetic distance between the cultivated peanut and wild diploid species was found to be closest for A. duranensis.Florida Agricultural Experiment Station, Journal Series No. R-01493     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci