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.     

RFLP variability in peanut (Arachis hypogaea L.) cultivars and wild species

Summary RFLP variability was studied in eight U.S. peanut cultivars, representing the four market types, and in 14 wild Arachis species accessions, using random genomic clones from a PstI library. Very low levels of RFLP variability were found among the allotetraploids, which included the U.S. cultivars and Arachis monticola, a wild species. The diploid wild species were very diverse, however. RFLP patterns of the allotetraploids were more complex than the diploids, and the two constituent genomes could usually be distinguished. On the basis of RFLP band sharing, A. ipaensis, A. duranensis, and A. spegazzinii appeared most closely related to the diploid progenitor species of the allotetraploids. A dendrogram of relationships among the diploid wild species was constructed based on band sharing.     

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.     

Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny

The economically important genus Arachis (Fabaceae) comprises 80 species restricted to South America. One monograph on the genus divided it into nine sections and included an intuitive assessment of evolutionary relationships. There is no comprehensive phylogenetic study of the genus. To test the current systematic treatment of the genus, we reconstructed a phylogeny for Arachis using nuclear ITS and plastid trnT–trnF sequences from 46 species representing all nine sections. ITS cloning of the allotetraploid species of section Arachis indicated the presence of A and B genome alleles and chimeric sequences. Our study revealed that species from section Extranervosae were the first emerging lineage in the genus, followed by sections Triseminatae and Caulorrhizae, and two terminal major lineages, which we refer to as erectoides and arachis. The lineage erectoides comprises members of sections Erectoides, Heteranthae, Procumbentes, Rhizomatosae, and Trierectoides. Species in the arachis lineage form two major clades, arachis I (B and D genomes species and the aneuploids) and arachis II (A genome species). Our results substantiated the sectional treatment of Caulorrhizae, Extranervosae, and Triseminatae, but demonstrated that sections Erectoides, Procumbentes, and Trierectoides are not monophyletic. A detailed study of the genus Arachis with denser taxon sampling, additional genomic regions, plus information from morphology and cytogenetics is needed for comprehensive assessment of its systematics.     

Genomic in situ hybridization inArachis (Fabaceae) identifies the diploid wild progenitors of cultivated (A. hypogaea) and related wild (A. monticola) peanut species

Genomic in situ hybridization offers a powerful tool for investigating genome organisation and evolution of taxa known, or suspected, to be allopolyploids. The question of the diploid progenitors of cultivated peanut (Arachis hypogaea, 2n=4x=40) has been the subject of numerous studies at cytogenetical, cytochemical, biochemical and molecular levels, but no definitive conclusions have been reached. The biotinylated total genomic DNA from potential diploidArachis species were separately hybridized in situ to root tip chromosomes ofA. hypogaea and wild speciesA. monticola (2n=4x=40) without or mixed with an excess of unlabelled DNA from the species not used as a probe. Among the range of different species combinations used, the strong and uniform signals given by labelledA. ipaensis DNA when hybridized toA. hypogaea andA. monticola in combination with unlabelledA. villosa DNA indicates that overall molecular composition of twenty chromosomes ofA. hypogaea andA. monticola is very similar toA. ipaensis chromosomes. ProbingA. hypogaea andA. monticola chromosomes with labelled genomic DNA fromA. villosa mixed with unlabelled DNA fromA. ipaensis likewise labelled strongly and uniformly the other twenty chromosomes. BarringA. ipaensis, all the diploidArachis species presently investigated had characteristic centromeric bands in the twenty chromosomes within the complement indicating a clear division ofA. ipaensis from other species. InA. hypogaea andA. monticola only twenty chromosomes showed centromeric bands. These results (i) confirm the allopolyploid nature ofA. hypogaea andA. monticola, (ii) strongly support the view that wildA. monticola and cultivatedA. hypogaea are very closely related, and (iii) indicate thatA. villosa andA. ipaensis are the diploid wild progenitors of the tetraploid species studied. The present results also reveal that the nucleolus organizing region (NOR) originating fromA. villosa alone is expressed in the two tetraploid species.     

Utilisation of wild relatives in the genetic improvement of Arachis hypogaea L.

Summary Cross-compatibility of species in section Arachis Krap. et Greg. nom. nud., and chromosome pairing and pollen fertility in their interspecific F₁ hybrids were studied to further understand the phylogenetic relationships among these species. Except those with A. batizocoi Krap. et Greg. nom. nud., hybrids between diploid species have near normal bivalent frequency (9.1–9.8) and moderate to high pollen fertility (60–91%). Hybrids between A. batizocoi and other species have low bivalent frequency (5.2–6.9) and very low pollen fertility (3–7%). These results confirm the earlier separation of these species into two groups based on karyomorphology and Mahalanobis D² calculated on arm ratios. These studies also provide a picture of relative affinities between A. batizocoi, the lone member of one cluster, and the other species, and among the rest of the species. They also indicate that the basic chromosome complement in the two groups of species is the same. Chromosome pairing in triploid hybrids, (A. hypogaea L. X diploid wild species), suggests that A. batizocoi is the closest diploid relative of A. hypogaea. It is closer to A. hypogaea subspecies fastigiata Waldron than to A. hypogaea subspecies hypogaea Krap. et. Rig. Other diploid species of the section Arachis are equidistant from A. hypogaea, and have the same genome which has strong homology to one of the genomes of A. hypogaea. Based on the present results, the two tetraploid species, A. monticola Krap. et Rig. and A. hypogaea can be recognised as two forms of the same species. Breeding implications have been discussed in the light of chromosome behaviour observed in hybrids of A. hypogaea X diploid species, and on the presumptions that A. hypogaea has an AABB genomic constitution, and that among the diploid species, the B genome is present in A. batizocoi while the A genome is common to the other diploid species of section Arachis.Submitted as Journal Article No. 328 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

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.     

A preliminary pachytene analysis of two species of Arachis L.

Summary The morphology of pachytene chromosomes was studied in A. glabrata Benth. and A. pusilla Benth. belonging respectively to the sections Rhizomatosae and Triseminale. These two species can not be crossed with the cultivated groundnut A. hypogaea L. All 20 chromosomes of A. glabrata could be identified individually and further classified into 5 basic types. The features that enabled the identification of chromosomes were: total length, arm ratios, nucleolus attachment and position and extent of heterochromatin. A simple key has been proposed for classifying different chromosomes to facilitate their easy identification. The genomes of A. glabrata did not resemble those of A. hypogaea except for the presence of an A chromosome, 2 euchromosomes and 2 nucleolus organisers. A. glabrata did not appear to be an amphidiploid but rather an allopolyploid hybrid. The genome of A. pusilla contained chromosomes unlike those of any other species of section Arachis. It was concluded that both these species are quite unrelated to other species of the section Arachis.     

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.     

A single dominant gene for Fusarium wilt resistance in protoplast-derived tomato plants

Summary Autotetraploids were established from 8 diploid wild species of section Arachis. In all the autotetraploids the chromosomes paired largely as bivalents even though they possess the ability to pair as multivalents. Pollen and pod fertility in the C₁ generation were not directly associated with chromosome pairing. The C₂ generation autotetraploids showed a gradual increase in bivalent associations and pollen and pod fertility. The identification of two genomes, A and B, in the diploid species and in the tetraploid, A. hypogaea, of the section Arachis, a fairly good crossability, and the type of chromosome associations observed in hybrids between A. hypogaea and the autotetraploids of wild Arachis species indicated good prospects of utilizing autotetraploids as genetic bridges in transferring desired traits from these taxa into groundnut.Submitted as Journal Article No. 516 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

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.     

RFLP AND CYTOGENETIC EVIDENCE ON THE ORIGIN AND EVOLUTION OF ALLOTETRAPLOID DOMESTICATED peanut,Arachis hypogaea (Leguminosae)

Nuclear restriction fragment length polymorphism (RFLP) analysis was used to determine the wild diploid Arachis species that hybridized to form tetraploid domesticated peanut. Results using 20 previously mapped cDNA clones strongly indicated A. duranensis as the progenitor of the A genome of domesticated peanut and A. ipaensis as the B genome parent. A large amount of RFLP variability was found among the various accessions of A. duranensis, and accessions most similar to the A genome of cultivated peanut were identified. Chloroplast DNA RFLP analysis determined that A. duranensis was the female parent of the original hybridization event. Domesticated peanut is known to have one genome with a distinctly smaller pair of chromosomes (“A”), and one genome that lacks this pair. Cytogenetic analysis demonstrated that A. duranensis has a pair of “A” chromosomes, and A. ipaensis does not. The cytogenetic evidence is thus consistent with the RFLP evidence concerning the identity of the progenitors. RFLP and cytogenetic evidence indicate a single origin for domesticated peanut in Northern Argentina or Southern Bolivia, followed by diversification under the influence of cultivation.     

Utilization of wild relatives in the genetic improvement of Arachis hypogaea L.

Summary Synthetic amphidiploids were established in 32 combinations involving 8 diploid wild species representing both A and B genomes of section Arachis. Bivalent and multivalent associations in the amphidiploids of 7 A genome species confirm that these species have identical genomes. Contrastingly, high bivalent frequencies in amphidiploids involving the A and B genome species suggest that A. batizocoi has a distinct B genome that is partially homologous to the other genome A represented in the rest of the species. Crossability, chromosome pairing and pollen and pod fertility in hybrids between A. hypogaea and amphidiploids have revealed that these amphidiploids can be used as a genetic bridge for the transfer of genes from the wild species into the cultivated groundnut.Submitted as Journal Article No. 530 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection

Section Arachis of the homonymous genus includes 29 wild diploid species and two allotetraploids (A. monticola and the domesticated peanut, A. hypogaea L.). Although, three different genomes (A, B and D) have been proposed for diploid species with x = 10, they are still not well characterized. Moreover, neither the relationships among species within each genome group nor between diploids and tetraploids (AABB) are completely resolved. To tackle these issues, particularly within the A genome, in this study the rRNA genes (5S and 18S–26S) and heterochromatic bands were physically mapped using fluorescent in situ hybridization (FISH) in 13 species of Arachis. These molecular cytogenetic landmarks have allowed individual identification of a set of chromosomes and were used to construct detailed FISH-based karyotypes for each species. The bulk of the chromosome markers mapped revealed that, although the A genome species have a common karyotype structure, the species can be arranged in three groups (La Plata River Basin, Chiquitano, and Pantanal) on the basis of the variability observed in the heterochromatin and 18S–26S rRNA loci. Notably, these groups are consistent with the geographical co-distribution of the species. This coincidence is discussed on the basis of the particular reproductive traits of the species such as autogamy and geocarpy. Combined with geographic distribution of the taxa, the cytogenetic data provide evidence that A. duranensis is the most probable A genome ancestor of tetraploid species. It is expected that the groups of diploid species established, and their relation with the cultigen, may aid to rationally select wild species with agronomic traits desirable for peanut breeding programs.     

Phylogenetic relations in section Arachis based on seed protein profile

Summary Seed protein profiles of nine diploid species (2n = 20), ten tetraploid accessions, two synthetic amphidiploids and two autotetraploids (2n = 40) were studied using SDS-polyacrylamide gel electrophoresis. While the general profiles suggested considerable homology among these taxa in spite of speciation and ploidy differences, appreciable genetic differences were present to support the existing genomic divisions and sub-divisions in the section Arachis. A high degree of relationship was indicated between the two diploid species (A. duranensis containing the A genome and A. batizocoi (ICG 8210) containing the B genome) and tetraploids A. monticola/ A. hypogaea (2n = 40) containing AABB genome. Similar relationships were recorded between the AABB synthetic amphidiploid and the profile obtained from the mixture of protein of A. duranensis and A. batizocoi, suggesting that these two diploid species were the donors of the A and B genome, respectively, to tetraploid A. monticola/A. hypogaea.Submitted as Journal Article No. 1114 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Profiling microRNA expression in Arabidopsis pollen using microRNA array and real-time PCR

Background

Arachis hypogaea (peanut) is an important crop worldwide, being mostly used for edible oil production, direct consumption and animal feed. Cultivated peanut is an allotetraploid species with two different genome components, A and B. Genetic linkage maps can greatly assist molecular breeding and genomic studies. However, the development of linkage maps for A. hypogaea is difficult because it has very low levels of polymorphism. This can be overcome by the utilization of wild species of Arachis, which present the A- and B-genomes in the diploid state, and show high levels of genetic variability.

Results

In this work, we constructed a B-genome linkage map, which will complement the previously published map for the A-genome of Arachis, and produced an entire framework for the tetraploid genome. This map is based on an F₂ population of 93 individuals obtained from the cross between the diploid A. ipaënsis (K30076) and the closely related A. magna (K30097), the former species being the most probable B genome donor to cultivated peanut. In spite of being classified as different species, the parents showed high crossability and relatively low polymorphism (22.3%), compared to other interspecific crosses. The map has 10 linkage groups, with 149 loci spanning a total map distance of 1,294 cM. The microsatellite markers utilized, developed for other Arachis species, showed high transferability (81.7%). Segregation distortion was 21.5%. This B-genome map was compared to the A-genome map using 51 common markers, revealing a high degree of synteny between both genomes.

Conclusion

The development of genetic maps for Arachis diploid wild species with A- and B-genomes effectively provides a genetic map for the tetraploid cultivated peanut in two separate diploid components and is a significant advance towards the construction of a transferable reference map for Arachis. Additionally, we were able to identify affinities of some Arachis linkage groups with Medicago truncatula, which will allow the transfer of information from the nearly-complete genome sequences of this model legume to the peanut crop.     

Variation of isozyme patterns among Arachis species

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

Genetic 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.     

Early Reproductive Ontogeny in Interspecific Crosses of Arachis hypogaea and Section Arachis Species

Wild Arachis species have been recognized as sources of resistanceto pests and pathogens that infect A. hypogaea L. and causesubstantial yield losses. However, utilization of these geneticresources for crop improvement has been difficult. This studywas conducted to (a) understand the processes of early embryogrowth and development in four Arachis species, two A. hypogaeacultivars and their hybrids and (b) identify parental compatibilitiesin reciprocal crosses of A. hypogaea. The results indicatedthat delayed fertilization beyond 24 h, coupled with slow proembryogrowth, leads to embryo abortion in many interspecific crosses.For example, in female A. cardenasii crosses, lack of or delayedfertilization leads to failure to obtain hybrids. When A. batizocoiwas used as a female parent, delayed fertilization and the inabilityof quiescent proembryos to resume growth after soil penetrationcaused abortion. Embryos of A. hypogaea x A. glandulifera crossesdeveloped normally during the first 21 d after fertilization,but then aborted at a later time. In this study, A. hypogaeawas always a better female parent than the wild Arachis species.Increasing the number of pollinations per cross, using the cultivatedspecies as the female parent, utilizing different A. hypogaeavarieties, and embryo rescue techniques are suggested to improvethe probability of obtaining interspecific hybrids in Arachis.Copyright1995, 1999 Academic Press Peanut, interspecific hybrids, Arachis, wild species, incompatibilities