Guillermo Seijo

Genomic relationships between the cultivated peanut (Arachis hypogaea, Leguminosae) and its close relatives revealed by double GISH.

Arachis hypogaea is a natural, well-established allotetraploid (AABB) with 2n = 40. However, researchers disagree on the diploid genome donor species and on whether peanut originated by a single or multiple events of polyploidization. Here we provide evidence on the genetic origin of peanut and on the involved wild relatives using double GISH (genomic in situ hybridization). Seven wild diploid species (2n = 20), harboring either the A or B genome, were tested. Of all genomic DNA probe combinations assayed, A. duranensis (A genome) and A. ipaensis (B genome) appeared to be the best candidates for the genome donors because they yielded the most intense and uniform hybridization pattern when tested against the corresponding chromosome subsets of A. hypogaea. A similar GISH pattern was observed for all varieties of the cultigen and also for A. monticola. These results suggest that all presently known subspecies and varieties of A. hypogaea have arisen from a unique allotetraploid plant population, or alternatively, from different allotetraploid populations that originated from the same two diploid species. Furthermore, the bulk of the data demonstrated a close genomic relationship between both tetraploids and strongly supports the hypothesis that A. monticola is the immediate wild antecessor of A. hypogaea.

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.

An overview of peanut and its wild relatives

Abstract The legume Arachis hypogaea, commonly known as peanut or groundnut, is a very importantfood crop throughout the tropics and sub-tropics. The genus is endemic to South Americabeing mostly associated with the savannah-like Cerrado. All species in the genus are unusualamong legumes in that they produce their fruit below the ground. This profoundly influencestheir biology and natural distributions. The species occur in diverse habitats including grass-lands, open patches of forest and even in temporarily flooded areas. Based on a number ofcriteria,includingmorphologyandsexualcompatibilities,the80describedspeciesarearrangedinnineinfragenerictaxonomicsections.Whilemostwildspeciesarediploid,cultivatedpeanutisa tetraploid. It is of recent origin and has an AABB-type genome. The most probable ancestralspecies are Arachis duranensis and Arachis ipae¨nsis, which contributed the A and B genomecomponents, respectively. Although cultivated peanut is tetraploid, genetically it behaves as adiploid,theAandBchromosomesonlyrarelypairingduringmeiosis.Althoughmorphologicallyvariable, cultivated peanut has a very narrow genetic base. For some traits, such as disease andpest resistance, this has been a fundamental limitation to crop improvement using only culti-vated germplasm. Transfer of some wild resistance genes to cultivated peanut has beenachieved, for instance, the gene for resistance to root-knot nematode. However, a wider useof wild species in breeding has been hampered by ploidy and sexual incompatibility barriers,by linkage drag, and historically, by a lack of the tools needed to conveniently confirm hybrididentities and track introgressed chromosomal segments. In recent years, improved knowledgeof species relationships has been gained by more detailed cytogenetic studies and molecularphylogenies. This knowledge, together with new tools for genetic and genomic analysis, willhelp in the more efficient use of peanut’s genetic resources in crop improvement.

Genetic and geographic origin of domesticated peanut as evidenced by 5S rDNA and chloroplast DNA sequences

The history of the cultivated peanut involves natural evolution and human domestication. Despite the economic importance of peanuts and the many studies carried out on their cytology and genetic variability, current knowledge on the origin of the cultigen is still very limited compared with other major crops. In this context, we analyzed the polymorphisms of some non-coding cpDNA regions and the non-transcribed spacer of the nuclear 5S rDNA of the six botanical varieties of the two subspecies of the cultigen, of the wild tetraploid A. monticola, and of the nine diploid species so far proposed as the most probable relatives of the peanut, to gain more insight into the genetic and geographic origin of this legume crop. The analysis showed complete homology in the sequences of all the peanut and A. monticola samples. These results strongly suggest that the six botanical varieties of the cultigen have a single genetic origin and that A. monticola should be regarded as the immediate tetraploid ancestor from which A. hypogaea has arisen upon domestication. Here we provide results from the first sequence-based analysis in which the maternal (A. duranensis) and paternal (A. ipaënsis) wild diploid species of the AABB tetraploids of Arachis were unequivocally identified. Not only that, but the combination of cpDNA and NTS 5S rDNA identified the population of A. duranensis from Río Seco, Salta, Argentina, and the only known population of A. ipaënsis from Villa Montes, Tarija, Bolivia, as those to which the genome donors of the peanut could have belonged.

The repetitive component of the A genome of peanut (Arachis hypogaea) and its role in remodelling intergenic sequence space since its evolutionary divergence from the B genome.

BACKGROUND AND AIMS Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes. METHODS The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues. KEY RESULTS BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity. CONCLUSIONS A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.

Cytogenetic analysis of Lathyrus japonicus Willd.(Leguminosae)

Abstract L. japonicus belongs to section Orobus and grows in most of the seashores of the Northern Hemisphere and as adventitious in Chile. In this report a detailed analysis of karyotype and meiotic behaviour was carried out in order to provide data for phylogenetic analysis. This species presented a symmetrical karyotype with 2n=14, 6m+8sm chromosomes. The longest and the shortest chromosomes of the complement were metacentrics while the sm were very similar and medium in size. NORs, determined by argentic staining, were localized in secondary constriction of the long arm of the longest m chromosome. The symmetrical karyotype is in accordance with the ancestral morphological characters showed by L. japonicus. Meiotic behaviour was regular with a chiasma frequency of 2.5 per bivalent at metaphase I. This fact agrees with the 98.2% of pollen stainability estimation. However, in ana-telophase I a bridge and fragment (<1%) were observed, while in interphase only fragments were detected (1%). In addition, out of plate chromosomes appeared in metaphase I and II (2%). These chromosomes segregated independently, formed micronuclei in telophase II and they became micromicrospores at the tetrad stage. These micromicrospores correspond to the micro pollen grains observed in anthesis.

Genome re-assignment of Arachis trinitensis (Sect. Arachis, Leguminosae) and its implications for the genetic origin of cultivated peanut

The karyotype structure of Arachis trinitensis was studied by conventional Feulgen staining, CMA/DAPI banding and rDNA loci detection by fluorescence in situ hybridization (FISH) in order to establish its genome status and test the hypothesis that this species is a genome donor of cultivated peanut. Conventional staining revealed that the karyotype lacked the small “A chromosomes” characteristic of the A genome. In agreement with this, chromosomal banding showed that none of the chromosomes had the large centromeric bands expected for A chromosomes. FISH revealed one pair each of 5S and 45S rDNA loci, located in different medium-sized metacentric chromosomes. Collectively, these results suggest that A. trinitensis should be removed from the A genome and be considered as a B or non-A genome species. The pattern of heterochromatic bands and rDNA loci of A. trinitensis differ markedly from any of the complements of A. hypogaea, suggesting that the former species is unlikely to be one of the wild diploid progenitors of the latter.

Characterization of Brazilian accessions of wild Arachis species of section Arachis (Fabaceae) using heterochromatin detection and fluorescence in situ hybridization (FISH).

The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species.

Cytogeographic Analysis of Southern South American Species of Stemodia (Scrophulariaceae)

Chromosome numbers for 52 populations representing eight South American species of Stemodia (Scrophulariaceae) were determined. The numbers 2n = 22 found in S. hassleriana and S. palustris, and 2n = 44 in S. lobelioides are the first records for the species, while those found in S. hyptoides (2n = 22, 44) and S. stricta (2n = 22) constitute new cytotypes for those species. The basic chromosome number X = 11 was confirmed for the New World species. Chromosome numbers indicate the existence of a polyploid series in S. hyptoides with 2n = 22, 44, 66. Moreover, the existence of at least three different ploidy levels, both within and among species, indicates that polyploidy has been one of the mechanisms involved in the evolution of the genus. The geographical distribution of different species and cytotypes are analysed and discussed in the light of their extant morphological variation and taxonomic implications.

Cytological Features of Peanut Genome

This chapter aims to update the chromosomal features evaluated by classical and molecular cytogenetic techniques. Karyotype variability detected within and among species was very useful to unravel the taxonomy of the genus and to establish relationships among species. This chapter includes analyses of chromosome morphology, heterochromatin, rDNA loci, as well as dispersed and clustered repetitive sequences. A critical review of the genome sizes of Arachis species is also provided. The usefulness of chromosome data is presented in three examples. The first one deals with the origin of the cultivated peanut. Molecular cytogenetics evidenced that the varieties of A. hypogaea may have had a single genetic origin, that A. monticola is a direct tetraploid ancestor of peanut, and that A. duranensis (A genome) and A. ipaensis (B genome) are the diploid progenitors of the AABB tetraploids. The second one pointed to the analysis of the origin of the rhizomatous tetraploids and their relation to the unique diploid species (A. burkartii) of section Rhizomatosae. The cytogenetic data suggest that A. burkartii has to be discarded as a genome donor of the tetraploids, and that the latter may have had independent origins involving different species. The third one concerns the species of section Arachis, and how the chromosome data aided in the establishment of the genome groups (A, B, D, F, G, and K).