H. T. Stalker

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.

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.

Genomic affinities in Arachis section Arachis (Fabaceae): molecular and cytogenetic evidence

Section Arachis is the largest of nine sections in the genus Arachis and includes domesticated peanut, A. hypogaea L. Most species are diploids (x=10) with two tetraploids and a few aneuploids. Three genome types have been recognized in this section (A, B and D), but the genomes are not well characterized and relationships of several newly described species are uncertain. To clarify genomic relationships in section Arachis, cytogenetic information and molecular data from amplified fragment length polymorphism (AFLP) and the trnT-F plastid region were used to provide an additional insight into genome composition and species relationships. Cytogenetic information supports earlier observations on genome types of A. cruziana, A. herzogii, A. kempff-mercadoi and A. kuhlmannii but was inconclusive about the genome composition of A. benensis, A. hoehnei, A. ipaensis, A. palustris, A. praecox and A. williamsii. An AFLP dendrogram resolved species into four major clusters and showed A. hypogaea grouping closely with A. ipaensis and A. williamsii. Sequence data of the trnT-F region provided genome-specific information and showed for the first time that the B and D genomes are more closely related to each other than to the A genome. Integration of information from cytogenetics and biparentally and maternally inherited genomic regions show promise in understanding genome types and relationships in Arachis.

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.

Diversity of seed storage protein patterns in wild peanut (Arachis, Fabaceae) species

Abstract55 accessions of wild peanuts (Arachis spp.) introduced from South America were analyzed for seed storage protein composition using SDS-PAGE electrophoresis. The objectives of the study were to evaluate variability within sect.Arachis and to classify taxa based on protein composition. 25 different band positions were resolved. Individual accessions had 11 to 18 bands which included the conarachin region (MW > 50 kD), two to five bands in the acidic arachin region (MW 38–49.9 kD), three to seven in the intermediate MW region (23 to 37.9 kD), two to five bands in the basic arachin region (18–22.9 kD), and one to three bands in the low MW protein region (14–17.9 kD). These data were utilized in a principal coordinate analysis based on the matrix of genetic distances between all pairs of the 55 accessions. Several groups of accessions conformed to expected species classification includingA. batizocoi, A. stenosperma, andA. monticola; whileA. duranensis, A. cardenasii, A. helodes, andA. correntina did not form good groups. The study showed that great diversity exists for protein profiles and seed storage proteins have potential for aiding species classification and for serving as markers for interspecific hybridization studies.

A RAPD-based Linkage Map of Peanut Based on a Backcross Population Between the Two Diploid Species Arachis stenosperma and A. cardenasii

Abstract A molecular linkage map based on an interspecific diploid backcross population [Arachis stenosperma × (A. stenosperma × A. cardenasii)] was constructed utilizing RAPD and RFLP markers. One hundred sixty-seven RAPD loci and 39 RFLPs were mapped to 11 linkage groups, covering a total genetic length of 800 cM. Clusters of 2 to18 markers were observed in most linkage groups. Twenty seven percent of the markers showed segregation distortion and mapped to four regions. Thirty-nine RFLP markers shared with a previously published linkage map, based on an A. stenosperma × A. cardenasii F2 population, and six RAPD markers were used to establish correspondence between maps and to compare recombination frequencies between common markers. A generalized reduction in the recombination fraction was observed in the backcross map compared to the F2 map. All common markers mapped to the same linkage groups and mostly in the same order in both maps.

Assessment of oil content and fatty acid composition variability in different peanut subspecies and botanical varieties

Within the cultivated peanut species (Arachis hypogaea L.), there are two subspecies compris- ing six botanical varieties, and the effect of botanical taxon on oil content and fatty acid com- position variability is unclear. To gauge the variability, 83 peanut accessions were analyzed for oil content (expressed at 0% moisture) and fatty acid composition. We found that within the subsp. hypogaea, var. hypogaea contained a much higher amount of oil in seeds than did the var. hirsuta Kohler (520 vs. 473 g/kg, P , 0.05); within the subsp. fastigiata Waldron, the vars. aequatoriana Krapov. & W.C. Gregory and vulgaris Harz contained a similar amount of oil in seeds (491 g/kg), not significantly different from other botanical varieties, but var. fastigiata contained a higher amount of oil (500 g/kg) than the var. peruviana Krapov. & W.C. Gregory (483 g/kg). In terms of the fatty acid composition, oil from seeds of var. hypogaea contained much more oleic acid than did var. hirsuta (491 vs. 377 g/kg, P , 0.05), but much less palmitic acid (97 vs. 138 g/kg, P , 0.05%) and linoleic acid (308 vs. 402 g/kg, P , 0.05). Oil from seeds of var. vulgaris contained much more oleic acid than did var. aequatoriana (437 vs. 402 g/kg, P , 0.05), but much less linoleic acid (346 vs. 380 g/kg, P , 0.05). Significant negative correlations of oleic with palmitic and linoleic acids were detected. The information on the oil content and fatty acid composition variability among botanical varieties would be useful for peanut breeders seeking germplasm containing both high oil content and proper fatty acid composition.

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.

Molecular analysis of Arachis interspecific hybrids

Incorporation of genetic resistance against several biotic stresses that plague cultivated peanut, Arachis hypogaea (2n=4x=40), is an ideal option to develop disease resistant and ecologically safe peanut varieties. The primary gene pool of peanut contains many diploid wild species (2n=2x=20) of Arachis, which have high levels of disease and insect resistances. However, transfer of resistant genes from these species into A. hypogaea is difficult due to ploidy level differences and genomic incompatibilities. This study was conducted to monitor alien germplasm transmission, using Random Amplified Polymorphic DNA (RAPD) markers, from two diploid wild species, A. cardenasii and A. batizocoi, into A. hypogaea. Triploid interspecific hybrids were produced by crossing two A. hypogaea cultivars (NC 6 and Argentine) with the two species and by colchicine-treating vegetative meristems, fertility was restored at the hexaploid (Co) level in the four hybrids. Hexaploids were allowed to self-pollinate for four generations, each referred to as a cycle (C1, C2, C3, and C4). At each cycle, a backcross was made with the respective A. hypogaea cultivar as the maternal parent and only lineages tracing back to a single hexaploid hybrid were used for RAPD analysis. Analysis of mapped, species-specific RAPD markers in BC1F1 to BC1F3 hybrids indicated that alien germplasm retention decreased every generation of inbreeding, especially in Argentine and in A. batizocoi crosses. A similar trend was also observed for every cycle in BC1F2 and BC1F3 families, possibly, due to the loss of alien chromosomes following selfing of hexaploids. RAPD marker analysis of 40–chromosome interspecific hybrid derivatives from the four crosses supported previous reports that reciprocal recombination and/or translocations are the predominant mechanisms for exchange of chromosomal segments. No evidence was found for preferential transfer of alien chromosomal regions to specific linkage groups. The implications for developing disease resistant peanut breeding lines are discussed in light of these findings.

An analysis of the B genome speciesArachis batizocoi (Fabaceae)

Arachis batizocoiKrap. & Greg. is a suggested B genome donor to the cultivated peanut,A. hypogaea L. Until recently, only one accession of this species was available in U.S.A. germplasm collections for analyses and species variability had not been documented. The objective of this study was to determine the intraspecific variability ofA. batizocoi to better understand phylogenetic relationships in sect.Arachis. Five accessions of the species were used for morphological and cytological studies and then F1 intraspecific hybrids analyzed. Some variation was observed among accessions—for example, differences in seed size, plant height and branch length. The somatic chromosomes of accessions 9484, 30079, and 30082 were nearly identical, whereas, the karyotypes of accessions 30081 and 30097 have several distinct differences. For example, 30081 had significantly more asymmetrical chromosomes 2 and 6 and more median chromosomes 7 and 10, and 30097 had significantly more asymmetrical chromosomes 3 and 10 and more median chromosomes 1 and 5 than accessions 9484, 30079, and 30082. All F1 hybrids among accessions were highly fertile. Meiotic observations indicated that hybrids among accessions 9484, 30079, or 30082 had mostly bivalents. However, quadrivalents were observed when either 30081 or 30097 was crossed with the above three accessions and 30081 × 30097 had quadrivalents, hexavalents and octavalents. The presence of translocations is the most likely cause of multivalent formation inA. batizocoi hybrids. Cytological evolution via translocations has apparently been an important mechanism for differentiation in the species.