A high-density genetic map of Arachis duranensis, a diploid ancestor of cultivated peanut

Species relationships

A preliminary study of SSR marker variation among 37 A. duranensis accessions using 556 markers indicated that the species is highly polymorphic at the molecular level and individual accessions could be separated based on a cluster analysis (Figure 1). Interestingly, we found that A. ipaensis, the proposed B-genome (BB) progenitor species, clustered with the A-genome (AA) species A. stenosperma and not with the B-genome species A. batizocoi. Recent molecular cytogenetic analysis of A- and non-A- (i.e., B-) genome species suggests that karyotype diversity among non-A-genome species is extensive enough to support separation into additional genome classes where A. ipaensis remains in B sensu stricto while A. batizocoi is placed into a separate group [25]. Therefore, A. batizocoi is less typical of B-genome species.

The number of polymorphic SSR markers between paired A. duranensis accessions ranged from 160 to 375 out of 556, which is 29 to 67% of the total number of SSR markers screened. This is a significant amount of variation, which indicates the high genetic diversity within the species. Based on cluster analysis, success of crosses, and fertility of F₁s, accessions PI 475887 and Grif 15036 were selected for subsequent mapping studies using 94 F₂ progenies. Screening of the parental accessions with 2,138 SSR markers derived from A. hypogaea EST sequences resulted in 1,768 (82.7%) that were scorable (detected by ABI3730XL genotyping systems) and 896 (41.9%) that were polymorphic (Guo Y et al: Comparative mapping in intraspecific populations uncovers a high degree of macrosynteny between A- and B-genome diploid species of peanut, Submitted). The same markers were used to create a map between two A. batizocoi accessions and to determine syntenic relationships between the A and B genome species (Guo Y et al: Comparative mapping in intraspecific populations uncovers a high degree of macrosynteny between A-and B-genome diploid species of peanut, submitted).

Arachis duranensis genetic map

The total number of published SSR markers has now risen beyond the 2,847 cataloged in a related paper by Guo et al. (Guo Y et al: Comparative mapping in intraspecific populations uncovers a high degree of macrosynteny between A-and B-genome diploid species of peanut, submitted) to around 6,000 [26]. Those most recently reported include: 14 by Gimenes et al. [27]; 51 by Mace et al. [28]; 188 by Proite et al. [29]; 104 by Cuc et al. [30]; 138 by Yuan et al. [31]; 33 by Song et al. [32]; 123 by Wang et al. [33]; 290 by Liang et al. [34]; and 1,571 by Koilkonda et al. [35]. Five hundred and ninety-eight of these markers are included in the A. duranensis map (Figure 2). Of the 34 genomic SSR markers mapped in the current study (Table 1), 24 were mapped previously in an interspecific population between A. duranensis and A. stenosperma[6, 36]. These markers served to anchor and align the current and previously published peanut maps (Figure 2). Linkage group assignments of all markers were consistent between the current map and that of Bertioli et al. [36] except for the marker GM117 (AC3C02 on map in reference 36 derived from GenBank accession DQ099133) that was localized on chromosome 2A (the ‘A’ following a chromosome number is presented in this study to represent chromosomes in the A genome of peanut) in their interspecific map, while mapping to chromosome 10A in the A. duranensis intraspecific map. Although detailed information for parental alleles in the study by Bertioli et al. [36] was not presented, GM117 amplified only one locus from each parent in both their population and ours. It is, therefore, unlikely that the marker location discrepancy was due to mapping of multiple loci and perhaps could reflect a small chromosomal rearrangement. Chromosomal rearrangements are not unexpected based on previous cytological observations in the genus [24, 37].

The A. duranensis map produced in this study contained 1,724 markers combined into 10 linkage groups with a total genetic distance of 1081.3 cM. MSTMap, a software program that accommodates large numbers of markers and utilizes a “minimum spanning tree” algorithm, was used to construct an initial genetic map using only the codominant markers. The 1,673 codominant markers were distributed into 810 co-segregating groups (bins). Although this program has been reported to be accurate for large-scale mapping projects [38], few independent studies are available establishing consistency between MSTMap and other commonly used mapping software [39]. To confirm the linkage group assignments, marker orders, and genetic distances determined by alternative software, both codominant and dominant markers were mapped with Joinmap 3.0. Marker orders and genetic distances were highly consistent between MSTMap and Joinmap 3.0 (Additional file 2).

Significant segregation distortion (p = 0.05) was observed for 513 (29.8%) markers (Figure 2, Additional file 3). Chromosomes 4A and 9A carried particularly long segments of distorted segregation suggesting large-scale chromosomal selection in these regions. Guo et al. (Guo Y et al: Comparative mapping in intraspecific populations uncovers a high degree of macrosynteny between A-and B-genome diploid species of peanut, Submitted) found that a single linkage group (4/9B) in A. batizocoi was syntenic with chromosomes 4A and 9A of A. duranensis implicating inversion and reciprocal translocation events as the underlying chromosomal rearrangements in this B-genome species. Recombination frequencies were generally low in the central, presumably centromeric chromosomal regions of A. duranensis and increased toward the telomeres, a pattern typical of many plant species [40, 41]. More even distribution was observed along chromosome 3A and only slightly suppressed recombination was observed around the presumable location of the centromere (Figure 2).

The map produced in the current study is the first high-density map available in peanut, and because it was generated from a progenitor species of A. hypogaea, we anticipate that it will have significant applications for analyzing the cultivated genome. For example, the data generated in this map was used by Nagy et al. [43] to more precisely map the Rma gene for nematode resistance that originated from an introgression line between A. hypogaea and A. cardenasii. The A-genome SNP array also has been useful at the tetraploid level for genotyping a recombinant inbred line population derived from a cross between cultivated peanut and a synthetic A. ipaensis x A. duranensis tetraploid (Ozias-Akins, unpublished).

Gene annotation and comparative mapping

Homology search of the 1,724 mapped loci resulted in significant hits for 1,463 loci in at least one of the three databases: Medicago, Uniprot and GenBank NR database, and 580 of the mapped loci gave significant similarities in either of the two gene ontology databases: Medicago Gene Atlas and TAIR (Additional file 4). Altogether 1,366 gene ontology terms were assigned to the 580 genes. These were distributed among the three major gene ontology categories as follows: 521 molecular functions, 534 biological processes, and 311 cellular components (Additional file 4).

The sequences used to create the A. duranensis map also were compared to the genomes of two legumes where 995 loci on the A. duranensis map could be mapped to M. truncatula, and 2,711 matches could be found in G. max (with potentially two hits per mapped locus). While a majority of the dots in the synteny plots appear to be random (Figure 3), there are definite clusters of markers, and striking examples of colinearity (red arrows), especially for the comparisons to Glycine. Presumably there has been extensive single gene movement since the last common ancestors in one or both species, but many genes remain in the ancestral locations and can be detected. Overall, the synteny patterns for G. max showed the recent whole genome duplication within Glycine, with each location in peanut showing corresponding synteny at two locations in Glycine. Colinearity between Medicago and Arachis is much less conserved than between Arachis and Glycine. This could be due to extensive inversions in either genome, or more likely, due to preliminary ordering of sequences within the Medicago unfinished genome assembly. In general, the patterns showed strong synteny on the chromosomal ends in both genetic and physical distance, while the central regions of chromosomes tended to show less synteny. Presumably this could be attributed to pericentromeric heterochromatin which is known to define less recombinogenic regions where genomic rearrangements are more likely to persist [44]. Chromosome arms tend to be maintained as syntenic between Glycine and Arachis, but there is evidence that chromosome arms have been translocated in some cases so that synteny exists at the chromosome arm level, but less so at the whole chromosome level.

Plant materials

Thirty-seven accessions of A. duranensis, 14 accessions of A. stenosperma (A genome), one accession of A. ipaensis, and eight accessions of A. batizocoi (B genome) were obtained from the USDA or NCSU germplasm collections. Plants were then grown in greenhouses at the University of Georgia at Athens. The accessions evaluated are shown in Figure 1. Hybrids were made between three pairs of A. duranensis accessions, including PI 468200 × PI 468198, PI 468319 × PI 475885, and PI 475887 × Grif 15036. The hybrid combination PI 475887 × Grif 15036 was one of the most polymorphic as revealed by using a panel of SSR markers as described below and thus was selected for subsequent mapping. PI 475887 was originally collected by Krapovickas, Schinini, and Simpson near Salta, Argentina during 1980, and Grif 15036 was originally collected by Williams, Simpson, and Vargas near Boqueron, Paraguay during 2002 [22]. Crosses were made by manually emasculating flowers of the female parent PI 475887 during the late afternoon and pollinating stigmas between 8 and 10 am the following morning with pollen from the male parent Grif 15036. An F₂ population was developed by self-pollinating multiple F₁ individuals. The intraspecific F₂ population (n = 94) from a hybrid between two A. duranensis accessions was then used for mapping studies.

Molecular diversity between and within A- and B-genome diploid species

DNA was isolated from leaf samples of A. duranensis, A. ipaensis, A. stenosperma, and A. batizocoi accessions using a modified CTAB method [45, 46]. The 60 DNA samples were amplified using 709 different SSR primer pairs (GM1-GM709) that had been generated from sequences reported in the literature [6, 29, 47–53] and screened for polymorphisms. SSR markers were genotyped on an ABI3730XL Capillary DNA Sequencer (Applied Biosystems, Foster City, CA) as described in a related paper by Guo et al. (Guo Y et al: Comparative mapping in intraspecific populations uncovers a high degree of macrosynteny between A-and B-genome diploid species of peanut, Submitted) using forward primers labelled with FAM, HEX, or TAMRA fluorophores. Microsat [54] was used for construction of a distance matrix based on the proportion of shared bands (D = 1 - ps) from 556 primer pairs amplifying polymorphic fragments. The matrix was imported into Phylip v3.67 [55] for the construction of the neighbor-joining tree.

Marker development

Map construction

The program, MSTMap [39] was used to build a core genetic map including all codominant markers using the cut-off p-value of 10^-12 for clustering markers into linkage groups. The recombinant inbred line2 (RIL2) algorithm and Kosambi function were used to calculate genetic distances. The program Joinmap 3.0 [63] was used to localize the dominant markers and to confirm the marker order, a range of LOD scores of 5–16 was used to create groups. The Kosambi mapping function was used for map length estimations. Markers were tested for segregation distortion by the chi-square test. Graphic presentation of the map was drawn using Mapchart 2.0 software [64].

Gene annotation

The cDNA sequences included in the genetic map have been used to search for homologous genes in the Medicago (http://www.medicago.org), Uniprot (http://www.uniprot.org) and GenBank NR (http://www.ncbi.nlm.nih.gov/genbank) databases using various blast algorithms. Gene ontology annotations were also added by searching Medicago Gene Atlas (http://mtgea.noble.org) and The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org) databases. A significance threshold of E =1e-5 was applied in all inquiries.

Synteny between Arachis, Medicago, and Glycine

The EST sequences used for marker-development were compared to the whole genome sequences of Glycine max and Medicago truncatula to establish synteny. Sequences for the genomes G. max V5 and M. truncatula MT3.0 were obtained through http://www.phytozome.net. The sequences associated with each locus on the A. duranensis peanut map (Additional file 1 and Additional file 5) were searched against the respective whole genome sequences using blastn and E < =1e-6. For comparison to Medicago, only the best match was retained because diploid peanut and M. truncatula are at the same relative ploidy level. However for Glycine, the two best matches for each peanut sequence were retained because of the recent polyploidy within soybean and the high level of retention of duplicated genes in the species. Blast hits to scaffolds or Bacterial Artificial Chromosomes (BACs) not anchored to the chromosomal assembly in the target genomes were discarded. Plotting the data and processing of blast results were performed with Visual Basic programs written for this study.