Charles E. Simpson

Identification of peanut (Arachis hypogaea L.) RAPD markers diagnostic of root-knot nematode (Meloidogyne arenaria (Neal) Chitwood) resistance

DNA markers linked to a root-knot nematode resistance gene derived from wild peanut species have been identified. The wild diploid peanut accessions K9484 (Arachis batizocoi Krapov. & W. C. Gregory), GKP10017, (A. cardenasii Krapov & W. C. Gregory), and GKP10602 (A. diogoi Hoehne) possess genes for ressitance to Meloidogyne arenaria. These three accessions and A. hypogaea cv. Florunner were crossed to generate the hybrid resistant breeding line TxAg-7. This line was used as donor parent to develop a BC4F2 population segregating for resistance. Three RAPD markers associated with nematode resistance were identified in this population by bulked segregant analysis. Linkage was confirmed by screening 21 segregatingh BC4F2 and 63 BC5F2 single plants. Recombination between marker RKN410 and resistance, and between marker RKN440 and resistance, was estimated to be 5.4±1.9% and 5.8±2.1%, respectively, on a per-generation basis. These two markers identified a resistance gene derived from either A. cardenasii or A. diogoi, and were closely linked to each other. Recombination between a third marker, RKN229, inherited from A. cardenasii or A. diogoi, and resistance was 9.0±3.2% per generation. Markers RKN410 and RKN229 appeared to be linked genetically and flank the same resistance gene. All markers were confirmed by hybridization of cloned or gel-purified marker DNA to blots of PCR-amplified DNA. Pooled data on the segregation of BC5F2 plants was consistent with the presence of one resistance gene in the advanced breeding lines. Different distributions of resistance in the BC5F2 progeny and TxAG-7 suggest the presence of additional resistance genes in TxAG-7.

Comparisons of De Novo Transcriptome Assemblers in Diploid and Polyploid Species Using Peanut (Arachis spp.) RNA-Seq Data

The narrow genetic base and limited genetic information on Arachis species have hindered the process of marker-assisted selection of peanut cultivars. However, recent developments in sequencing technologies have expanded opportunities to exploit genetic resources, and at lower cost. To use the genetic information for Arachis species available at the transcriptome level, it is important to have a good quality reference transcriptome. The available Tifrunner 454 FLEX transcriptome sequences have an assembly with 37,000 contigs and low N50 values of 500-751bp. Therefore, we generated de novo transcriptome assemblies, with about 38 million reads in the tetraploid cultivar OLin, and 16 million reads in each of the diploids, A. duranensis K38901 and A. ipaënsis KGBSPSc30076 using three different de novo assemblers, Trinity, SOAPdenovo-Trans and TransAByss. All these assemblers can use single kmer analysis, and the latter two also permit multiple kmer analysis. Assemblies generated for all three samples had N50 values ranging from 1278–1641 bp in Arachis hypogaea (AABB), 1401–1492 bp in Arachis duranensis (AA), and 1107–1342 bp in Arachis ipaënsis (BB). Comparison with legume ESTs and protein databases suggests that assemblies generated had more than 40% full length transcripts with good continuity. Also, on mapping the raw reads to each of the assemblies generated, Trinity had a high success rate in assembling sequences compared to both TransAByss and SOAPdenovo-Trans. De novo assembly of OLin had a greater number of contigs (67,098) and longer contig length (N50 = 1,641) compared to the Tifrunner TSA. Despite having shorter read length (2×50) than the Tifrunner 454FLEX TSA, de novo assembly of OLin proved superior in comparison. Assemblies generated to represent different genome combinations may serve as a valuable resource for the peanut research community.

Expressed variants of Δ12-fatty acid desaturase for the high oleate trait in spanish market-type peanut lines

Increasing the oleic to linoleic acid ratio (O/L) in peanut has positiveeffects on peanut quality and its nutritional value. Δ12-Fattyacid desaturases (Δ12-Fad) have been targeted as logicalcandidates controlling the high oleate trait. A previous study using genomicDNA identified an insertion and a polymorphism resulting in an amino acid changeassociated with the high oleate trait in Spanish-type peanut cultivars. Theobjectives of this research were to use RT-PCR to confirm that the SingleNucleotide Polymorphims (SNPs) identified by analysis of genomic DNA wereexpressed, and to determine if expression patterns for Δ12-Fadwere the same in both seeds and leaves. A polymorphic region of theΔ12-Fad containing a series of nucleotide changes wasamplified, cloned, and sequenced from mRNA of 155 clones of two parental linesand their independent derived backcross lines (IDBLs). The latter differed intheir oleic to linoleic ratio. Data indicated that the “A”insertion and the amino acid change were expressed in both leaf and seed tissue of thehigh and low-intermediate O/L genotypes. It is postulated that several copiesof the Δ12-Fad are present in the genome. It is reasonable toconclude that total activity, and ultimately the O/L ratio, is dependent on thenumber of functional copies. The results provide the basis for an assay toscreen for the high O/L ratio at the molecular level. We also report thepresence of another isozyme of Δ12-Fad with high homology tosoybean isozyme 2 that was expressed in seeds.

Identification of Peanut Hybrids Using Microsatellite Markers and Horizontal Polyacrylamide Gel Electrophoresis

In peanut hybridization, distinguishing inadvertent selfs from the true hybrids may be difficult. In this study, to differentiate between selfs and hybrids, DNA was extracted from leaf tissue of F1 or F2 plants, and SSR markers were amplified and bands separated by a novel submarine horizontal polyacrylamide gel electrophoresis (H-PAGE). By comparing the resulting banding patterns to those of the parents, 70% of the putative hybrids were shown to be true hybrids on the basis of possessing a marker allele from the male parent. The H-PAGE gels gave better band separation and differentiation of selfed progenies than agarose gels, and were compatible with the common horizontal agarose gel units. This method provides a quick assay to distinguish hybrids from inadvertent selfs, and should result in greater efficiency and more effective use of resources in peanut breeding programs.

Biogeography of wild Arachis (Leguminosae):distribution and environmental characterisation

Geographic Information System (GIS) tools are applied to a comprehensive database of 3514 records of wild Arachis species to assist in the conservation and utilisation of the species by: (a) determining the distributional range of species and their abundance; (b) characterising species environments; (c) determining the geographical distribution of species richness; and (d) determining the extent to which species are associated with river basins. Distributional ranges, climatic variables and indices of endemism for each species are tabulated. A. duranensis Krapov. & W.C. Gregory, the most probable donor of the A genome to the cultivated peanut, is distributed in close proximity to both the proposed donor of the B genome, A. ipaënsis, and the closest wild relative of the cultigen, A. monticola Krapov. & Rigoni. This region in the eastern foothills of the Andes and the adjoining chaco regions of Argentina, Bolivia and Paraguay, is a key area for further exploration for wild Arachis. An area of particularly high species richness occurs in the State of Mato Grosso, close to the Gran Pantanal in southwest Brazil. Seventy-one percent of the species were found to have some degree of association with water catchment areas, although in most cases it was difficult to determine whether this was due to climatic adaptation reasons, restricted dispersal due to geocarpic habit, or the role of watercourses as a principal dispersal agent. In only two cases could climatic adaptation be eliminated as the reason for species distribution.

Successful crosses between fungal-resistant wild species of Arachis (section Arachis) and Arachis hypogaea

Peanut (Arachis hypogaea) is the fifth most produced oil crop worldwide. Besides lack of water, fungal diseases are the most limiting factors for the crop. Several species of Arachis are resistant to certain pests and diseases. This study aimed to successfully cross the A-genome with B-K-A genome wild species previously selected for fungal disease resistance, but that are still untested. We also aimed to polyplodize the amphihaploid chromosomes; cross the synthetic amphidiploids and A. hypogaea to introgress disease resistance genes into the cultivated peanut; and analyze pollen viability and morphological descriptors for all progenies and their parents. We selected 12 A-genome accessions as male parents and three B-genome species, one K-genome species, and one A-genome species as female parents. Of the 26 distinct cross combinations, 13 different interspecific AB-genome and three AA-genome hybrids were obtained. These sterile hybrids were polyploidized and five combinations produced tetraploid flowers. Next, 16 combinations were crossed between A. hypogaea and the synthetic amphidiploids, resulting in 11 different hybrid combinations. Our results confirm that it is possible to introgress resistance genes from wild species into the peanut using artificial hybridization, and that more species than previously reported can be used, thus enhancing the genetic variability in peanut genetic improvement programs.

Advanced Backcross Quantitative Trait Loci (QTL) Analysis of Oil Concentration and Oil Quality Traits in Peanut (Arachis hypogaea L.)

Peanut seed oil is an important commodity worldwide and breeding efforts have been to improve both the quality and quantity of oil produced. Identifying sources of variation and elucidating the genetics of oil concentration and quality in peanut is essential to advancing the development of improved genotypes. The objective of this study was to discover QTLs for oil traits in an advanced backcross population derived from a cross between a wild-species derived amphidiploid, TxAG-6, and a cultivated genotype, Florunner. A BC1F1 population was developed for genetic mapping and an advanced backcross BC3F6 population was phenotyped in three environments and genotyped using SSR markers. Composite interval mapping results identified three genomic regions associated with oil concentration in a combined analysis. Marker PM36, associated with oil concentration and multiple fatty acids in this study, mapped directly to a HD-ZIP transcription factor in diploid Arachis genome sequences. For fatty acid concentrations, results suggested 17 QTLs identified in two or more environments, 15 of which were present across environments. Fourteen genomic regions on 13 linkage groups contained significant QTLs for more than one trait, suggesting that same genes or gene families are responsible for multiple phenotypes. QTLs and the genes identified in this study could be effective tools in marker-assisted breeding targeted at pyramiding seed oil alleles from wild-species while minimizing introgression of non-target chromatin.

Generation means analysis of oil concentration in peanut.

The current interest in biodiesel production has resulted in a concurrent interest in increasing the oil concentration in high-yielding cultivars, which could make peanuts (Arachis hypogaea.) more desirable as a biofuel source. Currently, peanut seed is approximately 450 to 500 g kg−1 oil on a dry weight basis, depending upon location grown, and there is relatively little genetic variation for oil concentration among adapted high-yielding cultivated peanut genotypes. Thus, identifying sources of variation and elucidating the genetics of oil concentration in peanut is essential to advancing the development of high oil genotypes. The objective of this study was to determine the types of gene action governing the inheritance of oil concentration in peanut by generation means analysis. The F1, F2, and backcross generations of two different runner peanut crosses segregating for oil concentration were evaluated in College Station, Texas, in 2010. Significant differences in oil concentration among the generations were detected, and generation means analysis revealed significant additive, dominance, and epistatic effects for oil concentration in both crosses. The broad-sense heritability estimates were 0.85 and 0.78, and narrow-sense heritability estimates were 0.55 and 0.53 for each of the crosses. Our data indicate that transgressive segregants for high oil were observed, and there is sufficient additive variation present to improve the oil concentration of current runner cultivars.

Transcriptome Sequencing of Diverse Peanut (Arachis) Wild Species and the Cultivated Species Reveals a Wealth of Untapped Genetic Variability

To test the hypothesis that the cultivated peanut species possesses almost no molecular variability, we sequenced a diverse panel of 22 Arachis accessions representing Arachis hypogaea botanical classes, A-, B-, and K- genome diploids, a synthetic amphidiploid, and a tetraploid wild species. RNASeq was performed on pools of three tissues, and de novo assembly was performed. Realignment of individual accession reads to transcripts of the cultivar OLin identified 306,820 biallelic SNPs. Among 10 naturally occurring tetraploid accessions, 40,382 unique homozygous SNPs were identified in 14,719 contigs. In eight diploid accessions, 291,115 unique SNPs were identified in 26,320 contigs. The average SNP rate among the 10 cultivated tetraploids was 0.5, and among eight diploids was 9.2 per 1000 bp. Diversity analysis indicated grouping of diploids according to genome classification, and cultivated tetraploids by subspecies. Cluster analysis of variants indicated that sequences of B genome species were the most similar to the tetraploids, and the next closest diploid accession belonged to the A genome species. A subset of 66 SNPs selected from the dataset was validated; of 782 SNP calls, 636 (81.32%) were confirmed using an allele-specific discrimination assay. We conclude that substantial genetic variability exists among wild species. Additionally, significant but lesser variability at the molecular level occurs among accessions of the cultivated species. This survey is the first to report significant SNP level diversity among transcripts, and may explain some of the phenotypic differences observed in germplasm surveys. Understanding SNP variants in the Arachis accessions will benefit in developing markers for selection.

Generation Means Analysis of Fatty Acid Composition in Peanut

Optimizing the chemical composition of peanut (Arachis hypogaea L.) oil is essential for the production of biodiesel. Specifically, increasing the ratio of oleic to linoleic acid (O/L) in peanut oil and reducing the long chain saturated fatty acid concentration (which includes arachidic, behenic, and lignoceric acids) produces high-quality, stable methyl esters for biodiesel. Therefore, elucidating the inheritance of these factors and their relationships in peanut populations segregating for high oil is critical. The F1, F2, and backcross generations derived from two crosses, both involving a high oil concentration, low O/L runner breeding line (31-08-05-02) and two high O/L, normal oil concentration, adapted runner genotypes (Tamrun OL01 and Tamrun OL07), were evaluated in College Station, Texas, in 2010. The results from generation means analysis confirm that the high-oleic trait is under simple genetic control and can be manipulated through breeding and selection. Most fatty acids were controlled primarily by additive gene action, which is highly selectable. Dominance effects also played an important part in the inheritance of most fatty acids. Additive × dominance interaction was significant in the inheritance of stearic and arachidic acids in the cross involving Tamrun OL07. Oil concentration was also negatively correlated with oleic acid concentration in the F2 generations of both crosses and positively correlated with arachidic acid in most of the segregating generations evaluated. Therefore, developing a peanut genotype high in oil and oleic acid concentration that has reduced long chain saturates will require the evaluation of large numbers of segregating progeny.