SNP genotyping reveals major QTLs for plant architectural traits between A-genome peanut wild species.

KEY MESSAGE: QTL mapping of important architectural traits was successfully applied to an A-genome diploid population using gene-specific variations. Peanut wild species are an important source of resistance to biotic and possibly abiotic stress; because these species differ from the cultigen in many traits, we have undertaken to identify QTLs for several plant architecture-related traits. In this study, we took recently identified SNPs, converted them into markers, and identified QTLs for architectural traits. SNPs from RNASeq data distinguishing two parents, A. duranensis (KSSc38901) and A. cardenasii (GKP10017), of a mapping population were identified using three references-A. duranensis V14167 genome sequence, and transcriptome sequences of A. duranensis KSSc38901 and OLin. More than 49,000 SNPs differentiated the parents, and 87.9% of the 190 SNP calls tested were validated. SNPs were then genotyped on 91 F2 lines using KASP chemistry on a Roche LightCycler 480 and a Fluidigm Biomark HD, and using SNPType chemistry on the Fluidigm Biomark HD. A linkage map was constructed having ten linkage groups, with 144 loci spanning a total map distance of 1040 cM. Comparison of the A-genome map to the A. duranensis genome sequence revealed a high degree of synteny. QTL analysis was also performed on the mapping population for important architectural traits. Fifteen definitive and 16 putative QTLs for petiole length, leaflet length and width, leaflet area, leaflet length/width ratio, main stem height, presence of flowers on the main stem, and seed mass were identified. Results demonstrate that SNPs identified from transcriptome sequencing could be converted to KASP or SNPType markers with a high success rate, and used to identify alleles with significant phenotypic effects, These could serve as information useful for introgression of alleles into cultivated peanut from wild species and have the potential to allow breeders to more easily fix these alleles using a marker-assisted backcrossing approach.

Next-generation transcriptome sequencing, SNP discovery and validation in four market classes of peanut, Arachis hypogaea L.

Single-nucleotide polymorphisms, which can be identified in the thousands or millions from comparisons of transcriptome or genome sequences, are ideally suited for making high-resolution genetic maps, investigating population evolutionary history, and discovering marker-trait linkages. Despite significant results from their use in human genetics, progress in identification and use in plants, and particularly polyploid plants, has lagged. As part of a long-term project to identify and use SNPs suitable for these purposes in cultivated peanut, which is tetraploid, we generated transcriptome sequences of four peanut cultivars, namely OLin, New Mexico Valencia C, Tamrun OL07 and Jupiter, which represent the four major market classes of peanut grown in the world, and which are important economically to the US southwest peanut growing region. CopyDNA libraries of each genotype were used to generate 2 × 54 paired-end reads using an Illumina GAIIx sequencer. Raw reads were mapped to a custom reference consisting of Tifrunner 454 sequences plus peanut ESTs in GenBank, compromising 43,108 contigs; 263,840 SNP and indel variants were identified among four genotypes compared to the reference. A subset of 6 variants was assayed across 24 genotypes representing four market types using KASP chemistry to assess the criteria for SNP selection. Results demonstrated that transcriptome sequencing can identify SNPs usable as selectable DNA-based markers in complex polyploid species such as peanut. Criteria for effective use of SNPs as markers are discussed in this context.

Lipid modulation contributes to heat stress adaptation in peanut.

At the cellular level, membrane damage is a fundamental cause of yield loss at high temperatures (HT). We report our investigations on a subset of a peanut (Arachis hypogaea) recombinant inbred line population, demonstrating that the membrane lipid remodeling occurring at HT is consistent with homeoviscous adaptation to maintain membrane fluidity. A major alteration in the leaf lipidome at HT was the reduction in the unsaturation levels, primarily through reductions of 18:3 fatty acid chains, of the plastidic and extra-plastidic diacyl membrane lipids. In contrast, levels of 18:3-containing triacylglycerols (TGs) increased at HT, consistent with a role for TGs in sequestering fatty acids when membrane lipids undergo remodeling during plant stress. Polyunsaturated acyl chains from membrane diacyl lipids were also sequestered as sterol esters (SEs). The removal of 18:3 chains from the membrane lipids decreased the availability of susceptible molecules for oxidation, thereby minimizing oxidative damage in membranes. Our results suggest that transferring 18:3 chains from membrane diacyl lipids to TGs and SEs is a key feature of lipid remodeling for HT adaptation in peanut. Finally, QTL-seq allowed the identification of a genomic region associated with heat-adaptive lipid remodeling, which would be useful for identifying molecular markers for heat tolerance.

A first insight into population structure and linkage disequilibrium in the US peanut minicore collection.

Knowledge of genetic diversity, population structure, and degree of linkage disequilibrium (LD) in target association mapping populations is of great importance and is a prerequisite for LD-based mapping. In the present study, 96 genotypes comprising 92 accessions of the US peanut minicore collection, a component line of the tetraploid variety Florunner, diploid progenitors A. duranensis (AA) and A. ipaënsis (BB), and synthetic amphidiploid accession TxAG-6 were investigated with 392 simple sequence repeat (SSR) marker bands amplified using 32 highly-polymorphic SSR primer pairs. Both distance- and model-based (Bayesian) cluster analysis revealed the presence of structured diversity. In general, the wild-species accessions and the synthetic amphidiploid grouped separately from most minicore accessions except for COC155, and were eliminated from most subsequent analyses. UPGMA analysis divided the population into four subgroups, two major subgroups representing subspecies fastigiata and hypogaea, a third group containing individuals from each subspecies or possibly of mixed ancestry, and a fourth group, either consisting of COC155 alone if wild species were excluded, or of COC155, the diploid species, and the synthetic amphidiploid. Model-based clustering identified four subgroups- one each for fastigiata and hypogaea subspecies, a third consisting of individuals of both subspecies or of mixed ancestry predominantly from Africa or Asia, and a fourth group, consisting of individuals predominantly of var fastigiata, peruviana, and aequatoriana accessions from South America, including COC155. Analysis of molecular variance (AMOVA) revealed statistically-significant (P < 0.0001) genetic variance of 16.87% among subgroups. A total of 4.85% of SSR marker pairs revealed significant LD (at r(2) ≥ 0.1). Of the syntenic marker pairs separated by distances < 10 cM, 11-20 cM, 21-50 cM, and > 50 cM, 19.33, 5.19, 6.25 and 5.29% of marker pairs were found in strong LD (P ≤ 0.01), in accord with LD extending to great distances in self pollinated crops. A threshold value of r(2) > 0.035 was found to distinguish mean r(2) values of linkage distance groups statistically from the mean r(2) values of unlinked markers; LD was found to extend to 10 cM over the entire minicore collection by this criterion. However, there were large differences in r(2) values among marker pairs even among tightly-linked markers. The implications of these findings with regard to the possibility of using association mapping for detection of genome-wide SSR marker-phenotype association are discussed.

Raman spectroscopy-based diagnostics of water deficit and salinity stresses in two accessions of peanut.

Water deficit and salinity are two major abiotic stresses that have tremendous effect on crop yield worldwide. Timely identification of these stresses can help limit associated yield loss. Confirmatory detection and identification of water deficit stress can also enable proper irrigation management. Traditionally, unmanned aerial vehicle (UAV)-based imaging and satellite-based imaging, together with visual field observation, are used for diagnostics of such stresses. However, these approaches can only detect salinity and water deficit stress at the symptomatic stage. Raman spectroscopy (RS) is a noninvasive and nondestructive technique that can identify and detect plant biotic and abiotic stress. In this study, we investigated accuracy of Raman-based diagnostics of water deficit and salinity stresses on two greenhouse-grown peanut accessions: tolerant and susceptible to water deficit. Plants were grown for 76 days prior to application of the water deficit and salinity stresses. Water deficit treatments received no irrigation for 5 days, and salinity treatments received 1.0 L of 240-mM salt water per day for the duration of 5-day sampling. Every day after the stress was imposed, plant leaves were collected and immediately analyzed by a hand-held Raman spectrometer. RS and chemometrics could identify control and stressed (either water deficit or salinity) susceptible plants with 95% and 80% accuracy just 1 day after treatment. Water deficit and salinity stressed plants could be differentiated from each other with 87% and 86% accuracy, respectively. In the tolerant accessions at the same timepoint, the identification accuracies were 66%, 65%, 67%, and 69% for control, combined stresses, water deficit, and salinity stresses, respectively. The high selectivity and specificity for presymptomatic identification of abiotic stresses in the susceptible line provide evidence for the potential of Raman-based surveillance in commercial-scale agriculture and digital farming.

Evaluation and Selection of Interspecific Lines of Groundnut (Arachis hypogaea L.) for Resistance to Leaf Spot Disease and for Yield Improvement.

Early and late leaf spot are two devastating diseases of peanut (Arachis hypogaea L.) worldwide. The development of a fertile, cross-compatible synthetic amphidiploid, TxAG-6 ([A. batizocoi × (A. cardenasii × A. diogoi)]4x), opened novel opportunities for the introgression of wild alleles for disease and pest resistance into commercial cultivars. Twenty-seven interspecific lines selected from prior evaluation of an advanced backcross population were evaluated for resistance to early and late leaf spot, and for yield in two locations in Ghana in 2006 and 2007. Several interspecific lines had early leaf spot scores significantly lower than the susceptible parent, indicating that resistance to leaf spot had been successfully introgressed and retained after three cycles of backcrossing. Time to appearance of early leaf spot symptoms was less in the introgression lines than in susceptible check cultivars, but the opposite was true for late leaf spot. Selected lines from families 43-08, 43-09, 50-04, and 60-02 had significantly reduced leaf spot scores, while lines from families 43-09, 44-10, and 63-06 had high pod yields. One line combined both resistance to leaf spot and high pod yield, and several other useful lines were also identified. Results suggest that it is possible to break linkage drag for low yield that accompanies resistance. However, results also suggest that resistance was diluted in many of the breeding lines, likely a result of the multigenic nature of resistance. Future QTL analysis may be useful to identify alleles for resistance and allow recombination and pyramiding of resistance alleles while reducing linkage drag.

A Proof-of-Principle Study of Non-invasive Identification of Peanut Genotypes and Nematode Resistance Using Raman Spectroscopy.

Identification of peanut cultivars for distinct phenotypic or genotypic traits whether using visual characterization or laboratory analysis requires substantial expertise, time, and resources. A less subjective and more precise method is needed for identification of peanut germplasm throughout the value chain. In this proof-of-principle study, the accuracy of Raman spectroscopy (RS), a non-invasive, non-destructive technique, in peanut phenotyping and identification is explored. We show that RS can be used for highly accurate peanut phenotyping via surface scans of peanut leaves and the resulting chemometric analysis: On average 94% accuracy in identification of peanut cultivars and breeding lines was achieved. Our results also suggest that RS can be used for highly accurate determination of nematode resistance and susceptibility of those breeding lines and cultivars. Specifically, nematode-resistant peanut cultivars can be identified with 92% accuracy, whereas susceptible breeding lines were identified with 81% accuracy. Finally, RS revealed substantial differences in biochemical composition between resistant and susceptible peanut cultivars. We found that resistant cultivars exhibit substantially higher carotenoid content compared to the susceptible breeding lines. The results of this study show that RS can be used for quick, accurate, and non-invasive identification of genotype, nematode resistance, and nutrient content. Armed with this knowledge, the peanut industry can utilize Raman spectroscopy for expedited breeding to increase yields, nutrition, and maintaining purity levels of cultivars following release.

Peanut Seed Coat Acts as a Physical and Biochemical Barrier against Aspergillus flavus Infection.

Aflatoxin contamination is a global menace that adversely affects food crops and human health. Peanut seed coat is the outer layer protecting the cotyledon both at pre- and post-harvest stages from biotic and abiotic stresses. The aim of the present study is to investigate the role of seed coat against A. flavus infection. In-vitro seed colonization (IVSC) with and without seed coat showed that the seed coat acts as a physical barrier, and the developmental series of peanut seed coat showed the formation of a robust multilayered protective seed coat. Radial growth bioassay revealed that both insoluble and soluble seed coat extracts from 55-437 line (resistant) showed higher A. flavus inhibition compared to TMV-2 line (susceptible). Further analysis of seed coat biochemicals showed that hydroxycinnamic and hydroxybenzoic acid derivatives are the predominant phenolic compounds, and addition of these compounds to the media inhibited A. flavus growth. Gene expression analysis showed that genes involved in lignin monomer, proanthocyanidin, and flavonoid biosynthesis are highly abundant in 55-437 compared to TMV-2 seed coats. Overall, the present study showed that the seed coat acts as a physical and biochemical barrier against A. flavus infection and its potential use in mitigating the aflatoxin contamination.

Use of Targeted Amplicon Sequencing in Peanut to Generate Allele Information on Allotetraploid Sub-Genomes.

The use of molecular markers in plant breeding has become a routine practice, but the cost per accession can be a hindrance to the routine use of Quantitative Trait Loci (QTL) identification in breeding programs. In this study, we demonstrate the use of targeted re-sequencing as a proof of concept of a cost-effective approach to retrieve highly informative allele information, as well as develop a bioinformatics strategy to capture the genome-specific information of a polyploid species. SNPs were identified from alignment of raw transcriptome reads (2 × 50 bp) to a synthetic tetraploid genome using BWA followed by a GATK pipeline. Regions containing high polymorphic SNPs in both A genome and B genomes were selected as targets for the resequencing study. Targets were amplified using multiplex PCR followed by sequencing on an Illumina HiSeq. Eighty-one percent of the SNP calls in diploids and 68% of the SNP calls in tetraploids were confirmed. These results were also confirmed by KASP validation. Based on this study, we find that targeted resequencing technologies have potential for obtaining maximum allele information in allopolyploids at reduced cost.

PeanutMap: an online genome database for comparative molecular maps of peanut.

BACKGROUND: Molecular maps have been developed for many species, and are of particular importance for varietal development and comparative genomics. However, despite the existence of multiple sets of linkage maps, databases of these data are lacking for many species, including peanut. DESCRIPTION: PeanutMap http://peanutgenetics.tamu.edu/cmap provides a web-based interface for viewing specific linkage groups of a map set. PeanutMap can display and compare multiple maps of a set based upon marker or trait correspondences, which is particularly important as cultivated peanut is a disomic tetraploid. The database can also compare linkage groups among multiple map sets, allowing identification of corresponding linkage groups from results of different research projects. Data from the two published peanut genome map sets, and also from three maps sets of phenotypic traits are present in the database. Data from PeanutMap have been incorporated into the Legume Information System website http://www.comparative-legumes.org to allow peanut map data to be used for cross-species comparisons. CONCLUSION: The utility of the database is expected to increase as several SSR-based maps are being developed currently, and expanded efforts for comparative mapping of legumes are underway. Optimal use of these data will benefit from the development of tools to facilitate comparative analysis.

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.

The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut.

Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ∼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanut's A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanut's subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.

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 500-751 bp. 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.

Molecular biogeographic study of recently described B- and A-genome Arachis species, also providing new insights into the origins of cultivated peanut.

The cultivated peanut Arachis hypogaea is a tetraploid, likely derived from A- and B-genome species. Reproductive isolation of the cultigen has resulted in limited genetic variability for important traits. Artificial hybridizations using selected diploid parents have introduced alleles from wild species, but improved understanding of recently classified B-genome accessions would aid future introgression work. To this end, 154 cDNA probes were used to produce 1887 RFLP bands scored on 18 recently classified or potential B-genome accessions and 16 previously identified species. One group of B-genome species consisted of Arachis batizocoi, Arachis cruziana, Arachis krapovickasii, and one potential additional species; a second consisted of Arachis ipaënsis, Arachis magna, and Arachis gregoryi. Twelve uncharacterized accessions grouped with A-genome species. Many RFLP markers diagnostic of A. batizocoi group specificity mapped to linkage group pair 2/12, suggesting selection or genetic control of chromosome pairing. The combination of Arachis duranensis and A. ipaënsis most closely reconstituted the marker haplotype of A. hypogaea, but differences allow for other progenitors or genetic rearrangements after polyploidization. From 2 to 30 alleles per locus were present, demonstrating section Arachis wild species variation of potential use for expanding the cultigen's genetic basis.

Identification of QTLs for pod and kernel traits in cultivated peanut by bulked segregant analysis

Bulked segregant analysis was used to identify simple sequence repeat (SSR) markers associated with pod and kernel traits in cultivated peanut, to permit rapid selection of superior quality genotypes in the breeding program. SSR markers linked to pod and kernel traits were identified in two DNA pools (high and low), which were established using selected F2:6 recombinant individuals resulting from a cultivated cross between a runner (Tamrun OL01) and a Spanish (BSS 56) peanut. To identify quantitative trait loci (QTLs) for pod and kernel-related traits, parents were screened initially with 112 SSR primer pairs. The survey revealed 8.9 percent polymorphism between parents. Of ten SSR primer pairs distinguishing the parents, five (PM375, PM36, PM45, pPGPseq8D9, and Ah-041) were associated with differences between bulks for seed length, pod length, number of pods per plant, 100-seed weight, maturity, or oil content. Association was confirmed by analysis of segregation among 88 F2:6 individuals in the RIL population. Phenotypic means associated with markers for three traits differed by more than 40 percent, indicating the presence of QTLs with major effects for number of pods per plant, plant weight, and pod maturity. The SSR markers can be used for marker assisted selection for quality and yield improvement in peanut. To the best of our knowledge, this is the first report on the identification of SSR markers linked to pod - and kernel- related traits in cultivated peanut.