The peanut (Arachis hypogaea) is an important oil crop. Breeding for high oil content is becoming increasingly important. Wild Arachis species have been reported to harbor genes for many valuable traits that may enable the improvement of cultivated Arachis hypogaea, such as resistance to pests and disease. However, only limited information is available on variation in oil content. In the present study, a collection of 72 wild Arachis accessions representing 19 species and 3 cultivated peanut accessions were genotyped using 136 genome-wide SSR markers and phenotyped for oil content over three growing seasons. The wild Arachis accessions showed abundant diversity across the 19 species. A. duranensis exhibited the highest diversity, with a Shannon-Weaver diversity index of 0.35. A total of 129 unique alleles were detected in the species studied. A. rigonii exhibited the largest number of unique alleles (75), indicating that this species is highly differentiated. AMOVA and genetic distance analyses confirmed the genetic differentiation between the wild Arachis species. The majority of SSR alleles were detected exclusively in the wild species and not in A. hypogaea, indicating that directional selection or the hitchhiking effect has played an important role in the domestication of the cultivated peanut. The 75 accessions were grouped into three clusters based on population structure and phylogenic analysis, consistent with their taxonomic sections, species and genome types. A. villosa and A. batizocoi were grouped with A. hypogaea, suggesting the close relationship between these two diploid wild species and the cultivated peanut. Considerable phenotypic variation in oil content was observed among different sections and species. Nine alleles were identified as associated with oil content based on association analysis, of these, three alleles were associated with higher oil content but were absent in the cultivated peanut. The results demonstrated that there is great potential to increase the oil content in A. hypogaea by using the wild Arachis germplasm.
Cultivated peanut (Arachis hypogaea) is one of the most widely grown grain legumes in the world, being valued for its high protein and unsaturated oil contents. Worldwide, the major constraints to peanut production are drought and fungal diseases. Wild Arachis species, which are exclusively South American in origin, have high genetic diversity and have been selected during evolution in a range of environments and biotic stresses, constituting a rich source of allele diversity. Arachis stenosperma harbors resistances to a number of pests, including fungal diseases, whilst A. duranensis has shown improved tolerance to water limited stress. In this study, these species were used for the creation of an extensive databank of wild Arachis transcripts under stress which will constitute a rich source for gene discovery and molecular markers development.
Transcriptome analysis of cDNA collections from A. stenosperma challenged with Cercosporidium personatum (Berk. and M.A. Curtis) Deighton, and A. duranensis submitted to gradual water limited stress was conducted using 454 GS FLX Titanium generating a total of 7.4 x 105 raw sequence reads covering 211 Mbp of both genomes. High quality reads were assembled to 7,723 contigs for A. stenosperma and 12,792 for A. duranensis and functional annotation indicated that 95% of the contigs in both species could be appointed to GO annotation categories. A number of transcription factors families and defense related genes were identified in both species. Additionally, the expression of five A. stenosperma Resistance Gene Analogs (RGAs) and four retrotransposon (FIDEL-related) sequences were analyzed by qRT-PCR. This data set was used to design a total of 2,325 EST-SSRs, of which a subset of 584 amplified in both species and 214 were shown to be polymorphic using ePCR.
This study comprises one of the largest unigene dataset for wild Arachis species and will help to elucidate genes involved in responses to biological processes such as fungal diseases and water limited stress. Moreover, it will also facilitate basic and applied research on the genetics of peanut through the development of new molecular markers and the study of adaptive variation across the genus.
The genus Arachis, originated in South America, is divided into nine taxonomical sections comprising of 80 species. Most of the Arachis species are diploids (2n = 2x = 20) and the tetraploid species (2n = 2x = 40) are found in sections Arachis, Extranervosae and Rhizomatosae. Diploid species have great potential to be used as resistance sources for agronomic traits like pests and diseases, drought related traits and different life cycle spans. Understanding of genetic relationships among wild species and between wild and cultivated species will be useful for enhanced utilization of wild species in improving cultivated germplasm. The present study was undertaken to evaluate genetic relationships among species (96 accessions) belonging to seven sections of Arachis by using simple sequence repeat (SSR) markers developed from Arachis hypogaea genomic library and gene sequences from related genera of Arachis.
The average transferability rate of 101 SSR markers tested to section Arachis and six other sections was 81% and 59% respectively. Five markers (IPAHM 164, IPAHM 165, IPAHM 407a, IPAHM 409, and IPAHM 659) showed 100% transferability. Cluster analysis of allelic data from a subset of 32 SSR markers on 85 wild and 11 cultivated accessions grouped accessions according to their genome composition, sections and species to which they belong. A total of 109 species specific alleles were detected in different wild species, Arachis pusilla exhibited largest number of species specific alleles (15). Based on genetic distance analysis, the A-genome accession ICG 8200 (A. duranensis) and the B-genome accession ICG 8206 (A. ipaënsis) were found most closely related to A. hypogaea.
A set of cross species and cross section transferable SSR markers has been identified that will be useful for genetic studies of wild species of Arachis, including comparative genome mapping, germplasm analysis, population genetic structure and phylogenetic inferences among species. The present study provides strong support based on both genomic and genic markers, probably for the first time, on relationships of A. monticola and A. hypogaea as well as on the most probable donor of A and B-genomes of cultivated groundnut.
Peanut (Arachis hypogaea L) is one of the widely cultivated and leading oilseed crops of the world and its yields are greatly affected by various biotic and abiotic stresses. Arachis diogoi, a wild relative of peanut, is an important source of genes for resistance against various stresses that affect peanut. In our previous study a thaumatin-like protein gene was found to be upregulated in a differential expression reverse transcription PCR (DDRT-PCR) study using the conidial spray of the late leaf spot pathogen, Phaeoisariopsis personata. In the present study, the corresponding full length cDNA was cloned using RACE-PCR and has been designated as AdTLP. It carried an open reading frame of 726 bp potentially capable of encoding a polypeptide of 241 amino acids with 16 conserved cysteine residues. The semi-quantitative RT-PCR analysis showed that the transcript level of AdTLP increased upon treatment with the late leaf spot pathogen of peanut, P. personata and various hormone treatments indicating its involvement in both, biotic and abiotic stresses. The antifungal activity of the purified recombinant protein was checked against different fungal pathogens, which showed enhanced anti-fungal activity compared to many other reported TLP proteins. The recombinant AdTLP-GFP fusion protein was found to be predominantly localized to extracellular spaces. Transgenic tobacco plants ectopically expressing AdTLP showed enhanced resistance to fungal pathogen, Rhizoctonia solani. The seedling assays showed enhanced tolerance of AdTLP transgenic plants against salt and oxidative stress. The transcript analysis of various defense related genes highlighted constitutively higher level expression of PR1a, PI-I and PI-II genes in transgenic plants. These results suggest that the AdTLP is a good candidate gene for enhancing stress resistance in crop plants.
Cultivated peanut (Arachis hypogaea) is an allotetraploid species whose ancestral genomes are most likely derived from the A-genome species, A. duranensis, and the B-genome species, A. ipaensis. The very recent (several millennia) evolutionary origin of A. hypogaea has imposed a bottleneck for allelic and phenotypic diversity within the cultigen. However, wild diploid relatives are a rich source of alleles that could be used for crop improvement and their simpler genomes can be more easily analyzed while providing insight into the structure of the allotetraploid peanut genome. The objective of this research was to establish a high-density genetic map of the diploid species A. duranensis based on de novo generated EST databases. Arachis duranensis was chosen for mapping because it is the A-genome progenitor of cultivated peanut and also in order to circumvent the confounding effects of gene duplication associated with allopolyploidy in A. hypogaea.
More than one million expressed sequence tag (EST) sequences generated from normalized cDNA libraries of A. duranensis were assembled into 81,116 unique transcripts. Mining this dataset, 1236 EST-SNP markers were developed between two A. duranensis accessions, PI 475887 and Grif 15036. An additional 300 SNP markers also were developed from genomic sequences representing conserved legume orthologs. Of the 1536 SNP markers, 1054 were placed on a genetic map. In addition, 598 EST-SSR markers identified in A. hypogaea assemblies were included in the map along with 37 disease resistance gene candidate (RGC) and 35 other previously published markers. In total, 1724 markers spanning 1081.3 cM over 10 linkage groups were mapped. Gene sequences that provided mapped markers were annotated using similarity searches in three different databases, and gene ontology descriptions were determined using the Medicago Gene Atlas and TAIR databases. Synteny analysis between A. duranensis, Medicago and Glycine revealed significant stretches of conserved gene clusters spread across the peanut genome. A higher level of colinearity was detected between A. duranensis and Glycine than with Medicago.
The first high-density, gene-based linkage map for A. duranensis was generated that can serve as a reference map for both wild and cultivated Arachis species. The markers developed here are valuable resources for the peanut, and more broadly, to the legume research community. The A-genome map will have utility for fine mapping in other peanut species and has already had application for mapping a nematode resistance gene that was introgressed into A. hypogaea from A. cardenasii.
Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively.
We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.
These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.
The genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives.
A total of 67 new microsatellite markers (mainly TTG motif) were developed for Arachis. Only three of these markers, however, were polymorphic in cultivated peanut. These three new markers plus five other markers characterized previously were evaluated for number of alleles per locus and gene diversity using 60 accessions of A. hypogaea. Genetic relationships among these 60 accessions and a sample of 36 wild accessions representative of section Arachis were estimated using allelic variation observed in a selected set of 12 SSR markers. Results showed that the Brazilian peanut germplasm collection has considerable levels of genetic diversity detected by SSR markers. Similarity groups for A. hypogaea accessions were established, which is a useful criteria for selecting parental plants for crop improvement. Microsatellite marker transferability was up to 76% for species of the section Arachis, but only 45% for species from the other eight Arachis sections tested. A new marker (Ah-041) presented a 100% transferability and could be used to classify the peanut accessions in AA and non-AA genome carriers.
The level of polymorphism observed among accessions of A. hypogaea analyzed with newly developed microsatellite markers was low, corroborating the accumulated data which show that cultivated peanut presents a relatively reduced variation at the DNA level. A selected panel of SSR markers allowed the classification of A. hypogaea accessions into two major groups. The identification of similarity groups will be useful for the selection of parental plants to be used in breeding programs. Marker transferability is relatively high between accessions of section Arachis. The possibility of using microsatellite markers developed for one species in genetic evaluation of other species greatly reduces the cost of the analysis, since the development of microsatellite markers is still expensive and time consuming. The SSR markers developed in this study could be very useful for genetic analysis of wild species of Arachis, including comparative genome mapping, population genetic structure and phylogenetic inferences among species.
Arachis hypogaea (peanut) is an important crop worldwide, being mostly used for edible oil production, direct consumption and animal feed. Cultivated peanut is an allotetraploid species with two different genome components, A and B. Genetic linkage maps can greatly assist molecular breeding and genomic studies. However, the development of linkage maps for A. hypogaea is difficult because it has very low levels of polymorphism. This can be overcome by the utilization of wild species of Arachis, which present the A- and B-genomes in the diploid state, and show high levels of genetic variability.
In this work, we constructed a B-genome linkage map, which will complement the previously published map for the A-genome of Arachis, and produced an entire framework for the tetraploid genome. This map is based on an F2 population of 93 individuals obtained from the cross between the diploid A. ipaënsis (K30076) and the closely related A. magna (K30097), the former species being the most probable B genome donor to cultivated peanut. In spite of being classified as different species, the parents showed high crossability and relatively low polymorphism (22.3%), compared to other interspecific crosses. The map has 10 linkage groups, with 149 loci spanning a total map distance of 1,294 cM. The microsatellite markers utilized, developed for other Arachis species, showed high transferability (81.7%). Segregation distortion was 21.5%. This B-genome map was compared to the A-genome map using 51 common markers, revealing a high degree of synteny between both genomes.
The development of genetic maps for Arachis diploid wild species with A- and B-genomes effectively provides a genetic map for the tetraploid cultivated peanut in two separate diploid components and is a significant advance towards the construction of a transferable reference map for Arachis. Additionally, we were able to identify affinities of some Arachis linkage groups with Medicago truncatula, which will allow the transfer of information from the nearly-complete genome sequences of this model legume to the peanut crop.
The construction of genetic linkage maps for cultivated peanut (Arachis hypogaea L.) has and continues to be an important research goal to facilitate quantitative trait locus (QTL) analysis and gene tagging for use in a marker-assisted selection in breeding. Even though a few maps have been developed, they were constructed using diploid or interspecific tetraploid populations. The most recently published intra-specific map was constructed from the cross of cultivated peanuts, in which only 135 simple sequence repeat (SSR) markers were sparsely populated in 22 linkage groups. The more detailed linkage map with sufficient markers is necessary to be feasible for QTL identification and marker-assisted selection. The objective of this study was to construct a genetic linkage map of cultivated peanut using simple sequence repeat (SSR) markers derived primarily from peanut genomic sequences, expressed sequence tags (ESTs), and by "data mining" sequences released in GenBank.
Three recombinant inbred lines (RILs) populations were constructed from three crosses with one common female parental line Yueyou 13, a high yielding Spanish market type. The four parents were screened with 1044 primer pairs designed to amplify SSRs and 901 primer pairs produced clear PCR products. Of the 901 primer pairs, 146, 124 and 64 primer pairs (markers) were polymorphic in these populations, respectively, and used in genotyping these RIL populations. Individual linkage maps were constructed from each of the three populations and a composite map based on 93 common loci were created using JoinMap. The composite linkage maps consist of 22 composite linkage groups (LG) with 175 SSR markers (including 47 SSRs on the published AA genome maps), representing the 20 chromosomes of A. hypogaea. The total composite map length is 885.4 cM, with an average marker density of 5.8 cM. Segregation distortion in the 3 populations was 23.0%, 13.5% and 7.8% of the markers, respectively. These distorted loci tended to cluster on LG1, LG3, LG4 and LG5. There were only 15 EST-SSR markers mapped due to low polymorphism. By comparison, there were potential synteny, collinear order of some markers and conservation of collinear linkage groups among the maps and with the AA genome but not fully conservative.
A composite linkage map was constructed from three individual mapping populations with 175 SSR markers in 22 composite linkage groups. This composite genetic linkage map is among the first "true" tetraploid peanut maps produced. This map also consists of 47 SSRs that have been used in the published AA genome maps, and could be used in comparative mapping studies. The primers described in this study are PCR-based markers, which are easy to share for genetic mapping in peanuts. All 1044 primer pairs are provided as additional files and the three RIL populations will be made available to public upon request for quantitative trait loci (QTL) analysis and linkage map improvement.
The complex, tetraploid genome structure of peanut (Arachis hypogaea) has obstructed advances in genetics and genomics in the species. The aim of this study is to understand the genome structure of Arachis by developing a high-density integrated consensus map. Three recombinant inbred line populations derived from crosses between the A genome diploid species, Arachis duranensis and Arachis stenosperma; the B genome diploid species, Arachis ipaënsis and Arachis magna; and between the AB genome tetraploids, A. hypogaea and an artificial amphidiploid (A. ipaënsis × A. duranensis)4×, were used to construct genetic linkage maps: 10 linkage groups (LGs) of 544 cM with 597 loci for the A genome; 10 LGs of 461 cM with 798 loci for the B genome; and 20 LGs of 1442 cM with 1469 loci for the AB genome. The resultant maps plus 13 published maps were integrated into a consensus map covering 2651 cM with 3693 marker loci which was anchored to 20 consensus LGs corresponding to the A and B genomes. The comparative genomics with genome sequences of Cajanus cajan, Glycine max, Lotus japonicus, and Medicago truncatula revealed that the Arachis genome has segmented synteny relationship to the other legumes. The comparative maps in legumes, integrated tetraploid consensus maps, and genome-specific diploid maps will increase the genetic and genomic understanding of Arachis and should facilitate molecular breeding.
Arachis spp.; comparative genomics; genetic linkage map; integrated consensus map; legume genome
The genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species.
Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair Ah11 from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci.
These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species.
Cultivated peanut or groundnut (Arachis hypogaea L.) is an important oilseed crop with an allotetraploid genome (AABB, 2n = 4x = 40). Both the low level of genetic variation within the cultivated gene pool and its polyploid nature limit the utilization of molecular markers to explore genome structure and facilitate genetic improvement. Nevertheless, a wealth of genetic diversity exists in diploid Arachis species (2n = 2x = 20), which represent a valuable gene pool for cultivated peanut improvement. Interspecific populations have been used widely for genetic mapping in diploid species of Arachis. However, an intraspecific mapping strategy was essential to detect chromosomal rearrangements among species that could be obscured by mapping in interspecific populations. To develop intraspecific reference linkage maps and gain insights into karyotypic evolution within the genus, we comparatively mapped the A- and B-genome diploid species using intraspecific F2 populations. Exploring genome organization among diploid peanut species by comparative mapping will enhance our understanding of the cultivated tetraploid peanut genome. Moreover, new sources of molecular markers that are highly transferable between species and developed from expressed genes will be required to construct saturated genetic maps for peanut.
A total of 2,138 EST-SSR (expressed sequence tag-simple sequence repeat) markers were developed by mining a tetraploid peanut EST assembly including 101,132 unigenes (37,916 contigs and 63,216 singletons) derived from 70,771 long-read (Sanger) and 270,957 short-read (454) sequences. A set of 97 SSR markers were also developed by mining 9,517 genomic survey sequences of Arachis. An SSR-based intraspecific linkage map was constructed using an F2 population derived from a cross between K 9484 (PI 298639) and GKBSPSc 30081 (PI 468327) in the B-genome species A. batizocoi. A high degree of macrosynteny was observed when comparing the homoeologous linkage groups between A (A. duranensis) and B (A. batizocoi) genomes. Comparison of the A- and B-genome genetic linkage maps also showed a total of five inversions and one major reciprocal translocation between two pairs of chromosomes under our current mapping resolution.
Our findings will contribute to understanding tetraploid peanut genome origin and evolution and eventually promote its genetic improvement. The newly developed EST-SSR markers will enrich current molecular marker resources in peanut.
Peanut (Arachis hypogaea); SSR; Genetic linkage map; Intraspecific cross; EST
Peanut (Arachis hypogaea L.) ranks fifth among the world oil crops and is widely grown in India and neighbouring countries. Due to
its large and unknown genome size, studies on genomics and genetic modification of peanut are still scanty as compared to other
model crops like Arabidopsis, rice, cotton and soybean. Because of its favourable cultivation in semi-arid regions, study on abiotic
stress responsive genes and its regulation in peanut is very much important. Therefore, we aim to identify and annotate the abiotic
stress responsive candidate genes in peanut ESTs. Expression data of drought stress responsive corresponding genes and EST
sequences were screened from dot blot experiments shown as heat maps and supplementary tables, respectively as reported by
Govind et al. (2009). Some of the screened genes having no information about their ESTs in above mentioned supplementary tables
were retrieved from NCBI. A phylogenetic analysis was performed to find a group of utmost similar ESTs for each selected gene.
Individual EST of the said group were further searched in peanut ESTs (1,78,490 whole EST sequences) using stand alone BLAST.
For the prediction as well as annotation of abiotic stress responsive selected genes, various tools (like Vec-Screen, Repeat Masker,
EST-Trimmer, DNA Baser, WISE2 and I-TASSER) were used. Here we report the predicted result of Contigs, domain as well as 3D
structure for HSP 17.3KDa protein, DnaJ protein and Type 2 Metallothionein protein.
Arachis hypogaea; EST; Gene annotation; Stress; Contigs
Background and Aims
The genus Arachis contains 80 described species. Section Arachis is of particular interest because it includes cultivated peanut, an allotetraploid, and closely related wild species, most of which are diploids. This study aimed to analyse the genetic relationships of multiple accessions of section Arachis species using two complementary methods. Microsatellites allowed the analysis of inter- and intraspecific variability. Intron sequences from single-copy genes allowed phylogenetic analysis including the separation of the allotetraploid genome components.
Intron sequences and microsatellite markers were used to reconstruct phylogenetic relationships in section Arachis through maximum parsimony and genetic distance analyses.
Although high intraspecific variability was evident, there was good support for most species. However, some problems were revealed, notably a probable polyphyletic origin for A. kuhlmannii. The validity of the genome groups was well supported. The F, K and D genomes grouped close to the A genome group. The 2n = 18 species grouped closer to the B genome group. The phylogenetic tree based on the intron data strongly indicated that A. duranensis and A. ipaënsis are the ancestors of A. hypogaea and A. monticola. Intron nucleotide substitutions allowed the ages of divergences of the main genome groups to be estimated at a relatively recent 2·3–2·9 million years ago. This age and the number of species described indicate a much higher speciation rate for section Arachis than for legumes in general.
The analyses revealed relationships between the species and genome groups and showed a generally high level of intraspecific genetic diversity. The improved knowledge of species relationships should facilitate the utilization of wild species for peanut improvement. The estimates of speciation rates in section Arachis are high, but not unprecedented. We suggest these high rates may be linked to the peculiar reproductive biology of Arachis.
Arachis; peanut; groundnut; intron sequences; single-copy genes; molecular phylogeny; microsatellites; genetic relationships; speciation rates; genome donors; molecular dating
Wild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield. The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. This can be achieved by comparing the expression ratio between a gene of interest and a reference gene which is constitutively expressed. Nowadays there is a lack of appropriate reference genes for both wild and cultivated Arachis. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut.
A set of ten reference genes were analyzed in four Arachis species (A. magna; A. duranensis; A. stenosperma and A. hypogaea) subjected to biotic (root-knot nematode and leaf spot fungus) and abiotic (drought) stresses, in two distinct plant organs (roots and leaves). By the use of three programs (GeNorm, NormFinder and BestKeeper) and taking into account the entire dataset, five of these ten genes, ACT1 (actin depolymerizing factor-like protein), UBI1 (polyubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L10) and UBI2 (ubiquitin/ribosomal protein S27a) emerged as top reference genes, with their stability varying in eight subsets. The former three genes were the most stable across all species, organs and treatments studied.
This first in-depth study of reference genes validation in wild Arachis species will allow the use of specific combinations of secure and stable reference genes in qRT-PCR assays. The use of these appropriate references characterized here should improve the accuracy and reliability of gene expression analysis in both wild and cultivated Arachis and contribute for the better understanding of gene expression in, for instance, stress tolerance/resistance mechanisms in plants.
Cultivated groundnut or peanut (Arachis hypogaea L.), an allotetraploid (2n = 4x = 40), is a self pollinated and widely grown crop in the semi-arid regions of the world. Improvement of drought tolerance is an important area of research for groundnut breeding programmes. Therefore, for the identification of candidate QTLs for drought tolerance, a comprehensive and refined genetic map containing 191 SSR loci based on a single mapping population (TAG 24 × ICGV 86031), segregating for drought and surrogate traits was developed. Genotyping data and phenotyping data collected for more than ten drought related traits in 2–3 seasons were analyzed in detail for identification of main effect QTLs (M-QTLs) and epistatic QTLs (E-QTLs) using QTL Cartographer, QTLNetwork and Genotype Matrix Mapping (GMM) programmes. A total of 105 M-QTLs with 3.48–33.36% phenotypic variation explained (PVE) were identified using QTL Cartographer, while only 65 M-QTLs with 1.3–15.01% PVE were identified using QTLNetwork. A total of 53 M-QTLs were such which were identified using both programmes. On the other hand, GMM identified 186 (8.54–44.72% PVE) and 63 (7.11–21.13% PVE), three and two loci interactions, whereas only 8 E-QTL interactions with 1.7–8.34% PVE were identified through QTLNetwork. Interestingly a number of co-localized QTLs controlling 2–9 traits were also identified. The identification of few major, many minor M-QTLs and QTL × QTL interactions during the present study confirmed the complex and quantitative nature of drought tolerance in groundnut. This study suggests deployment of modern approaches like marker-assisted recurrent selection or genomic selection instead of marker-assisted backcrossing approach for breeding for drought tolerance in groundnut.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-010-1517-0) contains supplementary material, which is available to authorized users.
Peanut; Drought tolerance; Genetic map; Molecular markers; Main-effect QTLs; Epistatic QTLs; Molecular breeding
Peanut (Arachis hypogaea L.) is an important crop economically and nutritionally, and is one of the most susceptible host crops to colonization of Aspergillus parasiticus and subsequent aflatoxin contamination. Knowledge from molecular genetic studies could help to devise strategies in alleviating this problem; however, few peanut DNA sequences are available in the public database. In order to understand the molecular basis of host resistance to aflatoxin contamination, a large-scale project was conducted to generate expressed sequence tags (ESTs) from developing seeds to identify resistance-related genes involved in defense response against Aspergillus infection and subsequent aflatoxin contamination.
We constructed six different cDNA libraries derived from developing peanut seeds at three reproduction stages (R5, R6 and R7) from a resistant and a susceptible cultivated peanut genotypes, 'Tifrunner' (susceptible to Aspergillus infection with higher aflatoxin contamination and resistant to TSWV) and 'GT-C20' (resistant to Aspergillus with reduced aflatoxin contamination and susceptible to TSWV). The developing peanut seed tissues were challenged by A. parasiticus and drought stress in the field. A total of 24,192 randomly selected cDNA clones from six libraries were sequenced. After removing vector sequences and quality trimming, 21,777 high-quality EST sequences were generated. Sequence clustering and assembling resulted in 8,689 unique EST sequences with 1,741 tentative consensus EST sequences (TCs) and 6,948 singleton ESTs. Functional classification was performed according to MIPS functional catalogue criteria. The unique EST sequences were divided into twenty-two categories. A similarity search against the non-redundant protein database available from NCBI indicated that 84.78% of total ESTs showed significant similarity to known proteins, of which 165 genes had been previously reported in peanuts. There were differences in overall expression patterns in different libraries and genotypes. A number of sequences were expressed throughout all of the libraries, representing constitutive expressed sequences. In order to identify resistance-related genes with significantly differential expression, a statistical analysis to estimate the relative abundance (R) was used to compare the relative abundance of each gene transcripts in each cDNA library. Thirty six and forty seven unique EST sequences with threshold of R > 4 from libraries of 'GT-C20' and 'Tifrunner', respectively, were selected for examination of temporal gene expression patterns according to EST frequencies. Nine and eight resistance-related genes with significant up-regulation were obtained in 'GT-C20' and 'Tifrunner' libraries, respectively. Among them, three genes were common in both genotypes. Furthermore, a comparison of our EST sequences with other plant sequences in the TIGR Gene Indices libraries showed that the percentage of peanut EST matched to Arabidopsis thaliana, maize (Zea mays), Medicago truncatula, rapeseed (Brassica napus), rice (Oryza sativa), soybean (Glycine max) and wheat (Triticum aestivum) ESTs ranged from 33.84% to 79.46% with the sequence identity ≥ 80%. These results revealed that peanut ESTs are more closely related to legume species than to cereal crops, and more homologous to dicot than to monocot plant species.
The developed ESTs can be used to discover novel sequences or genes, to identify resistance-related genes and to detect the differences among alleles or markers between these resistant and susceptible peanut genotypes. Additionally, this large collection of cultivated peanut EST sequences will make it possible to construct microarrays for gene expression studies and for further characterization of host resistance mechanisms. It will be a valuable genomic resource for the peanut community. The 21,777 ESTs have been deposited to the NCBI GenBank database with accession numbers ES702769 to ES724546.
A single dominant gene for resistance to Meloidogyne arenaria was identified previously in two peanut cultivars, Arachis hypogaea 'COAN' and 'NemaTAM'. The interspecific Arachis hybrid TxAG-6 was the source of this resistance and the donor parent in a backcross breeding program to introgress resistance into cultivated peanut. To determine if other resistance genes were present in TxAG-6 and derived breeding populations from the third backcross generation (BC₃), F₂ individuals were evaluated for the resistance phenotype. The ratio of the resistant and susceptible individuals for all F₂ populations fit the expected ratio for resistance being governed by one dominant gene and one recessive gene. Evaluation of the F₃ generation from four susceptible F₂ individuals (two from TxAG-6 × A. hypogaea and two from the BC₃ population) confirmed that a recessive gene for resistance to M. arenaria was present in each of the tested populations. The identification of a second gene for resistance in the A. hypogaea germplasm may improve the durability of the resistance phenotype.
Arachis hypogaea; durable resistance; Meloidogyne arenaria; peanut; recessive inheritance; resistance genes; root-knot nematode
Two hundred thirty-five cultivated varieties, breeding lines and plant introductions of Arachis hypogaea and 12 accessions of wild Arachis spp. were tested for resistance to Meloidogyne hapla. Eight of the cultivated peanut lines were only moderately susceptible and four of the wild peanuts exhibited resistance. No resistance-breaking M. hapla populations were found among 10 geographical isolates tested.
northern root-knot nematode; Arachis hypogaea; Arachis spp.
Peanut (Arachis hypogaea) is an autogamous allotetraploid legume (2n = 4x = 40) that is widely cultivated as a food and oil crop. More than 6,000 DNA markers have been developed in Arachis spp., but high-density linkage maps useful for genetics, genomics, and breeding have not been constructed due to extremely low genetic diversity. Polymorphic marker loci are useful for the construction of such high-density linkage maps. The present study used in silico analysis to develop simple sequence repeat-based and transposon-based markers.
The use of in silico analysis increased the efficiency of polymorphic marker development by more than 3-fold. In total, 926 (34.2%) of 2,702 markers showed polymorphisms between parental lines of the mapping population. Linkage analysis of the 926 markers along with 253 polymorphic markers selected from 4,449 published markers generated 21 linkage groups covering 2,166.4 cM with 1,114 loci. Based on the map thus produced, 23 quantitative trait loci (QTLs) for 15 agronomical traits were detected. Another linkage map with 326 loci was also constructed and revealed a relationship between the genotypes of the FAD2 genes and the ratio of oleic/linoleic acid in peanut seed.
In silico analysis of polymorphisms increased the efficiency of polymorphic marker development, and contributed to the construction of high-density linkage maps in cultivated peanut. The resultant maps were applicable to QTL analysis. Marker subsets and linkage maps developed in this study should be useful for genetics, genomics, and breeding in Arachis. The data are available at the Kazusa DNA Marker Database (http://marker.kazusa.or.jp).
DNA marker; Genetic linkage map; Peanut (Arachis hypogaea); QTL analysis; Ratio of oleic/linoleic acid (O/L ratio)
Peanut (Arachis hypogaea L.) is widely used as a food and cash crop around the world. It is considered to be an allotetraploid (2n = 4x = 40) originated from a single hybridization event between two wild diploids. The most probable hypothesis gave A. duranensis as the wild donor of the A genome and A. ipaënsis as the wild donor of the B genome. A low level of molecular polymorphism is found in cultivated germplasm and up to date few genetic linkage maps have been published. The utilization of wild germplasm in breeding programs has received little attention due to the reproductive barriers between wild and cultivated species and to the technical difficulties encountered in making large number of crosses. We report here the development of a SSR based genetic map and the analysis of genome-wide segment introgressions into the background of a cultivated variety through the utilization of a synthetic amphidiploid between A. duranensis and A. ipaënsis.
Two hundred ninety eight (298) loci were mapped in 21 linkage groups (LGs), spanning a total map distance of 1843.7 cM with an average distance of 6.1 cM between adjacent markers. The level of polymorphism observed between the parent of the amphidiploid and the cultivated variety is consistent with A. duranensis and A. ipaënsis being the most probable donor of the A and B genomes respectively. The synteny analysis between the A and B genomes revealed an overall good collinearity of the homeologous LGs. The comparison with the diploid and tetraploid maps shed new light on the evolutionary forces that contributed to the divergence of the A and B genome species and raised the question of the classification of the B genome species. Structural modifications such as chromosomal segment inversions and a major translocation event prior to the tetraploidisation of the cultivated species were revealed. Marker assisted selection of BC1F1 and then BC2F1 lines carrying the desirable donor segment with the best possible return to the background of the cultivated variety provided a set of lines offering an optimal distribution of the wild introgressions.
The genetic map developed, allowed the synteny analysis of the A and B genomes, the comparison with diploid and tetraploid maps and the analysis of the introgression segments from the wild synthetic into the background of a cultivated variety. The material we have produced in this study should facilitate the development of advanced backcross and CSSL breeding populations for the improvement of cultivated peanut.
The peanut (Arachis hypogaea) is an important crop cultivated worldwide for oil production and food sources. Its complex genetic architecture (e.g., the large and tetraploid genome possibly due to unique cross of wild diploid relatives and subsequent chromosome duplication: 2n = 4x = 40, AABB, 2800 Mb) presents a major challenge for its genome sequencing and makes it a less-studied crop. Without a doubt, transcriptome sequencing is the most effective way to harness the genome structure and gene expression dynamics of this non-model species that has a limited genomic resource.
With the development of next generation sequencing technologies such as 454 pyro-sequencing and Illumina sequencing by synthesis, the transcriptomics data of peanut is rapidly accumulated in both the public databases and private sectors. Integrating 187,636 Sanger reads (103,685,419 bases), 1,165,168 Roche 454 reads (333,862,593 bases) and 57,135,995 Illumina reads (4,073,740,115 bases), we generated the first release of our peanut transcriptome assembly that contains 32,619 contigs. We provided EC, KEGG and GO functional annotations to these contigs and detected SSRs, SNPs and other genetic polymorphisms for each contig. Based on both open-source and our in-house tools, PeanutDB presents many seamlessly integrated web interfaces that allow users to search, filter, navigate and visualize easily the whole transcript assembly, its annotations and detected polymorphisms and simple sequence repeats. For each contig, sequence alignment is presented in both bird’s-eye view and nucleotide level resolution, with colorfully highlighted regions of mismatches, indels and repeats that facilitate close examination of assembly quality, genetic polymorphisms, sequence repeats and/or sequencing errors.
As a public genomic database that integrates peanut transcriptome data from different sources, PeanutDB (http://bioinfolab.muohio.edu/txid3818v1) provides the Peanut research community with an easy-to-use web portal that will definitely facilitate genomics research and molecular breeding in this less-studied crop.
Peanut; Arachis hypogaea; Transcriptome sequencing; Transcriptome assembly; Database; PeanutDB; SNP; SSR; Functional annotation
Due to its origin, peanut has a very narrow genetic background. Wild relatives can be a source of genetic variability for cultivated peanut. In this study, the transcriptome of the wild species Arachis stenosperma accession V10309 was analyzed.
ESTs were produced from four cDNA libraries of RNAs extracted from leaves and roots of A. stenosperma. Randomly selected cDNA clones were sequenced to generate 8,785 ESTs, of which 6,264 (71.3%) had high quality, with 3,500 clusters: 963 contigs and 2537 singlets. Only 55.9% matched homologous sequences of known genes. ESTs were classified into 23 different categories according to putative protein functions. Numerous sequences related to disease resistance, drought tolerance and human health were identified. Two hundred and six microsatellites were found and markers have been developed for 188 of these. The microsatellite profile was analyzed and compared to other transcribed and genomic sequence data.
This is, to date, the first report on the analysis of transcriptome of a wild relative of peanut. The ESTs produced in this study are a valuable resource for gene discovery, the characterization of new wild alleles, and for marker development. The ESTs were released in the [GenBank:EH041934 to EH048197].
Improving drought tolerance through gene manipulation has been of importance for modern agriculture, which requires identification and validation of candidate genes. Prospecting candidate genes from drought adapted crop species is of immense significance. To identify candidate stress responsive genes from adapted crop, we carried out expression analysis of a few drought responsive ESTs from Arachis hypogaea L. (peanut). The expression patterns of nine AhDR (Arachis hypogea drought responsive) clones were analysed under drought. Quantitative reverse transcription PCR analysis revealed stress responsive nature of the selected genes. The clones AhDR 118 (putative cyclin T-like), AhDR185 (aldehyde reductase-like), AhDR193 (cholin kinase-like) and AhDR 76 (proline amino peptidase-like) showed more than five fold increase in expression. Highly upregulated genes analysed for expression pattern against salinity at seedling level indicated that these genes provide cross protection. This paper is the first report indicating the association of peanut genes cyclin T, proline amino peptidase and choline kinase to drought tolerance, and the possible roles of these genes are discussed.
Arachis; Drought genes; Gene expression
One hundred and forty-six highly polymorphic simple sequence repeat (SSR) markers were used to assess the genetic diversity and population structure of 196 peanut (Arachis Hypogaea L.) cultivars which had been extensively planted in different regions in China. These SSR markers amplified 440 polymorphic bands with an average of 2.99, and the average gene diversity index was 0.11. Eighty-six rare alleles with a frequency of less than 1% were identified in these cultivars. The largest Fst or genetic distance was found between the cultivars that adapted to the south regions and those to the north regions in China. A neighbor-joining tree of cultivars adapted to different ecological regions was constructed based on pairwise Nei’s genetic distances, which showed a significant difference between cultivars from the south and the north regions. A model-based population structure analysis divided these peanut cultivars into five subpopulations (P1a, P1b, P2, P3a and P3b). P1a and P1b included most the cultivars from the southern provinces including Guangdong, Guangxi and Fujian. P2 population consisted of the cultivars from Hubei province and parts from Shandong and Henan. P3a and P3b had cultivars from the northern provinces including Shandong, Anhui, Henan, Hebei, Jiangsu and the Yangtze River region including Sichuan province. The cluster analysis, PCoA and PCA based on the marker genotypes, revealed five distinct clusters for the entire population that were related to their germplasm regions. The results indicated that there were obvious genetic variations between cultivars from the south and the north, and there were distinct genetic differentiation among individual cultivars from the south and the north. Taken together, these results provided a molecular basis for understanding genetic diversity of Chinese peanut cultivars.