Development and characterization of highly polymorphic long TC repeat microsatellite markers for genetic analysis of peanut

<p>Abstract</p> <p>Background</p> <p>Peanut (<it>Arachis hypogaea </it>L.) is a crop of economic and social importance, mainly in tropical areas, and developing countries. Its molecular breeding has been hindered by a shortage of polymorphic genetic markers due to a very narrow genetic base. Microsatellites (SSRs) are markers of choice in peanut because they are co-dominant, highly transferrable between species and easily applicable in the allotetraploid genome. In spite of substantial effort over the last few years by a number of research groups, the number of SSRs that are polymorphic for <it>A. hypogaea </it>is still limiting for routine application, creating the demand for the discovery of more markers polymorphic within cultivated germplasm.</p> <p>Findings</p> <p>A plasmid genomic library enriched for TC/AG repeats was constructed and 1401 clones sequenced. From the sequences obtained 146 primer pairs flanking mostly TC microsatellites were developed. The average number of repeat motifs amplified was 23. These 146 markers were characterized on 22 genotypes of cultivated peanut. In total 78 of the markers were polymorphic within cultivated germplasm. Most of those 78 markers were highly informative with an average of 5.4 alleles per locus being amplified. Average gene diversity index (GD) was 0.6, and 66 markers showed a GD of more than 0.5. Genetic relationship analysis was performed and corroborated the current taxonomical classification of <it>A. hypogaea </it>subspecies and varieties.</p> <p>Conclusions</p> <p>The microsatellite markers described here are a useful resource for genetics and genomics in <it>Arachis</it>. In particular, the 66 markers that are highly polymorphic in cultivated peanut are a significant step towards routine genetic mapping and marker-assisted selection for the crop.</p

ESTs from a wild Arachis species for gene discovery and marker development

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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]

Development of molecular markers for resistance gene analogs in wild Arachis spp.

O maior grupo de genes de resistência de plantas já clonados codifica para proteínas com um sítio de ligação a nucleotídios (NBS) na região N-terminal, e um domínio rico em repetições de leucina (LRR) na região C-terminal. Genes desta classe conferem resistência a diversos patógenos incluindo vírus, bactérias, fungos e nematóides. Para diferentes espécies do gênero Arachis, primers de "polymerase chain reaction" (PCR) degenerados foram construídos para a região NBS, e o produto de tradução putativo indicou similaridade com proteínas de resistência conhecidas sendo denominados análogos a genes de resistência (RGAs). Doze destes RGAs foram utilizados para o desenvolvimento de marcadores moleculares baseados em seus padrões de hibridização com DNA de Arachis spp. digerido com enzimas de restrição. Inicialmente, avaliou-se o polimorfismo de cada RGA como sonda nos parentais de uma população de mapeamento, contrastantes quanto à resistência as manchas foliares e nematóides das galhas, e no híbrido F1. Os RGAs, mesmo isolados de espécies diferentes do gênero Arachis apresentaram homologia com o DNA das espécies testadas, além de apresentarem múltiplas cópias e alto polimorfismo na progênie F2. Todas estas características tornam estes RGAs marcadores moleculares altamente informativos, sendo que alguns apresentaram segregação em "clusters" na F2, indicando que seus locos estão ligados. Estes marcadores serão incluídos em um mapa genético de Arachis spp., o que será de grande utilidade para os programas de melhoramento do amendoim (Arachis hypogaea) cultivado.The majority of cloned plant pathogen resistance genes (R genes) encode a putative nucleotide binding site (NBS) domain and a leucine-rich repeat (NBS-LRR genes). Genes of this NBS-LRR class confer resistance to diverse pathogens such as viruses, bacteria, fungi, nematodes and aphids. The conserved NBS domain was used to generate resistance gene analogues (RGAs) fragments by polymerase chain reaction (PCR) using degenerated primers in different Arachis species. Twelve of these RGAs were used to develop molecular markers based on their patterns of hybridisation to restricted Arachis spp. DNA. An initial step was the evaluation of the polymorphism generated by each RGA in genomic fragments of contrasting parents of a mapping population that segregates for resistance to leaf spot and nematodes, and of the F1 hybrid. The RGAs isolated from different Arachis species showed high homology to the DNA of the parents and hybrid, multiple copies in the genome and high polymorphism in the F2 generation. Therefore, they were considered highly informative markers, with some segregating in clusters in the F2. These RGAs will be included in the Arachis genetic map, which will be of paramount importance for the Arachis spp. breeding programs

Resistance to Thrips in Peanut and Implications for Management of Thrips and Thrips-Transmitted Orthotospoviruses in Peanut

Thrips are major pests of peanut (Arachis hypogaea L.) worldwide, and they serve as vectors of devastating orthotospoviruses such as Tomato spotted wilt virus (TSWV) and Groundnut bud necrosis virus (GBNV). A tremendous effort has been devoted to developing peanut cultivars with resistance to orthotospoviruses. Consequently, cultivars with moderate field resistance to viruses exist, but not much is known about host resistance to thrips. Integrating host plant resistance to thrips in peanut could suppress thrips feeding damage and reduce virus transmission, will decrease insecticide usage, and enhance sustainability in the production system. This review focuses on details of thrips resistance in peanut and identifies future directions for incorporating thrips resistance in peanut cultivars. Research on thrips–host interactions in peanut is predominantly limited to field evaluations of feeding damage, though, laboratory studies have revealed that peanut cultivars could differentially affect thrips feeding and thrips biology. Many runner type cultivars, field resistant to TSWV, representing diverse pedigrees evaluated against thrips in the greenhouse revealed that thrips preferred some cultivars over others, suggesting that antixenosis “non-preference” could contribute to thrips resistance in peanut. In other crops, morphological traits such as leaf architecture and waxiness and spectral reflectance have been associated with thrips non-preference. It is not clear if foliar morphological traits in peanut are associated with reduced preference or non-preference of thrips and need to be evaluated. Besides thrips non-preference, thrips larval survival to adulthood and median developmental time were negatively affected in some peanut cultivars and in a diploid peanut species Arachis diogoi (Hoehne) and its hybrids with a Virginia type cultivar, indicating that antibiosis (negative effects on biology) could also be a factor influencing thrips resistance in peanut. Available field resistance to orthotospoviruses in peanut is not complete, and cultivars can suffer substantial yield loss under high thrips and virus pressure. Integrating thrips resistance with available virus resistance would be ideal to limit losses. A discussion of modern technologies such as transgenic resistance, marker assisted selection and RNA interference, and future directions that could be undertaken to integrate resistance to thrips and to orthotospoviruses in peanut cultivars is included in this article

A linkage map for the B-genome of Arachis (Fabaceae) and its synteny to the A-genome

<p>Abstract</p> <p>Background</p> <p><it>Arachis hypogaea </it>(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 <it>A. hypogaea </it>is difficult because it has very low levels of polymorphism. This can be overcome by the utilization of wild species of <it>Arachis</it>, which present the A- and B-genomes in the diploid state, and show high levels of genetic variability.</p> <p>Results</p> <p>In this work, we constructed a B-genome linkage map, which will complement the previously published map for the A-genome of <it>Arachis</it>, and produced an entire framework for the tetraploid genome. This map is based on an F₂population of 93 individuals obtained from the cross between the diploid <it>A. ipaënsis </it>(K30076) and the closely related <it>A. magna </it>(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 <it>Arachis </it>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.</p> <p>Conclusion</p> <p>The development of genetic maps for <it>Arachis </it>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 <it>Arachis</it>. Additionally, we were able to identify affinities of some <it>Arachis </it>linkage groups with <it>Medicago truncatula</it>, which will allow the transfer of information from the nearly-complete genome sequences of this model legume to the peanut crop.</p

An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes

<p>Abstract</p> <p>Background</p> <p>Most agriculturally important legumes fall within two sub-clades of the Papilionoid legumes: the Phaseoloids and Galegoids, which diverged about 50 Mya. The Phaseoloids are mostly tropical and include crops such as common bean and soybean. The Galegoids are mostly temperate and include clover, fava bean and the model legumes <it>Lotus </it>and <it>Medicago </it>(both with substantially sequenced genomes). In contrast, peanut (<it>Arachis hypogaea</it>) falls in the Dalbergioid clade which is more basal in its divergence within the Papilionoids. The aim of this work was to integrate the genetic map of <it>Arachis </it>with <it>Lotus </it>and <it>Medicago </it>and improve our understanding of the <it>Arachis </it>genome and legume genomes in general. To do this we placed on the <it>Arachis </it>map, comparative anchor markers defined using a previously described bioinformatics pipeline. Also we investigated the possible role of transposons in the patterns of synteny that were observed.</p> <p>Results</p> <p>The <it>Arachis </it>genetic map was substantially aligned with <it>Lotus </it>and <it>Medicago </it>with most synteny blocks presenting a single main affinity to each genome. This indicates that the last common whole genome duplication within the Papilionoid legumes predated the divergence of <it>Arachis </it>from the Galegoids and Phaseoloids sufficiently that the common ancestral genome was substantially diploidized. The <it>Arachis </it>and model legume genomes comparison made here, together with a previously published comparison of <it>Lotus </it>and <it>Medicago </it>allowed all possible <it>Arachis-Lotus-Medicago </it>species by species comparisons to be made and genome syntenies observed. Distinct conserved synteny blocks and non-conserved regions were present in all genome comparisons, implying that certain legume genomic regions are consistently more stable during evolution than others. We found that in <it>Medicago </it>and possibly also in <it>Lotus</it>, retrotransposons tend to be more frequent in the variable regions. Furthermore, while these variable regions generally have lower densities of single copy genes than the more conserved regions, some harbor high densities of the fast evolving disease resistance genes.</p> <p>Conclusion</p> <p>We suggest that gene space in Papilionoids may be divided into two broadly defined components: more conserved regions which tend to have low retrotransposon densities and are relatively stable during evolution; and variable regions that tend to have high retrotransposon densities, and whose frequent restructuring may fuel the evolution of some gene families.</p

Identification of candidate genome regions controlling disease resistance in Arachis

<p>Abstract</p> <p>Background</p> <p>Worldwide, diseases are important reducers of peanut (<it>Arachis hypogaea</it>) yield. Sources of resistance against many diseases are available in cultivated peanut genotypes, although often not in farmer preferred varieties. Wild species generally harbor greater levels of resistance and even apparent immunity, although the linkage of agronomically un-adapted wild alleles with wild disease resistance genes is inevitable. Marker-assisted selection has the potential to facilitate the combination of both cultivated and wild resistance loci with agronomically adapted alleles. However, in peanut there is an almost complete lack of knowledge of the regions of the <it>Arachis </it>genome that control disease resistance.</p> <p>Results</p> <p>In this work we identified candidate genome regions that control disease resistance. For this we placed candidate disease resistance genes and QTLs against late leaf spot disease on the genetic map of the A-genome of <it>Arachis</it>, which is based on microsatellite markers and legume anchor markers. These marker types are transferable within the genus <it>Arachis </it>and to other legumes respectively, enabling this map to be aligned to other <it>Arachis </it>maps and to maps of other legume crops including those with sequenced genomes. In total, 34 sequence-confirmed candidate disease resistance genes and five QTLs were mapped.</p> <p>Conclusion</p> <p>Candidate genes and QTLs were distributed on all linkage groups except for the smallest, but the distribution was not even. Groupings of candidate genes and QTLs for late leaf spot resistance were apparent on the upper region of linkage group 4 and the lower region of linkage group 2, indicating that these regions are likely to control disease resistance.</p

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

<p>Abstract</p> <p>Background</p> <p>Wild peanut species (<it>Arachis </it>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 <it>Arachis</it>. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut.</p> <p>Results</p> <p>A set of ten reference genes were analyzed in four <it>Arachis </it>species (<it>A. magna</it>; <it>A. duranensis</it>; <it>A. stenosperma </it>and <it>A. hypogaea</it>) 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, <it>ACT1 </it>(actin depolymerizing factor-like protein), <it>UBI1 </it>(polyubiquitin), <it>GAPDH </it>(glyceraldehyde-3-phosphate dehydrogenase), <it>60S </it>(60S ribosomal protein L10) and <it>UBI2 </it>(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.</p> <p>Conclusions</p> <p>This first in-depth study of reference genes validation in wild <it>Arachis </it>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.</p

Legacy genetics of Arachis cardenasii in the peanut crop shows the profound benefits of international seed exchange

A great challenge for humanity is feeding its growing population while minimizing ecosystem damage and climate change. Here, we uncover the global benefits arising from the introduction of one wild species accession to peanut-breeding programs decades ago. This work emphasizes the importance of biodiversity to crop improvement: peanut cultivars with genetics from this wild accession provided improved food security and reduced use of fungicide sprays. However, this study also highlights the perilous consequences of changes in legal frameworks and attitudes concerning biodiversity. These changes have greatly reduced the botanical collections, seed exchanges, and international collaborations which are essential for the continued diversification of crop genetics and, consequently, the long-term resilience of crops against evolving pests and pathogens and changing climate.The narrow genetics of most crops is a fundamental vulnerability to food security. This makes wild crop relatives a strategic resource of genetic diversity that can be used for crop improvement and adaptation to new agricultural challenges. Here, we uncover the contribution of one wild species accession, Arachis cardenasii GKP 10017, to the peanut crop (Arachis hypogaea) that was initiated by complex hybridizations in the 1960s and propagated by international seed exchange. However, until this study, the global scale of the dispersal of genetic contributions from this wild accession had been obscured by the multiple germplasm transfers, breeding cycles, and unrecorded genetic mixing between lineages that had occurred over the years. By genetic analysis and pedigree research, we identified A. cardenasii–enhanced, disease-resistant cultivars in Africa, Asia, Oceania, and the Americas. These cultivars provide widespread improved food security and environmental and economic benefits. This study emphasizes the importance of wild species and collaborative networks of international expertise for crop improvement. However, it also highlights the consequences of the implementation of a patchwork of restrictive national laws and sea changes in attitudes regarding germplasm that followed in the wake of the Convention on Biological Diversity. Today, the botanical collections and multiple seed exchanges which enable benefits such as those revealed by this study are drastically reduced. The research reported here underscores the vital importance of ready access to germplasm in ensuring long-term world food security.Genome sequence, genotyping, pedigree information, and yield trial data have been deposited in National Center for Biotechnology Information (NCBI), PeanutBase, and USDA Data Repository (NCBI: JADQCP000000000) (14). Datasets S1–S6 are available at USDA Ag Data Commons: https://data.nal.usda.gov/dataset/data-legacy-genetics-arachis-cardenasii-peanut-crop-v2 (17). All other study data are included in the article and/or supporting information