Genome-wide transcriptome and physiological analyses provide new insights into peanut drought response mechanisms

Drought is one of the main constraints in peanut production in West Texas and eastern New Mexico regions due to the depletion of groundwater. A multi-seasonal phenotypic analysis of 10 peanut genotypes revealed C76-16 (C-76) and Valencia-C (Val-C) as the best and poor performers under deficit irrigation (DI) in West Texas, respectively. In order to decipher transcriptome changes under DI, RNAseq was performed in C-76 and Val-C. Approximately 369 million raw reads were generated from 12 different libraries of two genotypes subjected to fully irrigated (FI) and DI conditions, of which ~329 million (90.2%) filtered reads were mapped to the diploid ancestors of peanut. The transcriptome analysis detected 4,508 differentially expressed genes (DEGs), 1554 genes encoding transcription factors (TFs) and a total of 514 single nucleotide polymorphisms (SNPs) among the identified DEGs. The comparative analysis between the two genotypes revealed higher and integral tolerance in C-76 through activation of key genes involved in ABA and sucrose metabolic pathways. Interestingly, one SNP from the gene coding F-box protein (Araip.3WN1Q) and another SNP from gene coding for the lipid transfer protein (Aradu.03ENG) showed polymorphism in selected contrasting genotypes. These SNPs after further validation may be useful for performing early generation selection for selecting drought-responsive genotypes

Integration of novel SSR and gene-based SNP marker loci in the chickpea genetic map and establishment of new anchor points with Medicago truncatula genome

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago

Genome-Wide Identification, Characterization, and Expression Analysis of Small RNA Biogenesis Purveyors Reveal Their Role in Regulation of Biotic Stress Responses in Three Legume Crops

Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA) mediated defense in plants. Small RNAs (sRNAs) have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDR) constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes

Novel SSR Markers from BAC-End Sequences, DArT Arrays and a Comprehensive Genetic Map with 1,291 Marker Loci for Chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes

Allele diversity for abiotic stress responsive candidate genes in chickpea reference set using gene based SNP markers

Chickpea is an important food legume crop for the semi-arid regions, however, its productivity is adversely affected by various biotic and abiotic stresses. Identification of candidate genes associated with abiotic stress response will help breeding efforts aiming to enhance its productivity. With this objective, 10 abiotic stress responsive candidate genes were selected on the basis of prior knowledge of this complex trait. These 10 genes were subjected to allele specific sequencing across a chickpea reference set comprising 300 genotypes including 211 genotypes of chickpea mini core collection. A total of 1.3 Mbp sequence data were generated. Multiple sequence alignment (MSA) revealed 79 SNPs and 41 indels in nine genes while the CAP2 gene was found to be conserved across all the genotypes. Among 10 candidate genes, the maximum number of SNPs (34) was observed in abscisic acid stress and ripening (ASR) gene including 22 transitions, 11 transversions and one tri-allelic SNP. Nucleotide diversity varied from 0.0004 to 0.0029 while polymorphism information content (PIC) values ranged from 0.01 (AKIN gene) to 0.43 (CAP2 promoter). Haplotype analysis revealed that alleles were represented by more than two haplotype blocks, except alleles of the CAP2 and sucrose synthase (SuSy) gene, where only one haplotype was identified. These genes can be used for association analysis and if validated, may be useful for enhancing abiotic stress, including drought tolerance, through molecular breeding

Genome-wide transcriptome and physiological analyses provide new insights into peanut drought response mechanisms

Drought is one of the main constraints in peanut production in West Texas and eastern New Mexico regions due to the depletion of groundwater. A multi-seasonal phenotypic analysis of 10 peanut genotypes revealed C76-16 (C-76) and Valencia-C (Val-C) as the best and poor performers under deficit irrigation (DI) in West Texas, respectively. In order to decipher transcriptome changes under DI, RNAseq was performed in C-76 and Val-C. Approximately 369 million raw reads were generated from 12 different libraries of two genotypes subjected to fully irrigated (FI) and DI conditions, of which ~329 million (90.2%) filtered reads were mapped to the diploid ancestors of peanut. The transcriptome analysis detected 4,508 differentially expressed genes (DEGs), 1554 genes encoding transcription factors (TFs) and a total of 514 single nucleotide polymorphisms (SNPs) among the identified DEGs. The comparative analysis between the two genotypes revealed higher and integral tolerance in C-76 through activation of key genes involved in ABA and sucrose metabolic pathways. Interestingly, one SNP from the gene coding F-box protein (Araip.3WN1Q) and another SNP from gene coding for the lipid transfer protein (Aradu.03ENG) showed polymorphism in selected contrasting genotypes. These SNPs after further validation may be useful for performing early generation selection for selecting drought-responsive genotypes

Resistance to Aspergillus flavus in maize and peanut: Molecular biology, breeding, environmental stress, and future perspectives

The colonization of maize (Zea mays L.) and peanut (Arachis hypogaea L.) by the fungal pathogen Aspergillus flavus results in the contamination of kernels with carcinogenic mycotoxins known as aflatoxins leading to economic losses and potential health threats to humans. The regulation of aflatoxin biosynthesis in various Aspergillus spp. has been extensively studied, and has been shown to be related to oxidative stress responses. Given that environmental stresses such as drought and heat stress result in the accumulation of reactive oxygen species (ROS) within host plant tissues, host-derived ROS may play an important role in cross-kingdom communication between host plants and A. flavus. Recent technological advances in plant breeding have provided the tools necessary to study and apply knowledge derived from metabolomic, proteomic, and transcriptomic studies in the context of productive breeding populations. Here, we review the current understanding of the potential roles of environmental stress, ROS, and aflatoxin in the interaction between A. flavus and its host plants, and the current status in molecular breeding and marker discovery for resistance to A. flavus colonization and aflatoxin contamination in maize and peanut. We will also propose future directions and a working model for continuing research efforts linking environmental stress tolerance and aflatoxin contamination resistance in maize and peanut

Aspergillus flavus infection triggered immune responses and host-pathogen cross-talks in groundnut during in-vitro seed colonization

Abstract Aflatoxin contamination, caused by fungal pathogen Aspergillus flavus, is a major quality and health problem delimiting the trade and consumption of groundnut (Arachis hypogaea L.) worldwide. RNA-seq approach was deployed to understand the host-pathogen interaction by identifying differentially expressed genes (DEGs) for resistance to in-vitro seed colonization (IVSC) at four critical stages after inoculation in J 11 (resistant) and JL 24 (susceptible) genotypes of groundnut. About 1,344.04 million sequencing reads have been generated from sixteen libraries representing four stages in control and infected conditions. About 64% and 67% of quality filtered reads (1,148.09 million) were mapped onto A (A. duranensis) and B (A. ipaёnsis) subgenomes of groundnut respectively. About 101 million unaligned reads each from J 11 and JL 24 were used to map onto A. flavus genome. As a result, 4,445 DEGs including defense-related genes like senescence-associated proteins, resveratrol synthase, 9s-lipoxygenase, pathogenesis-related proteins were identified. In A. flavus, about 578 DEGs coding for growth and development of fungus, aflatoxin biosynthesis, binding, transport, and signaling were identified in compatible interaction. Besides identifying candidate genes for IVSC resistance in groundnut, the study identified the genes involved in host-pathogen cross-talks and markers that can be used in breeding resistant varieties

Promoting utilization of Saccharum spp. genetic resources through genetic diversity analysis and core collection construction.

Sugarcane (Saccharum spp.) and other members of Saccharum spp. are attractive biofuel feedstocks. One of the two World Collections of Sugarcane and Related Grasses (WCSRG) is in Miami, FL. This WCSRG has 1002 accessions, presumably with valuable alleles for biomass, other important agronomic traits, and stress resistance. However, the WCSRG has not been fully exploited by breeders due to its lack of characterization and unmanageable population. In order to optimize the use of this genetic resource, we aim to 1) genotypically evaluate all the 1002 accessions to understand its genetic diversity and population structure and 2) form a core collection, which captures most of the genetic diversity in the WCSRG. We screened 36 microsatellite markers on 1002 genotypes and recorded 209 alleles. Genetic diversity of the WCSRG ranged from 0 to 0.5 with an average of 0.304. The population structure analysis and principal coordinate analysis revealed three clusters with all S. spontaneum in one cluster, S. officinarum and S. hybrids in the second cluster and mostly non-Saccharum spp. in the third cluster. A core collection of 300 accessions was identified which captured the maximum genetic diversity of the entire WCSRG which can be further exploited for sugarcane and energy cane breeding. Sugarcane and energy cane breeders can effectively utilize this core collection for cultivar improvement. Further, the core collection can provide resources for forming an association panel to evaluate the traits of agronomic and commercial importance

Mapping of important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut (Arachis hypogaea L.).

A mapping population of recombinant inbred lines (RILs) derived from TMV 2 and its mutant, TMV 2-NLM was employed for mapping important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut. Single nucleotide polymorphism and copy number variation using RAD-Sequencing data indicated very limited polymorphism between TMV 2 and TMV 2-NLM. But phenotypically they differed significantly for many taxonomic and productivity traits. Also, the RIL population showed significant variation for a few additional agronomic traits. A genetic linkage map of 1,205.66 cM was constructed using 91 genic and non-genic Arachis hypogaea transposable element (AhTE) markers. Using single marker analysis and QTL analysis, the markers with high phenotypic variance explained (PVE) were identified for branching pattern (32.3%), number of primary and secondary branches (19.9% and 28.4%, respectively), protein content (26.4%), days to 50% flowering (22.0%), content of oleic acid (15.1%), test weight (13.6%) and pod width (12.0%). Three genic markers (AhTE0357, AhTE0391, AhTE0025) with Arachis hypogaea miniature inverted-repeat transposable element (AhMITE1) activity in the genes Araip.TG1BL (B02 chromosome), Aradu.7N61X (A09 chromosome) and Aradu.7065G (A07 chromosome), respectively showed strong linkage with these taxonomic, productivity and quality traits. Since TMV 2 and TMV 2-NLM differed subtly at DNA level, the background noise in detecting the marker-trait associations was minimum; therefore, the markers identified in this study for the taxonomic and productivity traits may be significant and useful in peanut molecular breeding