GWAS and bulked segregant analysis reveal the Loci controlling growth habit-related traits in cultivated Peanut (Arachis hypogaea L.).

BACKGROUND: Peanut (Arachis hypogaea L.) is a grain legume crop that originated from South America and is now grown around the world. Peanut growth habit affects the variety's adaptability, planting patterns, mechanized harvesting, disease resistance, and yield. The objective of this study was to map the quantitative trait locus (QTL) associated with peanut growth habit-related traits by combining the genome-wide association analysis (GWAS) and bulked segregant analysis sequencing (BSA-seq) methods. RESULTS: GWAS was performed with 17,223 single nucleotide polymorphisms (SNPs) in 103 accessions of the U.S. mini core collection genotyped using an Affymetrix version 2.0 SNP array. With a total of 12,342 high-quality polymorphic SNPs, the 90 suggestive and significant SNPs associated with lateral branch angle (LBA), main stem height (MSH), lateral branch height (LBL), extent radius (ER), and the index of plant type (IOPT) were identified. These SNPs were distributed among 15 chromosomes. A total of 597 associated candidate genes may have important roles in biological processes, hormone signaling, growth, and development. BSA-seq coupled with specific length amplified fragment sequencing (SLAF-seq) method was used to find the association with LBA, an important trait of the peanut growth habit. A 4.08 Mb genomic region on B05 was associated with LBA. Based on the linkage disequilibrium (LD) decay distance, we narrowed down and confirmed the region within the 160 kb region (144,193,467-144,513,467) on B05. Four candidate genes in this region were involved in plant growth. The expression levels of Araip.E64SW detected by qRT-PCR showed significant difference between 'Jihua 5' and 'M130'. CONCLUSIONS: In this study, the SNP (AX-147,251,085 and AX-144,353,467) associated with LBA by GWAS was overlapped with the results in BSA-seq through combined analysis of GWAS and BSA-seq. Based on LD decay distance, the genome range related to LBA on B05 was shortened to 144,193,467-144,513,467. Three candidate genes related to F-box family proteins (Araip.E64SW, Araip.YG1LK, and Araip.JJ6RA) and one candidate gene related to PPP family proteins (Araip.YU281) may be involved in plant growth and development in this genome region. The expression analysis revealed that Araip.E64SW was involved in peanut growth habits. These candidate genes will provide molecular targets in marker-assisted selection for peanut growth habits.

Transcriptomic and metabolomic joint analysis reveals distinct flavonoid biosynthesis regulation for variegated testa color development in peanut (Arachis hypogaea L.).

Peanut is one of the important oil and economic crops, among which the variegated testa peanut is a unique member. The molecular mechanisms underlying the pigment synthesis in variegated testa are still unclear. Differentially expressed genes (DEGs) in the flavonoid metabolism pathway in pigmented areas indicated that there were 27 DEGs highly related to the synthesis of variegated testa color among 1,050 DEGs. Of these 27, 13 were up-regulated and 14 were down-regulated, including 3 PALs, 1 C4H, 2 CHSs, 1 F3H, 1 F3'H, 2 DFRs, 2 LARs, 2 IAAs, 4 bHLHs, and 9 MYBs. GO (Gene Ontology) analysis indicated that DEGs were similarly enriched in three branches. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis suggested flavonoid biosynthesis is the most direct metabolic pathway for the synthesis of testa variegation. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) results showed that cyanidin and delphinidin were the primary metabolites that caused the color differences between the pigmented and the non-pigmented areas. Through the verification of 20 DEGs via qPCR, the results were consistent with transcriptome sequencing in four comparison groups. The results in this study lay the foundation for revealing the molecular regulation mechanisms of flavonoid synthesis in variegated testa peanut.

Transcriptome and Co-expression Network Analyses Reveal Differential Gene Expression and Pathways in Response to Severe Drought Stress in Peanut (Arachis hypogaea L.).

Drought is one of the major abiotic stress factors limiting peanut production. It causes the loss of pod yield during the pod formation stage. Here, one previously identified drought-tolerant cultivar, "L422" of peanut, was stressed by drought (35 ± 5%) at pod formation stage for 5, 7, and 9 days. To analyze the drought effects on peanut, we conducted physiological and transcriptome analysis in leaves under well-watered (CK1, CK2, and CK3) and drought-stress conditions (T1, T2, and T3). By transcriptome analysis, 3,586, 6,730, and 8,054 differentially expressed genes (DEGs) were identified in "L422" at 5 days (CK1 vs T1), 7 days (CK2 vs T2), and 9 days (CK3 vs T3) of drought stress, respectively, and 2,846 genes were common DEGs among the three-time points. Furthermore, the result of weighted gene co-expression network analysis (WGCNA) revealed one significant module that was closely correlated between drought stress and physiological data. A total of 1,313 significantly up-/down-regulated genes, including 61 transcription factors, were identified in the module at three-time points throughout the drought stress stage. Additionally, six vital metabolic pathways, namely, "MAPK signaling pathway-plant," "flavonoid biosynthesis," "starch and sucrose metabolism," "phenylpropanoid biosynthesis," "glutathione metabolism," and "plant hormone signal transduction" were enriched in "L422" under severe drought stress. Nine genes responding to drought tolerance were selected for quantitative real-time PCR (qRT-PCR) verification and the results agreed with transcriptional profile data, which reveals the reliability and accuracy of transcriptome data. Taken together, these findings could lead to a better understanding of drought tolerance and facilitate the breeding of drought-resistant peanut cultivars.

Analysis of Microbial Diversity and Community Structure of Peanut Pod and Its Surrounding Soil in Peanut Rot Epidemic Area.

Fungal communities are associated with healthy peanut crops and good crop production, through the regulation of pod rot disease. Rotted peanut pods and their surrounding soil samples were collected from locations in northern China. Fungal species were identified by next-generation sequencing, using the conserved sequences of their internal transcribed spacer regions. Results showed that rotted pod samples were rich in the phyla Ascomycota and Basidiomycota, and soil samples also contained these, plus Chytridiomycota and Zygomycota. There were regional variations in the species of fungi related to peanut pod rot and its surrounding soil, between locations. Fungal species of Cryptococcus and Fusarium were less abundant in soil samples than in rotted pod samples, and were the main pathogenic fungi identified in our study. Soil total carbon, nitrogen, and potassium had a strong influence on the fungal community, and total phosphorous and calcium ions, together with soil pH, had a modest influence. Only Mycosphaerella and Gibberella were not significantly affected by these factors. These findings may be of some help to control pod rot disease and reduce the production loss of peanut crops.

Molecular cloning and expression characterization of flavonol synthase genes in peanut (Arachis hypogaea).

Flavonol is an important functional bioactive substance in peanut seeds, and plays important roles responding to abiotic stress. The flavonol content is closely related to the activity and regulation of gene expression patterns of flavonol synthase (FLS). In this study, eight FLS genes, AhFLSs were cloned and their expression characterization in different peanut organ and seedling under different abiotic stress were conducted. The results showed that the expressions levels of AhFLSs were differed in all assayed peanut organs and seedlings under abiotic stress treatments. Expression levels of AhFLS2, AhFLS3, AhFLS4, and AhFLS6 were higher than those of other AhFLSs. The flavonol contents of peanut organs and seedlings under different abiotic stress were also determined using high performance liquid chromatography (HPLC). Dried mature peanut seeds were the organ tissue with the highest flavonol content, and flavonol content increased with seed development. Under abiotic stress treatments, the types of flavonols induced differed among stress treatments. Correlation analysis results suggested that eight AhFLS genes may have different functions in peanut. Moreover, changes in the expression of the eight genes appear to has substrate preference. These results can lay the foundation for the study of improving nutritional value of peanut seed and resistance of peanut plant.

Identification and expression analysis of WRKY gene family under drought stress in peanut (Arachis hypogaea L.).

WRKY transcription factors play crucial roles in regulation mechanism leading to the adaption of plants to the complex environment. In this study, AhWRKY family was comprehensively analyzed using bioinformatic approaches in combination with transcriptome sequencing data of the drought-tolerant peanut variety 'L422'. A total of 158 AhWRKY genes were identified and named according to their distribution on the chromosomes. Based on the structural features and phylogenetic analysis of AhWRKY proteins, the AhWRKY family members were classified into three (3) groups, of which group II included five (5) subgroups. Results of structure and conserved motifs analysis for the AhWRKY genes confirmed the accuracy of the clustering analysis. In addition, 12 tandem and 136 segmental duplication genes were identified. The results indicated that segmental duplication events were the main driving force in the evolution of AhWRKY family. Collinearity analysis found that 32 gene pairs existed between Arachis hypogaea and two diploid wild ancestors (Arachis duranensis and Arachis ipaensis), which provided valuable clues for phylogenetic characteristics of AhWRKY family. Furthermore, 19 stress-related cis-acting elements were found in the promoter regions. During the study of gene expression level of AhWRKY family members in response to drought stress, 73 differentially expressed AhWRKY genes were obtained to have been influenced by drought stress. These results provide fundamental insights for further study of WRKY genes in peanut drought resistance.

Construction of High-Density Genetic Map and Mapping Quantitative Trait Loci for Growth Habit-Related Traits of Peanut (Arachis hypogaea L.).

Plant growth habit is an important and complex agronomic trait and is associated with yield, disease resistance, and mechanized harvesting in peanuts. There are at least two distinct growth habits (erect and prostrate) and several intermediate forms existing in the peanut germplasm. A recombinant inbred line population containing 188 individuals was developed from a cross of "Jihua 5" and "M130" for genetically dissecting the architecture of the growth habit. A new high-density genetic linkage map was constructed by using specific locus amplified fragment sequencing technology. The map contains 2,808 single-nucleotide polymorphism markers distributed on 20 linkage groups with a total length of 1,308.20 cM and an average inter-marker distance of 0.47 cM. The quantitative trait locus (QTL) analysis of the growth habit-related traits was conducted based on phenotyping data from seven environments. A total of 39 QTLs for growth habit-related traits was detected on 10 chromosomes explaining 4.55-27.74% of the phenotypic variance, in which 6 QTLs were for lateral branch angle, 8 QTLs were for extent radius, 7 QTLs were for the index of plant type, 11 QTLs were for main stem height, and 7 QTLs were for lateral branch length. Among these QTLs, 12 were co-localized on chromosome B05 spanning an approximately 0.17 Mb physical interval in comparison with the allotetraploid reference genome of "Tifrunner." Analysis of the co-localized genome region has shown that the putative genes are involved in light and hormones and will facilitate peanut growth habit molecular breeding and study of peanut domestication.

Selection of suitable reference genes for abiotic stress-responsive gene expression studies in peanut by real-time quantitative PCR

Background: Because of its strong specificity and high accuracy, real-time quantitative PCR (RT-qPCR) has been a widely used method to study the expression of genes responsive to stress. It is crucial to have a suitable set of reference genes to normalize target gene expression in peanut under different conditions using RT-qPCR. In this study, 11 candidate reference genes were selected and examined under abiotic stresses (drought, salt, heavy metal, and low temperature) and hormone (SA and ABA) conditions as well as across different organ types. Three statistical algorithms (geNorm, NormFinder and BestKeeper) were used to evaluate the expression stabilities of reference genes, and the comprehensive rankings of gene stability were generated. Results: The results indicated that ELF1B and YLS8 were the most stable reference genes under PEG-simulated drought treatment. For high-salt treatment using NaCl, YLS8 and GAPDH were the most stable genes. Under CdCl2 treatment, UBI1 and YLS8 were suitable as stable reference genes. UBI1, ADH3, and ACTIN11 were sufficient for gene expression normalization in low-temperature experiment. All the 11 candidate reference genes showed relatively high stability under hormone treatments. For organs subset, UBI1, GAPDH, and ELF1B showed the maximum stability. UBI1 and ADH3 were the top two genes that could be used reliably in all the stress conditions assessed. Furthermore, the necessity of the reference genes screened was further confirmed by the expression pattern of AnnAhs. Conclusions: The results perfect the selection of stable reference genes for future gene expression studies in peanut and provide a list of reference genes that may be used in the future.

[Ecological adaptability evaluation of peanut cultivars based on biomass and nutrient accumulation].

To identify the good peanut cultivars with the properties of high yield, high nutrient use efficiency and wide adaptability, 19 selected peanut cultivars were planted in the low champaign area and piedmont plain area of Hebei Province. By using principal component analysis, the adaptability of these 19 cultivars was evaluated for different ecological regions through comparing their 16 main traits including biomass and nutrient parameters. According to the critical value of principal component (>1.0), the 16 biomass and nutrient characteristics were integrated into 4 principal components which accounted for 85% of the original information. The results indicated that there were obvious differences in yield and nutrient use efficiency for the peanut cultivars in different ecological regions. The 19 peanut cultivars were classified into 2 groups according to their ecological adaptability, and the cultivars from the group with wide adaptability could further be divided into 3 categories according to their yield and nutrient use efficiency. Among these cultivars, Yuhua 9719, Jihua 0212-4, Weihua 10, Yuhua 15, Puhua 28 and Jihua 10 were selected as the better peanut cultivars with the properties of high yield, high nutrient use efficiency and wide adaptability.

Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.).

Annexin, Ca(2+) or phospholipid binding proteins, with many family members are distributed throughout all tissues during plant growth and development. Annexins participate in a number of physiological processes, such as exocytosis, cell elongation, nodule formation in legumes, maturation and stress response. Six different full-length cDNAs and two partial-length cDNAs of peanut, (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7, AnnAh4 and AnnAh8) encoding annexin proteins, were isolated and characterized using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins were 36.0kDa with acidic pIs of 5.97-8.81. ANNAh1, ANNAh2, ANNAh3, ANNAh5, ANNAh6 and ANNAh7 shared sequence similarity from 35.76 to 66.35% at amino acid level. Phylogenetic analysis revealed their evolutionary relationships with corresponding orthologous sequences in soybean and deduced proteins in various plant species. Real-time quantitative assays indicated that these genes were differentially expressed in various organs. Transcript level analysis for six annexin genes under stress conditions showed that these genes were regulated by drought, salinity, heavy metal stress, low temperature and hormone. Additionally, the prediction of cis-regulatory element suggested that different cis-responsive elements including stress- and hormone-responsive-related elements could respond to various stress conditions. These results indicated that members of AnnAhs family may play important roles in the adaptation of peanut to various environmental stresses.