AhNPR3 regulates the expression of WRKY and PR genes, and mediates the immune response of the peanut (Arachis hypogaea L.).

Systemic acquired resistance is an essential immune response that triggers a broad-spectrum disease resistance throughout the plant. In the present study, we identified a peanut lesion mimic mutant m14 derived from an ethyl methane sulfonate-mutagenized mutant pool of peanut cultivar "Yuanza9102." Brown lesions were observed in the leaves of an m14 mutant from seedling stage to maturity. Using MutMap together with bulked segregation RNA analysis approaches, a G-to-A point mutation was identified in the exon region of candidate gene Arahy.R60CUW, which is the homolog of AtNPR3 (Nonexpresser of PR genes) in Arabidopsis. This point mutation caused a transition from Gly to Arg within the C-terminal transactivation domain of AhNPR3A. The mutation of AhNPR3A showed no effect in the induction of PR genes when treated with salicylic acid. Instead, the mutation resulted in upregulation of WRKY genes and several PR genes, including pathogenesis-related thaumatin- and chitinase-encoding genes, which is consistent with the resistant phenotype of m14 to leaf spot disease. Further study on the AhNPR3A gene will provide valuable insights into understanding the molecular mechanism of systemic acquired resistance in peanut. Moreover, our results indicated that a combination of MutMap and bulked segregation RNA analysis is an effective method for identifying genes from peanut mutants.

Marker-assisted backcrossing to improve seed oleic acid content in four elite and popular peanut (Arachis hypogaea L.) cultivars with high oil content.

High oleic acid composition is an important determinant of seed quality in peanut (Arachis hypogaea) in regard to its nutritional benefits for human health and prolonged shelf-life for peanut products. To improve the oleic acid content of popular peanut cultivars in China, four peanut cultivars of different market types were hybridized with high-oleic-acid donors and backcrossed for four generations as recurrent parents using fad2 marker-assisted backcross selection. Seed quality traits in advanced generations derived by selfing were assessed using near-infrared reflectance spectroscopy for detection of oleic acid and Kompetitive allele-specific PCR (KASP) screening of fad2 mutant markers. Twenty-four high-oleic-acid lines of BC4F4 and BC4F5 populations, with morphological features and agronomic traits similar to those of the recurrent parents, were obtained within 5 years. The genetic backgrounds of BC4F5 lines were estimated using the KASP assay, which revealed the genetic background recovery rate was 79.49%-92.31%. The superior lines raised are undergoing a multi-location test for cultivar registration and release. To our knowledge, this is the first application of single nucleotide polymorphism markers based on the high-throughput and cost-effective KASP assay for detection of fad2 mutations and genetic background evaluation in a peanut breeding program.

High-resolution chromosome painting with repetitive and single-copy oligonucleotides in Arachis species identifies structural rearrangements and genome differentiation.

BACKGROUND: Arachis contains 80 species that carry many beneficial genes that can be utilized in the genetic improvement of peanut (Arachis hypogaea L. 2n = 4x = 40, genome AABB). Chromosome engineering is a powerful technique by which these genes can be transferred and utilized in cultivated peanut. However, their small chromosomes and insufficient cytological markers have made chromosome identification and studies relating to genome evolution quite difficult. The development of efficient cytological markers or probes is very necessary for both chromosome engineering and genome discrimination in cultivated peanut. RESULTS: A simple and efficient oligonucleotide multiplex probe to distinguish genomes, chromosomes, and chromosomal aberrations of peanut was developed based on eight single-stranded oligonucleotides (SSONs) derived from repetitive sequences. High-resolution karyotypes of 16 Arachis species, two interspecific F1 hybrids, and one radiation-induced M1 plant were then developed by fluorescence in situ hybridization (FISH) using oligonucleotide multiplex, 45S and 5S rDNAs, and genomic in situ hybridization (GISH) using total genomic DNA of A. duranensis (2n = 2x = 20, AA) and A. ipaënsis (2n = 2x = 20, BB) as probes. Genomes, chromosomes, and aberrations were clearly identifiable in the established karyotypes. All eight cultivars had similar karyotypes, whereas the eight wild species exhibited various chromosomal variations. In addition, a chromosome-specific SSON library was developed based on the single-copy sequence of chromosome 6A of A. duranensis. In combination with repetitive SSONs and rDNA FISH, the single-copy SSON library was applied to identify the corresponding A3 chromosome in the A. duranensis karyotype. CONCLUSIONS: The development of repetitive and single-copy SSON probes for FISH and GISH provides useful tools for the differentiation of chromosomes and identification of structural chromosomal rearrangement. It facilitates the development of high-resolution karyotypes and detection of chromosomal variations in Arachis species. To our knowledge, the methodology presented in this study demonstrates for the first time the correlation between a sequenced chromosome region and a cytologically identified chromosome in peanut.

Development of an oligonucleotide dye solution facilitates high throughput and cost-efficient chromosome identification in peanut.

BACKGROUND: Development of oligonucleotide probes facilitates chromosome identification via fluorescence in situ hybridization (FISH) in many organisms. RESULTS: We report a high throughput and economical method of chromosome identification based on the development of a dye solution containing 2 × saline-sodium citrate (SSC) and oligonucleotide probes. Based on the concentration, staining time, and sequence effects of oligonucleotides, an efficient probe dye of peanut was developed for chromosome identification. To validate the effects of this solution, 200 slides derived from 21 accessions of the cultivated peanut and 30 wild Arachis species were painted to identify Arachis genomes and establish karyotypes. The results showed that one jar of dye could be used to paint 10 chromosome preparations and recycled at least 10 times to efficiently dye more than 100 slides. The A, B, K, F, E, and H genomes showed unique staining karyotype patterns and signal colors. CONCLUSIONS: Based on the karyotype patterns of Arachis genomes, we revealed the relationships among the A, B, K, F, E, and H genomes in genus Arachis, and demonstrated the potential for adoption of this oligonucleotide dye solution in practice.

Genetic Diversity, Population Structure, and Botanical Variety of 320 Global Peanut Accessions Revealed Through Tunable Genotyping-by-Sequencing.

Cultivated peanut (Arachis hypogaea L.) were classified into six botanical varieties according to the morphological characteristics. However, their genetic evolutionary relationships at the genome-wide level were still unclear. A total of 320 peanut accessions, including four of the six botanical varieties, and 37,128 high-quality single nucleotide polymorphisms (SNPs) detected by tunable genotyping-by-sequencing (tGBS) were used to reveal the evolutionary relationships among different botanical varieties and verify the phenotypic classification. A phylogenetic tree indicated that the tested accessions were grouped into three clusters. Almost all of the peanut accessions in cluster C1 belong to var. fastigiata, and clusters C2 and C3 mainly consisted of accessions from var. vulgaris and subsp. hypogaea, respectively. The results of a principal component analysis were consistent with relationships revealed in the phylogenetic tree. Population structure analysis showed that var. fastigiata and var. vulgaris were not separated when K = 2 (subgroup number), whereas they were clearly divided when K = 3. However, var. hypogaea and var. hirsuta could not be distinguished from each other all the way. The nucleotide diversity (π) value implied that var. vulgaris exhibited the highest genetic diversity (0.048), followed by var. fastigiata (0.035) and subsp. hypogaea (0.012), which is consistent with the result of phylogenetic tree. Moreover, the fixation index (FST) value confirmed that var. fastigiata and var. vulgaris were closely related to each other (FST = 0.284), while both of them were clearly distinct from var. hypogaea (FST > 0.4). The present study confirmed the traditional botanical classifications of cultivated peanut at the genome-wide level. Furthermore, the reliable SNPs identified in this study may be a valuable resource for peanut breeders.

Differential gene expression in leaf tissues between mutant and wild-type genotypes response to late leaf spot in peanut (Arachis hypogaea L.).

Late leaf spot (LLS) is a major foliar disease in peanut (A. hypogaea L.) worldwide, causing significant losses of potential yield in the absence of fungicide applications. Mutants are important materials to study the function of disease-related genes. In this study, the mutant line M14 was derived from cultivar Yuanza 9102 treated with EMS. Yuanza 9102 was selected from an interspecific cross of cultivar Baisha 1016 with A. diogoi, and is resistant to several fungal diseases. By contrast, the M14 was highly susceptible to late leaf spot. RNA-Seq analysis in the leaf tissues of the M14 and its wild type Yuanza 9102 under pathogen challenge showed 2219 differentially expressed genes including1317 up-regulated genes and 902 down-regulated genes. Of these genes, 1541, 1988, 1344, 643 and 533 unigenes were obtained and annotated by public protein databases of SwissPort, TrEMBL, gene ontology (GO), KEGG and clusters of orthologous groups (COG), respectively. Differentially expressed genes (DEGs) showed that expression of inducible pathogenesis-related (PR) proteins was significantly up-regulated; in the meantime DEGs related to photosynthesis were down-regulated in the susceptible M14 in comparison to the resistant WT. Moreover, the up-regulated WRKY transcription factors and down-regulated plant hormones related to plant growth were detected in the M14. The results suggest that down-regulated chloroplast genes, up-regulated WRKY transcription factors, and depressed plant hormones related to plant growth in the M14 might coordinately render the susceptibility though there was a significant high level of PRs. Those negative effectors might be triggered in the susceptible plant by fungal infection and resulted in reduction of photosynthesis and phytohormones and led to symptom formation.