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Key message: QTLs for growth habit are identified on Arahy.15 and Arahy.06 in peanut, and diagnostic markers are developed and validated for further use in marker-assisted breeding. Peanut is a unique legume crop because its pods develop and mature underground. The pegs derive from flowers following pollination, then reach the ground and develop into pods in the soil. Pod number per plant is influenced by peanut growth habit (GH) that has been categorized into four types, including erect, bunch, spreading and prostrate. Restricting pod development at the plant base, as would be the case for peanut plants with upright lateral branches, would decrease pod yield. On the other hand, GH characterized by spreading lateral branches on the ground would facilitate pod formation on the nodes, thereby increasing yield potential. We describe herein an investigation into the GH traits of 521 peanut recombinant inbred lines grown in three distinct environments. Quantitative trait loci (QTLs) for GH were identified on linkage group (LG) 15 between 203.1 and 204.2 cM and on LG 16 from 139.1 to 139.3 cM. Analysis of resequencing data in the identified QTL regions revealed that single nucleotide polymorphism (SNP) or insertion and/or deletion (INDEL) at Arahy15.156854742, Arahy15.156931574, Arahy15.156976352 and Arahy06.111973258 may affect the functions of their respective candidate genes, Arahy.QV02Z8, Arahy.509QUQ, Arahy.ATH5WE and Arahy.SC7TJM. These SNPs and INDELs in relation to peanut GH were further developed for KASP genotyping and tested on a panel of 77 peanut accessions with distinct GH features. This study validates four diagnostic markers that may be used to distinguish erect/bunch peanuts from spreading/prostrate peanuts, thereby facilitating marker-assisted selection for GH traits in peanut breeding. [ABSTRACT FROM AUTHOR]

For peanut, the lack of stable cytological markers is a barrier to tracking specific chromosomes, elucidating the genetic relationships between genomes and identifying chromosomal variations. Chromosome mapping using single-copy oligonucleotide (oligo) probe libraries has unique advantages for identifying homologous chromosomes and chromosomal rearrangements. In this study, we developed two whole-chromosome single-copy oligo probe libraries, LS-7A and LS-8A, based on the reference genome sequences of chromosomes 7A and 8A of Arachis duranensis. Fluorescence in situ hybridization (FISH) analysis confirmed that the libraries could specifically paint chromosomes 7 and 8. In addition, sequential FISH and electronic localization of LS-7A and LS-8A in A. duranensis (AA) and A. ipaensis (BB) showed that chromosomes 7A and 8A contained translocations and inversions relative to chromosomes 7B and 8B. Analysis of the chromosomes of wild Arachis species using LS-8A confirmed that this library could accurately and effectively identify A genome species. Finally, LS-7A and LS-8A were used to paint the chromosomes of interspecific hybrids and their progenies, which verified the authenticity of the interspecific hybrids and identified a disomic addition line. This study provides a model for developing specific oligo probes to identify the structural variations of other chromosomes in Arachis and demonstrates the practical utility of LS-7A and LS-8A. [ABSTRACT FROM AUTHOR]

Background: This study aims to decipher the genetic basis governing yield components and quality attributes of peanuts, a critical aspect for advancing molecular breeding techniques. Integrating genotype re-sequencing and phenotypic evaluations of seven yield components and two grain quality traits across four distinct environments allowed for the execution of a genome-wide association study (GWAS). Results: The nine phenotypic traits were all continuous and followed a normal distribution. The broad heritability ranged from 88.09 to 98.08%, and the genotype-environment interaction effects were all significant. There was a highly significant negative correlation between protein content (PC) and oil content (OC). The 10× genome re-sequencing of 199 peanut accessions yielded a total of 631,988 high-quality single nucleotide polymorphisms (SNPs), with 374 significant SNP loci identified in association with the nine traits of interest. Notably, 66 of these pertinent SNPs were detected in multiple environments, and 48 of them were linked to multiple traits of interest. Five loci situated on chromosome 16 (Chr16) exhibited pleiotropic effects on yield traits, accounting for 17.64–32.61% of the observed phenotypic variation. Two loci on Chr08 were found to be strongly associated with protein and oil contents, accounting for 12.86% and 14.06% of their respective phenotypic variations, respectively. Linkage disequilibrium (LD) block analysis of these seven loci unraveled five nonsynonymous variants, leading to the identification of one yield-related candidate gene and two quality-related candidate genes. The correlation between phenotypic variation and SNP loci in these candidate genes was validated by Kompetitive allele-specific PCR (KASP) marker analysis. Conclusions: Overall, molecular markers were developed for genetic loci associated with yield and quality traits through a GWAS investigation of 199 peanut accessions across four distinct environments. These molecular tools can aid in the development of desirable peanut germplasm with an equilibrium of yield and quality through marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress. [ABSTRACT FROM AUTHOR]

The capability of embryogenic callus induction is a prerequisite for in vitro plant regeneration. However, embryogenic callus induction is strongly genotype-dependent, thus hindering the development of in vitro plant genetic engineering technology. In this study, to examine the genetic variation in embryogenic callus induction rate (CIR) in peanut (Arachis hypogaea L.) at the seventh, eighth, and ninth subcultures (T7, T8, and T9, respectively), we performed genome-wide association studies (GWAS) for CIR in a population of 353 peanut accessions. The coefficient of variation of CIR among the genotypes was high in the T7, T8, and T9 subcultures (33.06%, 34.18%, and 35.54%, respectively), and the average CIR ranged from 1.58 to 1.66. A total of 53 significant single-nucleotide polymorphisms (SNPs) were detected (based on the threshold value −log10(p) = 4.5). Among these SNPs, SNPB03-83801701 showed high phenotypic variance and neared a gene that encodes a peroxisomal ABC transporter 1. SNPA05-94095749, representing a nonsynonymous mutation, was located in the Arahy.MIX90M locus (encoding an auxin response factor 19 protein) at T8, which was associated with callus formation. These results provide guidance for future elucidation of the regulatory mechanism of embryogenic callus induction in peanut. [ABSTRACT FROM AUTHOR]

Background: Pod shell thickness (PST) is an important agronomic trait of peanut because it affects the ability of shells to resist pest infestations and pathogen attacks, while also influencing the peanut shelling process. However, very few studies have explored the genetic basis of PST. Results: An F2 segregating population derived from a cross between the thick-shelled cultivar Yueyou 18 (YY18) and the thin-shelled cultivar Weihua 8 (WH8) was used to identify the quantitative trait loci (QTLs) for PST. On the basis of a bulked segregant analysis sequencing (BSA-seq), four QTLs were preliminarily mapped to chromosomes 3, 8, 13, and 18. Using the genome resequencing data of YY18 and WH8, 22 kompetitive allele-specific PCR (KASP) markers were designed for the genotyping of the F2 population. Two major QTLs (qPSTA08 and qPSTA18) were identified and finely mapped, with qPSTA08 detected on chromosome 8 (0.69-Mb physical genomic region) and qPSTA18 detected on chromosome 18 (0.15-Mb physical genomic region). Moreover, qPSTA08 and qPSTA18 explained 31.1–32.3% and 16.7–16.8% of the phenotypic variation, respectively. Fifteen genes were detected in the two candidate regions, including three genes with nonsynonymous mutations in the exon region. Two molecular markers (Tif2_A08_31713024 and Tif2_A18_7198124) that were developed for the two major QTL regions effectively distinguished between thick-shelled and thin-shelled materials. Subsequently, the two markers were validated in four F2:3 lines selected. Conclusions: The QTLs identified and molecular markers developed in this study may lay the foundation for breeding cultivars with a shell thickness suitable for mechanized peanut shelling. [ABSTRACT FROM AUTHOR]

Peanut (Arachis hypogaea L.) is an important oilseed crop worldwide. Improving its yield is crucial for sustainable peanut production to meet increasing food and industrial requirements. Deciphering the genetic control underlying peanut kernel weight and size, which are essential components of peanut yield, would facilitate high-yield breeding. A high-density single nucleotide polymorphism (SNP)-based linkage map was constructed using a recombinant inbred lines (RIL) population derived from a cross between the variety Yuanza9102 and a germplasm accession wt09-0023. Kernel weight and size quantitative trait loci (QTLs) were co-localized to a 0.16 Mb interval on Arahy07 using inclusive composite interval mapping (ICIM). Analysis of SNP, and Insertion or Deletion (INDEL) markers in the QTL interval revealed a gene encoding a pentatricopeptide repeat (PPR) superfamily protein as a candidate closely linked with kernel weight and size in cultivated peanut. Examination of the PPR gene family indicated a high degree of collinearity of PPR genes between A. hypogaea and its diploid progenitors, Arachis duranensis and Arachis ipaensis. The candidate PPR gene, Arahy.JX1V6X, displayed a constitutive expression pattern in developing seeds. These findings lay a foundation for further fine mapping of QTLs related to kernel weight and size, as well as validation of candidate genes in cultivated peanut. [ABSTRACT FROM AUTHOR]

Oligo probe staining is a low-cost and efficient chromosome identification technique. In this study, oligo genomic in situ hybridization (Oligo-GISH) technology was established in peanut. Peanut A and B subgenome-specific interspersed repeat (IR) oligo probe sets were developed based on clustering and electronic localization of tandem repeat sequences in the reference genome of Tifrunner. The Oligo-GISH kit was then used to perform staining of 15 Arachis species. The A-subgenome probe set stained the chromosomes of A- and E-genome Arachis species, the B-subgenome probe set stained those of B-, F-, K-, and E-genome species, and neither set stained those of H-genome species. These results indicate the relationships among the genomes of these Arachis species. The Oligo-GISH kit was also used for batch staining of the chromosomes of 389 seedlings from the irradiated M1 generation, allowing 67 translocation and deletion lines to be identified. Subsequent Oligo-FISH karyotyping, FISH using single-copy probe libraries, and trait investigation identified seven homozygous chromosomal variants from the M3 generation and suggested that there may be genes on chromosome 4B controlling seed number per pod. These findings demonstrate that the IR probe sets and method developed in this study can facilitate research on distant hybridization and genetic improvement in peanut. [ABSTRACT FROM AUTHOR]

Nitrogen, one of the essential elements, is a key determinant for improving peanut growth and yield. GATA zinc finger transcription factors have been found to be involved in regulation of nitrogen metabolism. However, a systematic characterization of the GATA gene family and patterns of their expression under different nitrogen levels remains elusive. In this study, a total of 45 GATA genes distributed among 17 chromosomes were identified in the peanut genome and classified into three subfamilies I, II and III with 26, 13 and 6 members, respectively, whose physicochemical characteristics, gene structures and conserved motifs were also analyzed. Furthermore, the optimal level of nitrogen fertilizer on the growth of peanut cultivar Yuhua 23 was determined by pod yield and value cost ratio from 2020 to 2022, and the results revealed that 150 kg hm−2 nitrogen was the best for cultivation of peanut Yuhua 23 because of its highest pod yield and relatively higher VCR of more than four. In addition, expression patterns of peanut GATA genes under different nitrogen levels were detected by real-time quantitative PCR and several GATA genes were significantly changed under a nitrogen level of 150 kg hm−2. Overall, the above results would be helpful for further understanding biological functions of the GATA gene family in cultivated peanut. [ABSTRACT FROM AUTHOR]

Peanut (Arachis hypogaea) is an important economic crop that is susceptible to various foliar diseases. Web blotch, which is caused by Phoma arachidicola, is one of the most serious peanut foliar diseases because it affects the leaves and results in substantial yield losses. In this study, the P. arachidicola infection process and the response of peanut plants to infection were revealed through a cytological study of resistant and susceptible peanut varieties. Conidial germination rates, appressorium formation and resistance responses, including papillae formation and the hypersensitive response (HR), were analysed at different infection stages. Notably, the basal infection response of susceptible varieties resulted in limited papillae formation and a single HR, which was followed by programmed cell death. The papillae formation frequency and HR frequency were significantly greater in the resistant varieties than in the susceptible varieties at 84 h postinoculation. The primary difference between the two resistant varieties was the superior HR of Zheng8903 compared with that of Yuhua15. The complex amphidiploid peanut genome may explain the complexity and diversity of the resistance responses. The interactions of functional orthologs should be clarified and perhaps modulated in future investigations. The results of this study revealed the differences in the resistance responses among peanut germplasm and may be relevant for the breeding of new peanut varieties highly resistant to P. arachidicola. [ABSTRACT FROM AUTHOR]

Key message: A major QTL, qBWA12, was fine mapped to a 216.68 kb physical region, and A12.4097252 was identified as a useful KASP marker for breeding peanut varieties resistant to bacterial wilt. Bacterial wilt, caused by Ralstonia solanacearum, is a major disease detrimental to peanut production in China. Breeding disease-resistant peanut varieties is the most economical and effective way to prevent the disease and yield loss. Fine mapping the QTLs for bacterial wilt resistance is critical for the marker-assisted breeding of disease-resistant varieties. A recombinant inbred population comprising 521 lines was used to construct a high-density genetic linkage map and to identify QTLs for bacterial wilt resistance following restriction-site-associated DNA sequencing. The genetic map, which included 5120 SNP markers, covered a length of 3179 cM with an average marker distance of 0.6 cM. Four QTLs for bacterial wilt resistance were mapped on four chromosomes. One major QTL, qBWA12, with LOD score of 32.8–66.0 and PVE of 31.2–44.8%, was stably detected in all four development stages investigated over the 3 trial years. Additionally, qBWA12 spanned a 2.7 cM region, corresponding to approximately 0.4 Mb and was fine mapped to a 216.7 kb region by applying KASP markers that were polymorphic between the two parents based on whole-genome resequencing data. In a large collection of breeding and germplasm lines, it was proved that KASP marker A12.4097252 can be applied for the marker-assisted breeding to develop peanut varieties resistant to bacterial wilt. Of the 19 candidate genes in the region covered by qBWA12, nine NBS-LRR genes should be further investigated regarding their potential contribution to the resistance of peanut against bacterial wilt. [ABSTRACT FROM AUTHOR]

Key message: Groundnut has entered now in post-genome era enriched with optimum genomic and genetic resources to facilitate faster trait dissection, gene discovery and accelerated genetic improvement for developing climate-smart varieties. Cultivated groundnut or peanut (Arachis hypogaea), an allopolyploid oilseed crop with a large and complex genome, is one of the most nutritious food. This crop is grown in more than 100 countries, and the low productivity has remained the biggest challenge in the semiarid tropics. Recently, the groundnut research community has witnessed fast progress and achieved several key milestones in genomics research including genome sequence assemblies of wild diploid progenitors, wild tetraploid and both the subspecies of cultivated tetraploids, resequencing of diverse germplasm lines, genome-wide transcriptome atlas and cost-effective high and low-density genotyping assays. These genomic resources have enabled high-resolution trait mapping by using germplasm diversity panels and multi-parent genetic populations leading to precise gene discovery and diagnostic marker development. Furthermore, development and deployment of diagnostic markers have facilitated screening early generation populations as well as marker-assisted backcrossing breeding leading to development and commercialization of some molecular breeding products in groundnut. Several new genomics applications/technologies such as genomic selection, speed breeding, mid-density genotyping assay and genome editing are in pipeline. The integration of these new technologies hold great promise for developing climate-smart, high yielding and more nutritious groundnut varieties in the post-genome era. [ABSTRACT FROM AUTHOR]

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 selling w'ere 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. [ABSTRACT FROM AUTHOR]

Abstract: Peanut genomics is very challenging due to its inherent problem of genetic architecture. Blockage of gene flow from diploid wild relatives to the tetraploid; cultivated peanut, recent polyploidization combined with self pollination, and the narrow genetic base of the primary genepool have resulted in low genetic diversity that has remained a major bottleneck for genetic improvement of peanut. Harnessing the rich source of wild relatives has been negligible due to differences in ploidy level as well as genetic drag and undesirable alleles for low yield. Lack of appropriate genomic resources has severely hampered molecular breeding activities, and this crop remains among the less-studied crops. The last five years, however, have witnessed accelerated development of genomic resources such as development of molecular markers, genetic and physical maps, generation of expressed sequenced tags (ESTs), development of mutant resources, and functional genomics platforms that facilitate the identification of QTLs and discovery of genes associated with tolerance/resistance to abiotic and biotic stresses and agronomic traits. Molecular breeding has been initiated for several traits for development of superior genotypes. The genome or at least gene space sequence is expected to be available in near future and this will further accelerate use of biotechnological approaches for peanut improvement. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]