QTL‐seq approach identified genomic regions and diagnostic markers for rust and late leaf spot resistance in groundnut (Arachis hypogaea L.)

Rust and late leaf spot (LLS) are the two major foliar fungal diseases in groundnut, and their co‐occurrence leads to significant yield loss in addition to the deterioration of fodder quality. To identify candidate genomic regions controlling resistance to rust and LLS, whole‐genome resequencing (WGRS)‐based approach referred as ‘QTL‐seq’ was deployed. A total of 231.67 Gb raw and 192.10 Gb of clean sequence data were generated through WGRS of resistant parent and the resistant and susceptible bulks for rust and LLS. Sequence analysis of bulks for rust and LLS with reference‐guided resistant parent assembly identified 3136 single‐nucleotide polymorphisms (SNPs) for rust and 66 SNPs for LLS with the read depth of ≥7 in the identified genomic region on pseudomolecule A03. Detailed analysis identified 30 nonsynonymous SNPs affecting 25 candidate genes for rust resistance, while 14 intronic and three synonymous SNPs affecting nine candidate genes for LLS resistance. Subsequently, allele‐specific diagnostic markers were identified for three SNPs for rust resistance and one SNP for LLS resistance. Genotyping of one RIL population (TAG 24 × GPBD 4) with these four diagnostic markers revealed higher phenotypic variation for these two diseases. These results suggest usefulness of QTL‐seq approach in precise and rapid identification of candidate genomic regions and development of diagnostic markers for breeding applications.     

Next‐generation sequencing identified genomic region and diagnostic markers for resistance to bacterial wilt on chromosome B02 in peanut (Arachis hypogaea L.)

Bacterial wilt, caused by Ralstonia solanacearum, is a devastating disease affecting over 350 plant species. A few peanut cultivars were found to possess stable and durable bacterial wilt resistance (BWR). Genomics‐assisted breeding can accelerate the process of developing resistant cultivars by using diagnostic markers. Here, we deployed sequencing‐based trait mapping approach, QTL‐seq, to discover genomic regions, candidate genes and diagnostic markers for BWR in a recombination inbred line population (195 progenies) of peanut. The QTL‐seq analysis identified one candidate genomic region on chromosome B02 significantly associated with BWR. Mapping of newly developed single nucleotide polymorphism (SNP) markers narrowed down the region to 2.07 Mb and confirmed its major effects and stable expressions across three environments. This candidate genomic region had 49 nonsynonymous SNPs affecting 19 putative candidate genes including seven putative resistance genes (R‐genes). Two diagnostic markers were successfully validated in diverse breeding lines and cultivars and could be deployed in genomics‐assisted breeding of varieties with enhanced BWR.     

Whole‐genome resequencing‐based QTL‐seq identified candidate genes and molecular markers for fresh seed dormancy in groundnut

The subspecies fastigiata of cultivated groundnut lost fresh seed dormancy (FSD) during domestication and human‐made selection. Groundnut varieties lacking FSD experience precocious seed germination during harvest imposing severe losses. Development of easy‐to‐use genetic markers enables early‐generation selection in different molecular breeding approaches. In this context, one recombinant inbred lines (RIL) population (ICGV 00350 × ICGV 97045) segregating for FSD was used for deploying QTL‐seq approach for identification of key genomic regions and candidate genes. Whole‐genome sequencing (WGS) data (87.93 Gbp) were generated and analysed for the dormant parent (ICGV 97045) and two DNA pools (dormant and nondormant). After analysis of resequenced data from the pooled samples with dormant parent (reference genome), we calculated delta‐SNP index and identified a total of 10,759 genomewide high‐confidence SNPs. Two candidate genomic regions spanning 2.4 Mb and 0.74 Mb on the B05 and A09 pseudomolecules, respectively, were identified controlling FSD. Two candidate genes—RING‐H2 finger protein and zeaxanthin epoxidase—were identified in these two regions, which significantly express during seed development and control abscisic acid (ABA) accumulation. QTL‐seq study presented here laid out development of a marker, GMFSD1, which was validated on a diverse panel and could be used in molecular breeding to improve dormancy in groundnut.     

Nested‐association mapping (NAM)‐based genetic dissection uncovers candidate genes for seed and pod weights in peanut (Arachis hypogaea)

Multiparental genetic mapping populations such as nested‐association mapping (NAM) have great potential for investigating quantitative traits and associated genomic regions leading to rapid discovery of candidate genes and markers. To demonstrate the utility and power of this approach, two NAM populations, NAM_Tifrunner and NAM_Florida‐07, were used for dissecting genetic control of 100‐pod weight (PW) and 100‐seed weight (SW) in peanut. Two high‐density SNP‐based genetic maps were constructed with 3341 loci and 2668 loci for NAM_Tifrunner and NAM_Florida‐07, respectively. The quantitative trait locus (QTL) analysis identified 12 and 8 major effect QTLs for PW and SW, respectively, in NAM_Tifrunner, and 13 and 11 major effect QTLs for PW and SW, respectively, in NAM_Florida‐07. Most of the QTLs associated with PW and SW were mapped on the chromosomes A05, A06, B05 and B06. A genomewide association study (GWAS) analysis identified 19 and 28 highly significant SNP–trait associations (STAs) in NAM_Tifrunner and 11 and 17 STAs in NAM_Florida‐07 for PW and SW, respectively. These significant STAs were co‐localized, suggesting that PW and SW are co‐regulated by several candidate genes identified on chromosomes A05, A06, B05, and B06. This study demonstrates the utility of NAM population for genetic dissection of complex traits and performing high‐resolution trait mapping in peanut.     

Utility of EST-derived SSR in cultivated peanut (Arachis hypogaea L.) and Arachis wild species

Background  

Lack of sufficient molecular markers hinders current genetic research in peanuts (Arachis hypogaea L.). It is necessary to develop more molecular markers for potential use in peanut genetic research. With the development of peanut EST projects, a vast amount of available EST sequence data has been generated. These data offered an opportunity to identify SSR in ESTs by data mining.     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

Fine mapping of qAHPS07 and functional studies of AhRUVBL2 controlling pod size in peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) is an important oil and cash crop. Pod size is one of the major traits determining yield and commodity characteristic of peanut. Fine mapping of quantitative trait locus (QTL) and identification of candidate genes associated with pod size are essential for genetic improvement and molecular breeding of peanut varieties. In this study, a major QTL related to pod size, qAHPS07, was fine mapped to a 36.46 kb interval on chromosome A07 using F₂, recombinant inbred line (RIL) and secondary F₂ populations. qAHPS07 explained 38.6%, 23.35%, 37.48%, 25.94% of the phenotypic variation for single pod weight (SPW), pod length (PL), pod width (PW) and pod shell thickness (PST), respectively. Whole genome resequencing and gene expression analysis revealed that a RuvB-like 2 protein coding gene AhRUVBL2 was the most likely candidate for qAHPS07. Overexpression of AhRUVBL2 in Arabidopsis led to larger seeds and plants than the wild type. AhRUVBL2-silenced peanut seedlings represented small leaves and shorter main stems. Three haplotypes were identified according to three SNPs in the promoter of AhRUVBL2 among 119 peanut accessions. Among them, SPW, PW and PST of accessions carrying Hap_ATT represent 17.6%, 11.2% and 26.3% higher than those carrying Hap_GAC,respectively. In addition, a functional marker of AhRUVBL2 was developed. Taken together, our study identified a key functional gene of peanut pod size, which provides new insights into peanut pod size regulation mechanism and offers practicable markers for the genetic improvement of pod size-related traits in peanut breeding.     

Quantification of the geocarposphere and rhizosphere effect of peanut (Arachis hypogaea L.)

Roots and pods of field-grown peanut (groundnut) (Arachis hypogaea L.) were sampled at the R3, R5, and R7 developmental stages and examined in comparison to root- and pod-free soil for microbial population densities to assess the geocarposphere and rhizosphere effects. G/ S (no. geocarposphere microorganisms/no. soil microorganisms) and R/S (no. rhizosphere microorganisms/no. soil microorganisms) ratios were calculated for total fungi,Asperigillus flavus, spore-forming bacilli, coryneform bacteria, fluorescent pseudomonads, and total bacteria isolated on low- and high-nutrient media. A clear geocarposphere effect was evidenced by increased population densities of bacteria and fungi associated with developing pods compared to soil. G/S and R/S ratios were generally greater than 1.0 for all groups of microorganisms except bacilli. G/S ratios were greater for total bacteria than for total fungi at two of the three sample times, suggesting that bacteria were stimulated more than fungi in the zone around developing pods. In contrast, R/S ratios, were higher for total fungi than for total bacteria at two of three sample times. The preferential association of fungi and bacteria with early developmental stages of the pod indicates that some microorganisms are particularly well adapted for colonization of the peanut geocarposphere. These microorganisms are logical candidates for evaluation as biological control candiates forA. flavus.     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.     

Development of SSR markers and identification of major quantitative trait loci controlling shelling percentage in cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Key message

A total of 204,439 SSR markers were developed in diploid genomes, and 25 QTLs for shelling percentage were identified in a RIL population across 4 years including five consistent QTLs.

Abstract

Cultivated peanut (Arachis hypogaea L.) is an important grain legume providing edible oil and protein for human nutrition. Genome sequences of its diploid ancestors, Arachis duranensis and A. ipaensis, were reported, but their SSRs have not been well exploited and utilized hitherto. Shelling percentage is an important economic trait and its improvement has been one of the major objectives in peanut breeding programs. In this study, the genome sequences of A. duranensis and A. ipaensis were used to develop SSR markers, and a mapping population (Yuanza 9102 × Xuzhou 68-4) with 195 recombinant inbred lines was used to map QTLs controlling shelling percentage. The numbers of newly developed SSR markers were 84,383 and 120,056 in the A. duranensis and A. ipaensis genomes, respectively. Genotyping of the mapping population was conducted with both newly developed and previously reported markers. QTL analysis using the phenotyping data generated in Wuhan across four consecutive years and genotyping data of 830 mapped loci identified 25 QTLs with 4.46–17.01% of phenotypic variance explained in the four environments. Meta-analysis revealed five consistent QTLs that could be detected in at least two environments. Notably, the consistent QTL cqSPA09 was detected in all four environments and explained 10.47–17.01% of the phenotypic variance. The segregation in the progeny of a residual heterozygous line confirmed that the cpSPA09 locus had additive effect in increasing shelling percentage. These consistent and major QTL regions provide opportunity not only for further gene discovery, but also for the development of functional markers for breeding.

     

Diversity characterization and association analysis of agronomic traits in a Chinese peanut (Arachis hypogaea L.) mini-core collection

Association mapping is a powerful approach for exploring the molecular basis of phenotypic variations in plants.A peanut(Arachis hypogaea L.)mini-core collection in China comprising 298 accessions was genotyped using 109 simple sequence repeat(SSR)markers,which identified 554 SSR alleles and phenotyped for 15 agronomic traits in three different environments,exhibiting abundant genetic and phenotypic diversity within the panel.A model-based structure analysis assigned all accessions to three groups.Most of the accessions had the relative kinship of less than 0.05,indicating that there were no or weak relationships between accessions of the mini-core collection.For 15 agronomic traits in the peanut panel,generally the Q t K model exhibited the best performance to eliminate the false associated positives compared to the Q model and the general linear model-simple model.In total,89SSR alleles were identified to be associated with 15 agronomic traits of three environments by the Q t K model-based association analysis.Of these,eight alleles were repeatedly detected in two or three environments,and 15 alleles were commonly detected to be associated with multiple agronomic traits.Simple sequence repeat allelic effects confirmed significant differences between different genotypes of these repeatedly detected markers.Our results demonstrate the great potential of integrating the association analysis and marker-assisted breeding by utilizing the peanut mini-core collection.     

Diversity characterization and association analysis of agronomic traits in a Chinese peanut （Arachis hypogaea L.） mini-core collection

Association mapping is a powerful approach for exploring the molecular basis of phenotypic variations in plants. A peanut （Arachis hypogaea L.） mini-core collection in China comprising 298 accessions was genotyped using lo9 simple sequence repeat （SSR） markers, which identified 554 SSR alleles and phenotyped for 15 agronomic traits in three different environments, exhibiting abundant genetic and phenotypic diversity within the panel. A model-based structure analysis assigned all accessions to three groups. Most of the accessions had the relative kinship of less than o.05, indicating that there were no or weak relationships between accessions of the mini- core collection. For 15 agronomic traits in the peanut panel, generally the Q ＋ K model exhibited the best performance to eliminate the false associated positives compared to the Q model and the general linear model-simple model. In total, 89 SSR alleles were identified to be associated with 15 agronomic traits of three environments by the Q＋K model-based association analysis. Of these, eight alleles were repeatedly detected in two or three environments, and 15 alleles were commonly detected to be associated with multiple agronomic traits. Simple sequence repeat allelic effects confirmed significant differences between different genotypes of these repeatedly detected markers. Our results demonstrate the great potential of integrating the association analysis and marker-assisted breeding by utilizing the peanut mini-core collection.     

In vitro regeneration of peanut (Arachis hypogaea L.) through organogenesis: Effect of culture temperature and silver nitrate

Summary The effect of culture temperature on the morphogenetic response of Arachis hypogaea was studied. Cotyledons were cultivated on MS medium supplemented with 110 μM 6-benzyladenine. Leaf explants were cultivated in the presence of the same growth regulator at 22 μM. Cultures were incubated at temperatures of 25, 28, and 35±5° C. Both direct organogenesis from cotyledons and development of organogenic calluses from leaves showed optimal rates at 35±5° C. The highest frequency of elongation of buds into shoots from leaf-derived calluses occurred in the presence of 5 μM AgNO₃. At the best culture temperature, an average of 95% of shoots formed roots on growth-regulator-free MS medium. Plants were successfully transferred to soil, showing normal phenotypes.     

Assessment of transpiration efficiency in peanut (Arachis hypogaea L.) under drought using a lysimetric system

Transpiration efficiency (TE) is an important trait for drought tolerance in peanut ( Arachis hypogaea L.). The variation in TE was assessed gravimetrically using a long time interval in nine peanut genotypes (Chico, ICGS 44, ICGV 00350, ICGV 86015, ICGV 86031, ICGV 91114, JL 24, TAG 24 and TMV 2) grown in lysimeters under well-watered or drought conditions. Transpiration was measured by regularly weighing the lysimeters, in which the soil surface was mulched with a 2-cm layer of polythene beads. TE in the nine genotypes used varied from 1.4 to 2.9 g kg⁻¹ under well-watered and 1.7 to 2.9 g kg⁻¹ under drought conditions, showing consistent variation in TE among genotypes. A higher TE was found in ICGV 86031 in both well-watered and drought conditions and lower TE was found in TAG-24 under both water regimes. Although total water extraction differed little across genotypes, the pattern of water extraction from the soil profile varied among genotypes. High water extraction within 24 days following stress imposition was negatively related to pod yield ( r ²   =   0.36), and negatively related to water extraction during a subsequent period of 32 days ( r ²   =   0.73). By contrast, the latter, i.e. water extraction during a period corresponding to grain filling (24 to 56 days after flowering) was positively related to pod yield ( r ²   =   0.36). TE was positively correlated with pod weight ( r ²   =   0.30) under drought condition. Our data show that under an intermittent drought regime, TE and water extraction from the soil profile during a period corresponding to pod filling were the most important components.