Microsatellites as DNA markers in cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Background  

Genomic research of cultivated peanut has lagged behind other crop species because of the paucity of polymorphic DNA markers found in this crop. It is necessary to identify additional DNA markers for further genetic research in peanut.     

A SSR-based composite genetic linkage map for the cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genome

Background  

The construction of genetic linkage maps for cultivated peanut (Arachis hypogaea L.) has and continues to be an important research goal to facilitate quantitative trait locus (QTL) analysis and gene tagging for use in a marker-assisted selection in breeding. Even though a few maps have been developed, they were constructed using diploid or interspecific tetraploid populations. The most recently published intra-specific map was constructed from the cross of cultivated peanuts, in which only 135 simple sequence repeat (SSR) markers were sparsely populated in 22 linkage groups. The more detailed linkage map with sufficient markers is necessary to be feasible for QTL identification and marker-assisted selection. The objective of this study was to construct a genetic linkage map of cultivated peanut using simple sequence repeat (SSR) markers derived primarily from peanut genomic sequences, expressed sequence tags (ESTs), and by "data mining" sequences released in GenBank.     

QTL mapping for bacterial wilt resistance in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious, global, disease of peanut (Arachis hypogaea L.), but it is especially destructive in China. Identification of DNA markers linked to the resistance to this disease will help peanut breeders efficiently develop resistant cultivars through molecular breeding. A F₂ population, from a cross between disease-resistant and disease-susceptible cultivars, was used to detect quantitative trait loci (QTL) associated with the resistance to this disease in the cultivated peanut. Genome-wide SNPs were identified from restriction-site-associated DNA sequencing tags using next-generation DNA sequencing technology. SNPs linked to disease resistance were determined in two bulks of 30 resistant and 30 susceptible plants along with two parental plants using bulk segregant analysis. Polymorphic SSR and SNP markers were utilized for construction of a linkage map and for performing the QTL analysis, and a moderately dense linkage map was constructed in the F₂ population. Two QTL (qBW-1 and qBW-2) detected for resistance to BW disease were located in the linkage groups LG1 and LG10 and account for 21 and 12 % of the bacterial wilt phenotypic variance. To confirm these QTL, the F₈ RIL population with 223 plants was utilized for genotyping and phenotyping plants by year and location as compared to the F₂ population. The QTL qBW-1 was consistent in the location of LG1 in the F₈ population though the QTL qBW-2 could not be clarified due to fewer markers used and mapped in LG10. The QTL qBW-1, including four linked SNP markers and one SSR marker within 14.4-cM interval in the F₈, was closely related to a disease resistance gene homolog and was considered as a candidate gene for resistance to BW. QTL identified in this study would be useful to conduct marker-assisted selection and may permit cloning of resistance genes. Our study shows that bulk segregant analysis of genome-wide SNPs is a useful approach to expedite the identification of genetic markers linked to disease resistance traits in the allotetraploidy species peanut.     

Isolation and expression analysis of LEA genes in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.     

Optimization of factors for efficient recovery of transgenic peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

De-embryonated cotyledon explants of peanut were co-cultivated under different conditions with Agrobacterium tumefaciens harbouring pIG121hm plasmid carrying intron-containing β-glucuronidase as a reporter while hygromycin phosphotransferase and neomycin phosphotransferase as selectable marker genes. Co-cultivation duration and temperature, various antioxidants and their concentrations, bacterial strains and explant characteristics (incised and non-incised) were examined either alone or in combinations for optimization of transient expression of the reporter gene. Up to 81% transformation was recorded when non-incised explants were co-cultivated with strain EHA101 for 5 days at 21°C on shoot induction medium containing 100 mg/L l-cysteine. Addition of the optimized concentration of augmentin (200 mg/L) along with cefotaxime (200 mg/L) to the shoot induction medium not only effectively eliminated bacterial growth, but also facilitated high frequency of shoot induction. The 40 mg/L hygromycin concentration prevented complete shoot regeneration of non-transgenic explants thus considered for the regeneration of transgenics. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. Survival rate of the grafted shoots was nearly 100% in glass-house conditions. The optimized protocol took around 3 months to generate healthy plants. Polymerase chain reaction, Southern blot hybridization, histochemical tests, segregation and hygromycin-leaf assays of selected transgenic plants showed integration of the transgene into peanut genome. No chimeras were noticed during the study.     

Efficient production of <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed roots and composite plants in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Recalcitrance of most large-seeded legumes, such as peanut, to regeneration and genetic transformation has hampered studies on gene function and efforts for genetic improvement. Agrobacterium rhizogenes-mediated transformation provides a system for rapid and efficient transformation of plant tissues. In this study, embryonic axes along with cotyledons of peanut were injected with a suspension culture of A. rhizogenes using microliter syringes. The influence of several factors such as plant genotype, A. rhizogenes culture stage, co-culture period of A. rhizogenes, and acetosyringone concentration in the co-cultivation medium have been evaluated. It is found that A. rhizogenes-mediated transformation of peanut is genotype-independent. Up to 61% transformation was recorded when embryonic axes were co-cultivated with 5 × 10⁷ A. rhizogenes cells from logarithmic phase for 2 days on co-culture medium containing 50 μmol l⁻¹ acetosyringone. Composite plants with transgenic roots were harvested after 45 days of treatment. Furthermore, this method was applied to assess the insecticidal activity of a synthetic cry8Ea1 gene against Holotrichia parallela in transgenic roots of peanut.     

Heat stress screening of peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) seedlings for acquired thermotolerance

Heat can be one of the major abiotic stresses that adversely affect crop production worldwide at different stages of development. As field screening for heat tolerance can be inconsistent and seasonally-limited, it is important to develop a reliable protocol under controlled conditions that allows simultaneous screening of multiple genotypes. The objective of this research was to develop a straightforward laboratory protocol using acquired thermotolerance (ATT) in peanut seedlings as a measure of one mechanism of heat stress tolerance. Sixteen genotypes, including selected accessions of the US peanut minicore collection along with standard checks, were evaluated for acquired themotolerance in two independent experiments. A change in the temperature sensitivity of chlorophyll accumulation was used as an indicator of acquired thermotolerance. Pre-incubation at 38°C for 4 h before the 30-min 50°C challenge triggered the acquired thermotolerance system of the leaf disks, resulting in chlorophyll accumulation upon exposure to light. There was considerable variation among genotypes for ATT in both experiments. Genotypic ranking for mean ATT values were highly correlated (0.949) in both experiments. The effect of seed weight on ATT was not significant. This method is relatively simple and inexpensive and can be used to screen a large number of genotypes.     

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.

     

Seed protein fraction electrophoresis in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) accessions and wild species

Total seed storage proteins were studied in 50 accessions of A. hypogaea (11 A. hypogaea ssp. hypogaea var hypogaea, 13 A. hypogaea ssp. hypogaea var hirsuta, 11 A. hypogaea ssp. fastigiata var fastigiata and 15 A. hypogaea ssp. fastigiata var. vulgaris accessions) in SDS PAGE. These accessions were also analysed for albumin and globulin seed protein fractions. Among the six seed protein markers presently used, it was found that globulin fraction showed maximum diversity (77.2%) in A. hypogaea accessions followed by albumin (52.3%), denatured total soluble protein fraction in embryo (33.3%) and cotyledon (28.5%). The cluster analysis based on combined data of cotyledons, embryos, albumins and globulins seed protein fractions demarcated the accessions of two subspecies hypogaea and fastigiata into two separate clusters supported by 51% bootstrap value, with few exceptions, suggesting the genotypes to be moderately diverse. Native and denatured total soluble seed storage proteins were also electrophoretically analysed in 27 wild Arachis species belonging to six sections of the genus. Cluster analysis using different methods were performed for different seed proteins data alone and also in combination. Section Caulorrhizae (C genome) and Triseminatae (T genome) formed one, distantly related group to A. hypogaea and other section Arachis species in the dendrogram based on denatured seed storage proteins data. The present analysis has maintained that the section Arachis species belong to primary and secondary genepools and, sections Procumbenetes and Erectoides belong to tertiary gene pools.     

Characterization and transferability of microsatellite markers of the cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis>)

Background  

The genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species.     

Mapping of a dominant rust resistance gene revealed two R genes around the major Rust_QTL in cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Key message

A consensus rust QTL was identified within a 1.25 cM map interval of A03 chromosome in cultivated peanut. This map interval contains a TIR–NB–LRR R gene and four pathogenesis-related genes.

Abstract

Disease resistance in plants is manifested due to the specific interaction between the R gene product and its cognate avirulence gene product (AVR) in the pathogen. Puccinia arachidis Speg. causes rust disease and inflicts economic damages to peanut. Till now, no experimental evidence is known for the action of R gene in peanut for rust resistance. A fine mapping approach towards the development of closely linked markers for rust resistance gene was undertaken in this study. Phenotyping of an RIL population at five environments for field rust score and subsequent QTL analysis has identified a 1.25 cM map interval that harbored a consensus major Rust_QTL in A03 chromosome. This Rust_QTL is flanked by two SSR markers: FRS72 and SSR_GO340445. Both the markers clearly identified strong association of the mapped region with rust reaction in both resistant and susceptible genotypes from a collection of 95 cultivated peanut germplasm. This 1.25 cM map interval contained 331.7 kb in the physical map of A. duranensis and had a TIR–NB–LRR category R gene (Aradu.Z87JB) and four glucan endo-1,3 β glucosidase genes (Aradu.RKA6 M, Aradu.T44NR, Aradu.IWV86 and Aradu.VG51Q). Another resistance gene analog was also found in the vicinity of mapped Rust_QTL. The sequence between SSR markers, FRS72 and FRS49, contains an LRR-PK (Aradu.JG217) which is equivalent to RHG4 in soybean. Probably, the protein kinase domain in AhRHG4 acts as an integrated decoy for the cognate AVR from Puccinia arachidis and helps the TIR–NB–LRR R-protein to initiate a controlled program cell death in resistant peanut plants.

     

Microsatellite identification and characterization in peanut (<Emphasis Type="Italic">A. hypogaea</Emphasis> L.)

A major constraint to the application of biotechnology to the improvement of the allotetraploid peanut, or groundnut (Arachis hypogaea L.), has been the paucity of polymorphism among germplasm lines using biochemical (seed proteins, isozymes) and DNA markers (RFLPs and RAPDs). Six sequence-tagged microsatellite (STMS) markers were previously available that revealed polymorphism in cultivated peanut. Here, we identify and characterize 110 STMS markers that reveal genetic variation in a diverse array of 24 peanut landraces. The simple-sequence repeats (SSRs) were identified with a probe of two 27,648-clone genomic libraries: one constructed using PstI and the other using Sau3AI/BamHI. The most frequent, repeat motifs identified were ATT and GA, which represented 29% and 28%, respectively, of all SSRs identified. These were followed by AT, CTT, and GT. Of the amplifiable primers, 81% of ATT and 70.8% of GA repeats were polymorphic in the cultivated peanut test array. The repeat motif AT showed the maximum number of alleles per locus (5.7). Motifs ATT, GT, and GA had a mean number of alleles per locus of 4.8, 3.8, and 3.6, respectively. The high mean number of alleles per polymorphic locus, combined with their relative frequency in the genome and amenability to probing, make ATT and GA the most useful and appropriate motifs to target to generate further SSR markers for peanut.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by J.S. Heslop-Harrison     

Molecular characterization and expression profiling of the phosphoenolpyruvate carboxylase genes in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled enzyme located at the core of plant carbohydrate metabolism. Plant PEPCs belong to a small multigene family encoding several plant-type PEPC genes, along with at least one distantly related bacterial-type PEPC gene. The PEPC genes have been intensively studied in Arabidopsis, but not in peanut (Arachis hypogaea L.). Previously, we isolated five PEPC genes (AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4 and AhPEPC5) from peanut. Here, due to the sequencing of the peanut genome, we analyzed the complexity of its PEPC gene family, including phylogenetic relationships, gene structure and chromosome mapping. The results showed that AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 encoded typical plant-type enzymes, while AhPEPC5 was a bacterial-type PEPC. The recombinant proteins of these genes were expressed in Escherichia coli, and the calculated molecular weights of the recombinant proteins were 110.8 kD (AhPEPC1), 110.7 kD (AhPEPC2), 110.3 kD (AhPEPC3), 110.8 kD (AhPEPC4), and 116.4 kD (AhPEPC5). The expression patterns of AhPEPC1-5 were analyzed under cold, salt and drought conditions. Our results indicated that the expression of AhPEPC3 was rapidly and substantially enhanced under abiotic stress, whereas the expression of AhPEPC1 and AhPEPC2 was slightly enhanced under certain stress conditions. Some genes were down-regulated in leaves under stress: AhPEPC1, AhPEPC4 and AhPEPC5 under salt stress and AhPEPC4 and AhPEPC5 under drought stress. These results suggest that peanut PEPC proteins may differ in their functions during acclimation to abiotic stresses.     

Drought-induced oxidative damage and antioxidant responses in peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) seedlings

Two cultivars of peanut (Arachis hypogaea L.) which were designated as resistant (Florispan) and sensitive (Gazipasa) according to their growth retardation under drought stress conditions were compared for their oxidative damage and antioxidant responses. Sixteen days-old peanut seedlings were subjected to PEG-6000 solutions of two different osmotic potentials; −0.4 and −0.8 MPa, and various growth parameters, photosystem II activity, changes in malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline levels, activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POX) and gluthatione reductase (GR) enzymes were determined. Both cultivars exhibited water deficit at −0.8 MPa osmotic potential of PEG-6000 and H₂O₂ levels significantly increased during exposure to −0.4 MPa osmotic potential. However, H₂O₂ levels were under control in both cultivars at exposure to −0.8 MPa osmotic potential. Significant proline accumulation was observed in the tissues of cv. Florispan at −0.8 MPa osmotic potential, whereas proline accumulation did not appear to be an essential part of the protection mechanism against drought in cv. Gazipasa. No significant variation in chlorophyll fluorescence values were detected in neither of the cultivars. Enzyme activity measurements revealed that Gazipasa copes well with lesser magnitudes of drought stress by increasing the activity of mainly APX, and during harsh stress conditions, only APX maintains its activity in the tissues. In cultivar Florispan, GR activity appears to take role in lesser magnitudes of drought stress, whereas CAT and APX activities appear to be very crucial antioxidative defenses during intense stress conditions. The results indicate that, the level of proline and activities of the enzymes CAT and APX are important mechanisms for the maintenance of drought tolerance in peanut plants.     

Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) genotypes

Cultivated peanut possesses an extremely narrow genetic basis. Polymorphism is considerably difficult to identify with the use of conventional biochemical and molecular tools. For the purpose of obtaining considerable DNA polymorphisms and fingerprinting cultivated peanut genotypes in a convenient manner, start codon targeted polymorphism technique was used to study genetic diversity and relatedness among 20 accessions of four major botanical varieties of peanut. Of 36 primers screened, 18 primers could produce unambiguous and reproducible bands. All 18 primers generated a total of 157 fragments, with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22%) were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. Polymorphism per primer ranged from 14.29 to 66.67%, with an average of 36.76%. The results revealed that not all accessions of the same variety were grouped together and high genetic similarity was detected among the tested genotypes based on cluster analysis and genetic distance analysis, respectively. Further, accession-specific markers were observed in several accessions. All these results demonstrated the following: (1) start codon targeted polymorphism technique can be utilized to identify DNA polymorphisms and fingerprint cultivars in domesticated peanut, and (2) it possesses considerable potential for studying genetic diversity and relationships among peanut accessions.     

Metabolomics of groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes under varying temperature regimes

Various metabolites were analyzed in groundnut genotypes grown under varying temperature regimes (based on date of sowing). Four contrasting groundnut genotypes viz. ICGS44 (high-temperature tolerant), AK159 and GG7 (moderately-high-temperature tolerant), and DRG1 (high-temperature sensitive) were grown at three different temperature regimes i.e., low (early date of sowing), normal (normal date of sowing) and high temperature (late date of sowing) under field conditions. Untargeted metabolomic analysis of leaf tissue was performed by GC–MS, while targeted metabolite profiling was carried out by HPLC (polyamines) and UPLC-MS/MS (phenolics) at both the pegging and pod filling stages. Untargeted metabolomic profiling revealed exclusive expression/induction of beta-d-galactofuranoside, l-threonine, hexopyranose, d-glucopyranose, stearic acid, 4-ketoglucose, d-gulose, 2-o-glycerol-alpha-d-galactopyranoside and serine in ICGS44 during the pegging stage under high-temperature conditions. During the pod filling stage at higher temperature, alpha-d-galactoside, dodecanedioic acid, 1-nonadecene, 1-tetradecene and beta-d-galactofuranose were found to be higher in both ICGS44 and GG7. Moreover, almost all the metabolites detected by GC–MS were found to be higher in GG7, except beta-d-galactopyranoside, beta-d-glucopyranose, inositol and palmitic acid. Accumulation of putrescine was observed to be higher during low-temperature stress, while agmatine showed constitutive expression in all the genotypes, irrespective of temperature regime and crop growth stage. Interestingly, spermidine was observed only in the high-temperature tolerant genotype ICGS44. In our study, we found a higher accumulation of cinnamic acid, caffeic acid, salicylic acid and vanillic acid in ICGS44 compared to that of other genotypes at the pegging stage, whereas catechin and epicatechin were found during the pod filling stage in response to high-temperature stress, suggesting their probable roles in heat-stress tolerance in groundnut.     

Transgenic Peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) Expressing the Urease Subunit B Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Helicobacter pylori (H. pylori) has been identified as the main pathogenic factors of chronic gastritis and peptic ulcer, and the Class I carcinogen of gastric cancer by WHO. Vaccine has become the most effective measure to prevent and cure H. pylori infection. The UreB is the most effective and common immunogen of all strains of H. pylori and may stimulate the immunoresponse protecting the human body against the challenge of H. pylori. UreB antigen gene was cloned into the binary vector pBI121 which contains a seed-specific promoter Oleosin of peanut and a kanamycin resistance gene, and then UreB gene was transformed into peanut embryo leaflets by Agrobacter-mediated method. The putative transgenic plants were examined for the presence of UreB in the nuclear genome of peanut plants by PCR analysis. Expression of UreB gene in plants was identified by RT-PCR and Western blot analysis. These results suggest that the UreB transgenic peanut can be potentially used as an edible vaccine for controlling H. pylori.