Effect of ACC oxidase gene AhACOs on salt tolerance of peanut / 生物工程学报

ACC oxidase (ACO) is one of the key enzymes that catalyze the synthesis of ethylene. Ethylene is involved in salt stress response in plants, and salt stress seriously affects the yield of peanut. In this study, AhACO genes were cloned and their functions were investigated with the aim to explore the biological function of AhACOs in salt stress response, and to provide genetic resources for the breeding of salt-tolerant varieties of peanut. AhACO1 and AhACO2 were amplified from the cDNA of salt-tolerant peanut mutant M29, respectively, and cloned into the plant expression vector pCAMBIA super1300. The recombinant plasmid was transformed into Huayu22 by pollen tube injection mediated by Agrobacterium tumefaciens. After harvest, the small slice cotyledon was separated from the kernel, and the positive seeds were screened by PCR. The expression of AhACO genes was analyzed by qRT-PCR, and the ethylene release was detected by capillary column gas chromatography. Transgenic seeds were sowed and then irrigated with NaCl solution, and the phenotypic changes of 21-day-seedings were recorded. The results showed that the growth of transgenic plants were better than that of the control group Huayu 22 upon salt stress, and the relative content of chlorophyll SPAD value and net photosynthetic rate (Pn) of transgenic peanuts were higher than those of the control group. In addition, the ethylene production of AhACO1 and AhACO2 transgenic plants were 2.79 and 1.87 times higher than that of control peanut, respectively. These results showed that AhACO1 and AhACO2 could significantly improve the salt stress tolerance of transgenic peanut.

Characterization of AhLea-3 and its enhancement of salt tolerance in transgenic peanut plants

BACKGROUND: Late embryogenesis abundant (LEA) proteins were reported to be related to adversity stress and drought tolerance. Lea-3 from Arachis hypogaea L. (AhLea-3) was previously found to be related to salt tolerance according to the result of transcriptome profiling and digital gene expression analysis. So, AhLea-3 was cloned and the salt tolerance was validated by transgenic peanut plants. RESULTS: AhLea-3 was isolated from M34, a salt-resistant mutant of peanut, with its cDNA as the template. AhLea-3 contains one intron and two extrons, and the full-length cDNA sequence contains 303 bp. AhLea3 was ligated to pCAMBIA1301 to obtain the overexpression vector pCAMBIA1301-AhLea-3, which was then transferred into peanut variety Huayu23. The expression level of AhLea-3, as determined by qRTPCR analysis, was >10 times higher in transgenic than in non-transgenic plants. Five days after they were irrigated with 250 mM NaCl, the transgenic plants showed less severe leaf wilting, higher activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), and lower malonic dialdehyde content than non-transgenic plants. Relative to non-transgenic plants, the transgenic plants had a higher photosynthetic net rate, stomatal conductance, and transpiration rate, and a lower intercellular CO2 concentration after salt stress treatment (250 mM NaCl). CONCLUSIONS: These results indicate that overexpression of AhLea-3 increased the salt tolerance of transgenic peanut plants. AhLea-3 might become a useful gene resource for the variety breeding of salinity tolerance in peanut.

Genome-wide development of polymorphic microsatellite markers and their application in peanut breeding program

BACKGROUND: Cultivated peanut (Arachis hypogaea. L) represents one of the most important oil crops in the world. Although much effort has been expended to characterize microsatellites or Simple Sequence Repeats (SSRs) in peanut, the quantity and quality of the markers in breeding applications remain limited. Here, genome-wide SSR characterization and marker development were performed using the recently assembled genome of the cultivar Tifrunner. RESULTS: In total, 512,900 microsatellites were identified from 2556.9-Mb genomic sequences. Based on the flanking sequences of the identified microsatellites, 7757 primer pairs (markers) were designed, and further evaluated in the assembled genomic sequences of the tetraploid Arachis cultivars, Tifrunner and Shitouqi, and the diploid ancestral species, A. duranensis and A. ipaensis. In silico PCR analysis showed that the SSR markers had high amplification efficiency and polymorphism in four Arachis genotypes. Notably, nearly 60% of these markers were single-locus SSRs in tetraploid Arachis species, indicating they are more specific in distinguishing the alleles of the A and B sub-genomes of peanut. In addition, two markers closely related with purple testa color and 27 markers near to FAD2 genes were identified, which could be used for breeding varieties with purple testa and high-oleic acid content, respectively. Moreover, the potential application of these SSR markers in tracking introgressions from Arachis wild relatives was discussed. CONCLUSIONS: This study reported the development of genomic SSRs from assembled genomic sequences of the tetraploid Arachis Tifrunner, which will be useful for diversity analysis, genetic mapping and functional genomics studies in peanut

Transcriptome and proteome analyses of resistant preharvest peanut seed coat in response to Aspergillus flavus infection

BACKGROUND: The infection of peanut (Arachis hypogaea L.) seed coat by the pathogenic fungus Aspergillus flavus has highly negative economic and health impacts. However, the molecular mechanism underlying such defense response remains poorly understood. This study aims to address this issue by profiling the transcriptomic and proteomic changes that occur during the infection of the resistant peanut cultivar J11 by A. flavus. RESULTS: Transcriptomic study led to the detection of 13,539 genes, among which 663 exhibited differential expression. Further functional analysis found the differentially expressed genes to encode a wide range of pathogenesis- and/or defense-related proteins such as transcription factors, pathogenesis-related proteins, and chitinases. Changes in the expression patterns of these genes might contribute to peanut resistance to A. flavus. On the other hand, the proteomic profiling showed that 314 of the 1382 detected protein candidates were aberrantly expressed as a result of A. flavus invasion. However, the correlation between the transcriptomic and proteomic data was poor. We further demonstrated by in vitro fungistasis tests that hevamine-A, which was enriched at both transcript and protein levels, could directly inhibit the growth of A. flavus. Conclusions: The results demonstrate the power of complementary transcriptomic and proteomic analyses in the study of pathogen defense and resistance in plants and the chitinase could play an important role in the defense response of peanut to A. flavus. The current study also constitutes the first step toward building an integrated omics data platform for the development of Aspergillus-resistant peanut cultivars

Transcriptome profiling and digital gene expression analysis of genes associated with salinity resistance in peanut

Background: Soil salinity can significantly reduce crop production, but the molecular mechanism of salinity tolerance in peanut is poorly understood. A mutant (S1) with higher salinity resistance than its mutagenic parent HY22 (S3) was obtained. Transcriptome sequencing and digital gene expression (DGE) analysis were performed with leaves of S1 and S3 before and after plants were irrigated with 250 mM NaCl. Results: A total of 107,725 comprehensive transcripts were assembled into 67,738 unigenes using TIGR Gene Indices clustering tools (TGICL). All unigenes were searched against the euKaryotic Ortholog Groups (KOG), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and these unigenes were assigned to 26 functional KOG categories, 56 GO terms, 32 KEGG groups, respectively. In total 112 differentially expressed genes (DEGs) between S1 and S3 after salinity stress were screened, among them, 86 were responsive to salinity stress in S1 and/or S3. These 86 DEGs included genes that encoded the following kinds of proteins that are known to be involved in resistance to salinity stress: late embryogenesis abundant proteins (LEAs), major intrinsic proteins (MIPs) or aquaporins, metallothioneins (MTs), lipid transfer protein (LTP), calcineurin B-like protein-interacting protein kinases (CIPKs), 9-cis-epoxycarotenoid dioxygenase (NCED) and oleosins, etc. Of these 86 DEGs, 18 could not be matched with known proteins. Conclusion: The results from this study will be useful for further research on the mechanism of salinity resistance and will provide a useful gene resource for the variety breeding of salinity resistance in peanut.

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.

Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

Background: Cultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by theΔ12 fatty acid desaturase (FAD) encoded byAhFAD2AandAhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C→A:T) ofAhFAD2Aand an "A" insertion ofAhFAD2Bresulted in high-oleic acid phenotype. Detection ofAhFAD2mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detectAhFAD2genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detectionAhFAD2genotype of large number of breeding materials. Results: Here, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC4F2seeds was aabb. Conclusions: Due to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable fordeterminingAhFAD2genotype than other methods.

Identification of groundnut (Arachis hypogaea) SSR markers suitable for multiple resistance traits QTL mapping in African germplasm

Background This study aimed to identify and select informative Simple Sequence Repeat (SSR) markers that may be linked to resistance to important groundnut diseases such as Early Leaf Spot, Groundnut Rosette Disease, rust and aflatoxin contamination. To this end, 799 markers were screened across 16 farmer preferred and other cultivated African groundnut varieties that are routinely used in groundnut improvement, some with known resistance traits. Results The SSR markers amplified 817 loci and were graded on a scale of 1 to 4 according to successful amplification and ease of scoring of amplified alleles. Of these, 376 markers exhibited Polymorphic Information Content (PIC) values ranging from 0.06 to 0.86, with 1476 alleles detected at an average of 3.7 alleles per locus. The remaining 423 markers were either monomorphic or did not work well. The best performing polymorphic markers were subsequently used to construct a dissimilarity matrix that indicated the relatedness of the varieties in order to aid selection of appropriately diverse parents for groundnut improvement. The closest related varieties were MGV5 and ICGV-SM 90704 and most distant were Chalimbana and 47-10. The mean dissimilarity value was 0.51, ranging from 0.34 to 0.66. Discussion Of the 376 informative markers identified in this study, 139 (37%) have previously been mapped to the Arachis genome and can now be employed in Quantitative Trait Loci (QTL) mapping and the additional 237 markers identified can be used to improve the efficiency of introgression of resistance to multiple important biotic constraints into farmer-preferred varieties of Sub-Saharan Africa.

Isolation and characterization of drought-responsive genes from peanut roots by suppression subtractive hybridization

Background Peanut (Arachis hypogaea L.) is an important economic and oilseed crop. Long-term rainless conditions and seasonal droughts can limit peanut yields and were conducive to preharvest aflatoxin contamination. To elucidate the molecular mechanisms by which peanut responds and adapts to water limited conditions, we isolated and characterized several drought-induced genes from peanut roots using a suppression subtractive hybridization (SSH) technique. Results RNA was extracted from peanut roots subjected to a water stress treatment (45% field capacity) and from control plants (75% field capacity), and used to generate an SSH cDNA library. A total of 111 non-redundant sequences were obtained, with 80 unique transcripts showing homology to known genes and 31 clones with no similarity to either hypothetical or known proteins. GO and KEGG analyses of these differentially expressed ESTs indicated that drought-related responses in peanut could mainly be attributed to genes involved in cellular structure and metabolism. In addition, we examined the expression patterns of seven differentially expressed candidate genes using real-time reverse transcription-PCR (qRT-PCR) and confirmed that all were up-regulated in roots in response to drought stress, but to differing extents. Conclusions We successfully constructed an SSH cDNA library in peanut roots and identified several drought-related genes. Our results serve as a foundation for future studies into the elucidation of the drought stress response mechanisms of peanut.

Isolation and analysis of differentially expressed genes from peanut in response to challenge with Ralstonia solanacearum

Background: Bacterial wilt caused by Ralstonia solanacearum is the most devastating disease in peanut. Planting resistant peanut cultivars is deemed as the sole economically viable means for effective control of the disease. To understand the molecular mechanism underlying resistance and facilitate breeding process, differences in gene expression between seeds of Rihua 1 (a Virginia type peanut variety resistant to bacterial wilt) inoculated with the bacterial pathogen suspension (10(9) cfu ml-1) and seeds of the same cultivar treated with water (control), were studied using the GenefishingTM technology. Results: A total of 25 differentially expressed genes were isolated. Expression of genes encoding cyclophilin and ADP-ribosylation factor, respectively, were further studied by real time RT-PCR, and full length cDNAs of both genes were obtained by rapid amplification of cDNA ends. Conclusions: The study provided candidate genes potentially useful for breeding peanut cultivars with both high yield and bacterial wilt resistance, although confirmation of their functions through transgenic studies is still needed.

Cloning and expression analysis of peanut (Arachis hypogaea L. ) CHI gene

Chalcone isomerase (CHI) is the key enzyme that catalyzes chalcone into (2S)-flavanol or (2S)-5-desoxidation flavanol. The full length cDNA (1050 bp) of AhCHI (Arachis hypogaea CHI gene) was cloned by large scale EST sequencing using a peanut immature seed cDNA library. Sequence analysis results indicated that it was a type I CHI gene (with the accession number JN660794). The ORF of AhCHI was 768 bp, encoding a peptide of 255 amino acids with a pI of 5.189. Sequence alignment showed that the coding region of AhCHI gene is highly conserved to compare with CHI genes from other plant species. Peanut cDNA microarray and semi-quantitative RT-PCR analysis indicated that AhCHI was highly expressed in pegs. The expression level in flower and root was higher than the expression level in stem and leaf. AhCHI was expressed in a high level in seeds with a purple seed coat, while its expression was low in seed with white seed coat.

Cloning and analysis of a NBS-LRR disease resistance gene candidate PnAG1 from peanut (Arachis hypogaea L. )

Background: Based on the conserved sequences of a known NBS resistance gene, a pair of degenerate primers was designed to amplify the NBS-LRR resistance gene from peanut using PCR and RACE methods. Results: Analyzing the amino acid sequence by BLAST on NCBI, which was deduced from the 1088bp-long gene named PnAG1-2, showed that it had a certain homology with some resistance proteins, among which Arachis cardenasii resistance protein gene had the highest homology (66 percent). Relative quantification PCR analysis indicated that PnAG1-2 gene expresses more in J11 (an A. flavus-resistant variety) than in JH1012 (an A. flavus-susceptible variety) when the harvest time was coming. Conclusions: In this study, the NBS-LRR resistance sequence was successfully cloned from peanut and prokaryotic expression was done on the gene, which provided a foundation for cultivating anti-A. flavus peanut varieties.

Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L. )

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).

Sodium azide mutagenesis resulted in a peanut plant with elevated oleate content

Screening of peanut seeds resulting from 0.39 percent sodium azide treatment with NIRS calibration equation for bulk seed samples identified a plant with more than 60 percent oleate. Oleate content in individual seeds of the plant, as predicted by NIRS calibration equation for intact single peanut seeds, ranged from 50.05 percent ~ 68.69 percent. Three seeds with >60 percent oleate thus identified were further confirmed by gas chromatography. Multiple sequence alignments of the FAD2B gene from Huayu 22 (wild type) and peanut seeds with elevated oleate (mutant type) revealed a C281T transition in the coding region causing an I94T substitution in the oleoyl-PC desaturase, which may be responsible for reduction in the enzyme activity.

A real-time PCR genotyping assay to detect FAD2A SNPs in peanuts (Arachis hypogaea L)

The high oleic (C18:1) phenotype in peanuts has been previously demonstrated to result from a homozygous recessive genotype (ol1ol1ol2ol2) in two homeologous fatty acid desaturase genes (FAD2A and FAD2B) with two key SNPs. These mutant SNPs, specifically G448A in FAD2A and 442insA in FAD2B, significantly limit the normal function of the desaturase enzyme activity which converts oleic acid into linoleic acid by the addition of a second double bond in the hydrocarbon chain. Previously, a genotyping assay was developed to detect wild type and mutant alleles in FAD2B. A real-time PCR assay has now been developed to detect wild type and mutant alleles (G448A) in FAD2A using either seed or leaf tissue. This assay was demonstrated to be applicable for the detection of homozygous and heterozygous samples. The FAD2A genotyping assay was validated by employing gas chromatography (GC) to determine total fatty acid composition and by genotyping peanut lines that have been well characterized. Overall, development of rapid assays such as real-time PCR which can identify key genotypes associated with important agronomic traits such as oleic acid, will improve breeding efficiency by targeting desirable genotypes at early stages of development.

Development of trinucleotide (GGC)n SSR markers in peanut (Arachis hypogaea L)

Cultivated peanut (Arachis hypogaea L.) is an oilseed crop of economic importance. It is native to South America, and it is grown extensively in the semi-arid tropics of Asia, Africa, and Latin America. Given an extremely narrow genetic base, efforts are being made to develop simple sequence repeat (SSR) markers to provide useful genetic and genomic tools for the peanut research community. A SSR-enriched library to isolate trinucleotide (GGC)n SSRs in peanut was constructed. A total of 143 unique sequences containing (GGC)n repeats were identified. One hundred thirty eight primer pairs were successfully designed at the flanking regions of SSRs. A suitable polymerase was chosen to amplify these GC-rich sequences. Although a low level of polymorphism was observed in cultivated peanut by these new developed SSRs, a high level of transferability to wild species would be beneficial to increasing the number of SSRs in wild species.

Novel protocol to identify true hybrids in normal oleate x high oleate crosses in peanut

A novel hybrid identification protocol was developed for F0:1 peanut seeds resulting from crosses between normal oleate cultivars with wild type FAD2B gene and high oleate genotypes with an A insertion in FAD2B gene. Presence of a series of overlapped peaks in trace file of the PCR product amplified with bF19/R1 primers was an indication of hybridity. This protocol may facilitate high oleate breeding and genetic studies in peanut.

Simple method to prepare DNA templates from a slice of peanut cotyledonary tissue for polymerase chain reaction

An efficient DNA extraction method was developed for peanut seed, where only 3-5 mg cotyledonary tissue was enough for more than 50 PCR reactions with a reaction volume of 15 ul. Both low copy number and high copy number DNA sequences were successfully amplified. Processing one seed sample only took about half an hour. Sampling had no significant effects on germination and development. The DNA extraction method makes it possible to identify transformants and conduct molecular marker studies prior to sowing, and thus may greatly hasten research progress.

EST sequencing and SSR marker development from cultivated peanut (Arachis hypogaea L)

Making use of the gene resources of wild type peanuts is a way to increase the genetic diversity of the cultivars. Marker assisted selection (MAS) could shorten the process of inter-specific hybridization and provide a possible way to remove the undesirable traits. However, the limited number of molecular markers available in peanut retarded its MAS process. We started a peanut ESTs (Expressed Sequence Tags) project aiming at cloning genes with agronomic importance and developing molecular markers. In this study we found 610 ESTs that contained one or more SSRs from 12,000 peanut ESTs. The most abundant SSRs in peanut are trinucleotides (66.3 percent) SSRs and followed by dinucleotide (28.8 percent) SSRs. AG/TC (10.7 percent) repeat was the most abundant and followed by CT/GA (9.0 percent), CTT/GAA (7.4 percent), and AAG/TTC (7.3 percent) repeats. Ninety-four SSR containing ESTs were randomly selected for primer design and synthesis, of which 33 pairs could generate good amplification and were used for polymorphism assessment. Results showed that polymorphism was very low in cultivars, while high level of polymorphism was revealed in wild type peanuts.

Seleção de genótipos resistentes de amendoinzeiro a Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) com base em análises multivariadas / Selection of resistant peanut genotypes to Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) supported by multivariate analysis

The velvetbean caterpillar Anticarsia gemmatalis Hübner attacks peanut leaves, and the use of resistant varieties has directly contributed to ecological and economic aspects of pest control. The aim of this work was to select resistant peanut genotypes to A. gemmatalis using cluster analyses (dendogram obtained by Ward's methods and K-means) and Principal Components analysis for data interpretation. The evaluated genotypes were: IAC 5, IAC 8112, IAC 22 and IAC Tatu ST with upright growth habit, and IAC 147, IAC 125, IAC Caiapó and IAC Runner 886 with runner growth habit, and soybean genotype BR 16 as a susceptible control. The biological parameters: leaf consumption, larval (4º instar) and pupal (24h old) weight, larval and pupal development time and adult longevity were evaluated at laboratory conditions. The genotypes IAC 147 and IAC Runner 886 were resistant to A. gemmatalis in both cluster tests, grouping apart from most of the other genotypes. Both dendrogram and K-means methods provided satisfactory biological explanation, and they can be complementary used together with Principal Component and vice-versa. These results suggest that cluster analyses may be an important statistical tool in the selection of host plant resistance.