De novo full length transcriptome analysis of Arachis glabrata provides insights into gene expression dynamics in response to biotic and abiotic stresses.

The perennial ornamental peanut Arachis glabrata represents one of the most adaptable wild Arachis species. This study used PacBio combined with BGISEQ-500 RNA-seq technology to study the transcriptome and gene expression dynamics of A. glabrata. Of the total 109,747 unique transcripts obtained, >90,566 transcripts showed significant homology to known proteins and contained the complete coding sequence (CDS). RNA-seq revealed that 1229, 1039, 1671, 3923, 1521 and 1799 transcripts expressed specifically in the root, stem, leaf, flower, peg and pod, respectively. We also identified thousands of differentially expressed transcripts in response to drought, salt, cold and leaf spot disease. Furthermore, we identified 30 polyphenol oxidase encoding genes associated with the quality of forage, making A. glabrata suitable as a forage crop. Our findings presented the first transcriptome study of A. glabrata which will facilitate genetic and genomics studies and lays the groundwork for a deeper understanding of the A. glabrata genome.

Identification of the Eutrema salsugineum EsMYB90 gene important for anthocyanin biosynthesis.

BACKGROUND: Anthocyanins contribute to coloration and antioxidation effects in different plant tissues. MYB transcription factors have been demonstrated to be a key regulator for anthocyanin synthesis in many plants. However, little information was available about the MYB genes in the halophyte species Eutrema salsugineum. RESULT: Here we report the identification of an important anthocyanin biosynthesis regulator EsMYB90 from Eutrema salsugineum, which is a halophyte tolerant to multiple abiotic stresses. Our phylogenetic and localization analyses supported that EsMYB90 is an R2R3 type of MYB transcription factor. Ectopic expression of EsMYB90 in tobacco and Arabidopsis enhanced pigmentation and anthocyanin accumulation in various organs. The transcriptome analysis revealed that 42 genes upregulated by EsMYB90 in 35S:EsMYB90 tobacco transgenic plants are required for anthocyanin biosynthesis. Moreover, our qRT-PCR results showed that EsMYB90 promoted expression of early (PAL, CHS, and CHI) and late (DFR, ANS, and UFGT) anthocyanin biosynthesis genes in stems, leaves, and flowers of 35S:EsMYB90 tobacco transgenic plants. CONCLUSIONS: Our results indicated that EsMYB90 is a MYB transcription factor, which regulates anthocyanin biosynthesis genes to control anthocyanin biosynthesis. Our work provides a new tool to enhance anthocyanin production in various plants.

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

Whole-genome resequencing-based QTL-seq identified AhTc1 gene encoding a R2R3-MYB transcription factor controlling peanut purple testa colour.

Peanut (Arachis hypogaea. L) is an important oil crop worldwide. The common testa colours of peanut varieties are pink or red. But the peanut varieties with dark purple testa have been focused in recent years due to the potential high levels of anthocyanin, an added nutritional value of antioxidant. However, the genetic mechanism regulating testa colour of peanut is unknown. In this study, we found that the purple testa was decided by the female parent and controlled by a single major gene named AhTc1. To identify the candidate gene controlling peanut purple testa, whole-genome resequencing-based approach (QTL-seq) was applied, and a total of 260.9 Gb of data were generated from the parental and bulked lines. SNP index analysis indicated that AhTc1 located in a 4.7 Mb region in chromosome A10, which was confirmed by bulked segregant RNA sequencing (BSR) analysis in three segregation populations derived from the crosses between pink and purple testa varieties. Allele-specific markers were developed and demonstrated that the marker pTesta1089 was closely linked with purple testa. Further, AhTc1 encoding a R2R3-MYB gene was positional cloned. The expression of AhTc1 was significantly up-regulated in the purple testa parent YH29. Overexpression of AhTc1 in transgenic tobacco plants led to purple colour of leaves, flowers, pods and seeds. In conclusion, AhTc1, encoding a R2R3-MYB transcription factor and conferring peanut purple testa, was identified, which will be useful for peanut molecular breeding selection for cultivars with purple testa colour for potential increased nutritional value to consumers.

Expression of AtLEC2 and AtIPTs promotes embryogenic callus formation and shoot regeneration in tobacco.

BACKGROUND: LEAFY COTYLEDON 2 (LEC2) acts throughout embryo morphogenesis and maturation phase to maintain embryogenic identity. Our previous study stated that Arabidopsis thaliana LEC2 (AtLEC2) driven by glucocorticoid receptor-dexamethasone (GR-DEX) inducible system (AtLEC2-GR) triggers embryogenic callus formation in tobacco (Nicotiana tabacum). RESULTS: In this study, the adenosine phosphate isopentenyltransferase genes AtIPT3, AtIPT7 and the tRNA isopentenyltransferase gene AtIPT9 were overexpressed in the AtLEC2-GR transgenic background. In the AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR seedlings, high-quality embryogenic callus was obtained under the DEX condition, and the shoot regeneration efficiency was 2 to 3.5 folds higher than AtLEC2-GR alone on hormone free medium without DEX. Transcriptome analyses showed that up-regulated BBM, L1L, ABI3, and FUS3 might function during embryogenic callus formation. However, at the shoot regeneration stage, BBM, L1L, ABI3, and FUS3 were down-regulated and Type-B ARRs were up-regulated, which might contribute to the increased shoot regeneration rate. CONCLUSIONS: A novel system for inducing shoot regeneration in tobacco has been developed using the GR-DEX system. Induced expression of AtLEC2 triggers embryogenic callus formation and overexpression of AtIPT7 or AtIPT9 improves shoot regeneration without exogenous cytokinin.

The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication.

High oil and protein content make tetraploid peanut a leading oil and food legume. Here we report a high-quality peanut genome sequence, comprising 2.54 Gb with 20 pseudomolecules and 83,709 protein-coding gene models. We characterize gene functional groups implicated in seed size evolution, seed oil content, disease resistance and symbiotic nitrogen fixation. The peanut B subgenome has more genes and general expression dominance, temporally associated with long-terminal-repeat expansion in the A subgenome that also raises questions about the A-genome progenitor. The polyploid genome provided insights into the evolution of Arachis hypogaea and other legume chromosomes. Resequencing of 52 accessions suggests that independent domestications formed peanut ecotypes. Whereas 0.42-0.47 million years ago (Ma) polyploidy constrained genetic variation, the peanut genome sequence aids mapping and candidate-gene discovery for traits such as seed size and color, foliar disease resistance and others, also providing a cornerstone for functional genomics and peanut improvement.

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.

Induced expression of AtLEC1 and AtLEC2 differentially promotes somatic embryogenesis in transgenic tobacco plants.

Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene's induction of somatic embryogenesis.

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.