Physiological Characteristic Changes and Transcriptome Analysis of Maize (Zea mays L.) Roots under Drought Stress.

Water deficit is a key limiting factor for limiting yield in maize (Zea mays L.). It is crucial to elucidate the molecular regulatory networks of stress tolerance for genetic enhancement of drought tolerance. The mechanism of drought tolerance of maize was explored by comparing physiological and transcriptomic data under normal conditions and drought treatment at polyethylene glycol- (PEG-) induced drought stress (5%, 10%, 15%, and 20%) in the root during the seedling stage. The content of saccharide, SOD, CAT, and MDA showed an upward trend, proteins showed a downward trend, and the levels of POD first showed an upward trend and then decreased. Compared with the control group, a total of 597, 2748, 6588, and 5410 differentially expressed genes were found at 5%, 10%, 15%, and 20% PEG, respectively, and 354 common DEGs were identified in these comparisons. Some differentially expressed genes were remarkably enriched in the MAPK signaling pathway and plant hormone signal transduction. The 50 transcription factors (TFs) divided into 15 categories were screened from the 354 common DEGs during drought stress. Auxin response factor 10 (ARF10), auxin-responsive protein IAA9 (IAA9), auxin response factor 14 (ARF14), auxin-responsive protein IAA1 (IAA1), auxin-responsive protein IAA27 (IAA27), and 1 ethylene response sensor 2 (ERS2) were upregulated. The two TFs, including bHLH 35 and bHLH 96, involved in the MAPK signal pathway and plant hormones pathway, are significantly upregulated in 5%, 10%, 15%, and 20% PEG stress groups. The present study provides greater insight into the fundamental transcriptome reprogramming of grain crops under drought.

Genome-wide association study using specific-locus amplified fragment sequencing identifies new genes influencing nitrogen use efficiency in rice landraces.

Nitrogen is essential for crop production. It is a critical macronutrient for plant growth and development. However, excessive application of nitrogen fertilizer is not only a waste of resources but also pollutes the environment. An effective approach to solving this problem is to breed rice varieties with high nitrogen use efficiency (NUE). In this study, we performed a genome-wide association study (GWAS) on 419 rice landraces using 208,993 single nucleotide polymorphisms (SNPs). With the mixed linear model (MLM) in the Tassel software, we identified 834 SNPs associated with root surface area (RSA), root length (RL), root branch number (RBN), root number (RN), plant dry weight (PDW), plant height (PH), root volume (RL), plant fresh weight (PFW), root fractal dimension (RFD), number of root nodes (NRN), and average root diameter (ARD), with a significant level of p < 2.39×10-7. In addition, we found 49 SNPs that were correlated with RL, RBN, RN, PDW, PH, PFW, RFD, and NRN using genome-wide efficient mixed-model association (GEMMA), with a significant level of p < 1×10-6. Additionally, the final results for eight traits associated with 193 significant SNPs by using multi-locus random-SNP-effect mixed linear model (mrMLM) model and 272 significant SNPs associated with 11 traits by using IIIVmrMLM. Within the linkage intervals of significantly associated SNP, we identified eight known related genes to NUE in rice, namely, OsAMT2;3, OsGS1, OsNR2, OsNPF7.4, OsPTR9, OsNRT1.1B, OsNRT2.3, and OsNRT2.2. According to the linkage disequilibrium (LD) decay value of this population, there were 75 candidate genes within the 150-kb regions upstream and downstream of the most significantly associated SNP (Chr5_29804690, Chr5_29956584, and Chr10_17540654). These candidate genes included 22 transposon genes, 25 expressed genes, and 28 putative functional genes. The expression levels of these candidate genes were measured by real-time quantitative PCR (RT-qPCR), and the expression levels of LOC_Os05g51700 and LOC_Os05g51710 in C347 were significantly lower than that in C117; the expression levels of LOC_Os05g51740, LOC_Os05g51780, LOC_Os05g51960, LOC_Os05g51970, and LOC_Os10g33210 were significantly higher in C347 than C117. Among them, LOC_Os10g33210 encodes a peptide transporter, and LOC_Os05g51690 encodes a CCT domain protein and responds to NUE in rice. This study identified new loci related to NUE in rice, providing new genetic resources for the molecular breeding of rice landraces with high NUE.

Genome-wide characterization and expression analysis of α-amylase and ß-amylase genes underlying drought tolerance in cassava.

BACKGROUND: Starch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to both biological and abiotic stresses. The α-amylase (AMY) and the ß-amylase (BAM) are important enzymes that catalyze the hydrolysis of plant starch. Cassava (Manihot esculenta Crantz) is treated as one of the most drought-tolerant crops. However, the mechanisms of how AMY and BAM respond to drought in cassava are still unknown. RESULTS: Six MeAMY genes and ten MeBAM genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. Tandem and fragment replications play important roles in the amplification of MeAMY and MeBAM genes. Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at the N-terminus, which may have transcription factor functions. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are proven as critical genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity were significantly altered in cassava under different levels of drought stress. CONCLUSIONS: These results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

Beneficial shift of rhizosphere soil nutrients and metabolites under a sugarcane/peanut intercropping system.

Intercropping systems have been studied as a sustainable agricultural planting pattern to increase soil quality and crop yields. However, the relationships between metabolites and soil physicochemical properties remain poorly understood under sugarcane/peanut intercropping system. Thus, we determined the rhizosphere soil physicochemical properties, and analyzed rhizosphere soil metabolites and root metabolites by metabolomics method under monoculture and intercropping patterns of sugarcane and peanut. The results showed that pH, the contents of total phosphorus (P), total potassium (K), available nitrogen (N), available phosphorus (P), and available potassium (K) were higher in rhizosphere soil of intercropping peanut than monoculture peanut, and the content of total P was higher in rhizosphere soil of intercropping sugarcane than monoculture sugarcane. Sugarcane/peanut intercropping also significantly increased the activities of acid phosphatase and urease in rhizosphere soil. The metabolomics results showed that 32 metabolites, mainly organic acids and their derivatives (25.00%), nucleotides and their metabolites (18.75%), were detected in root and rhizosphere soil samples. In the MP-S (rhizosphere soil of monoculture peanut) vs. IP-S (rhizosphere soil of intercropping peanut) comparison, 47 differential metabolites (42 upregulated) were screened, including glycerolipids (19.15%), organic acids and their derivatives (17.89%), and amino acids and their metabolites (12.77%). In the MS-S (rhizosphere soil of monoculture sugarcane) vs. IS-S (rhizosphere soil of intercropping sugarcane) comparison, 51 differential metabolites (26 upregulated) were screened, including heterocyclic compounds (15.69%), glycerolipids (11.76%), and organic acids and their derivatives (9.80%). The metabolite species from MP-S, MS-S, IP-S, and IS-S were similar, but some metabolite contents were significantly different, such as adenine, adenosine, maltotriose, thermozeaxanthin-13 and PE-NMe (20:0/24:0). Adenine and adenosine were detected in root and rhizosphere soils, and their levels were increased in the intercropping treatment, which were mainly related to enhanced purine metabolism in root and rhizosphere soils under the sugarcane/peanut intercropping system. Importantly, adenine and adenosine were significantly positively correlated with total P and total K contents, acid phosphatase and urease activities, and pH. This study clarified that the sugarcane/peanut intercropping system could improve soil nutrients and enzymes and was related to purine metabolism.

MicroSugar: A database of comprehensive miRNA target prediction framework for sugarcane (Saccharum officinarum L.).

microRNA (miRNA) is a group of small non-coding RNA that plays important role in post-transcription of gene expression. With the studies about miRNA increase in sugarcane, the researchers lack an exhaustive resource to achieve the data. To fill this gap, we developed MicroSugar, a database that supported mRNA and miRNA annotation for sugarcane (http://suc.gene-db.com). MicroSugar is an integrated resource developed for 194,528 genes including 80,746 unigenes from long reads of Pacbio platform and 468 miRNAs from 72 samples. Internode elongation (jointing) is the key biological characteristic for the growth of sugarcane tillers into sugarcane stems. The present study combined the sequencing data from the different stages in internode elongation of stem and tiller. In total, the 14,300 3' untranslated region (UTR) sequences were extracted from the gene sequences and 3019 mRNAs as target of 327 miRNA were identified by miRanda algorithm and Spearman's Rho of expression levels. To determine the gene functions regulated by these miRNAs, the gene ontology enrichment analysis was performed and it confirmed that the over-represented Gene Ontology (GO) terms were associated with organism formation indicating the growth controlling function by miRNAs in sugarcane. Moreover, MicroSugar is a comprehensive and integrated database with a user-friendly responsive template. By browsing, searching and downloading of the nucleotide sequences, expression and miRNA targets, the user can retrieve information promptly. The database provides a valuable resource to facilitate the understanding of miRNA in sugarcane development and growth which will contribute to the study of sugarcane and other plants.

WRKY Genes Improve Drought Tolerance in Arachis duranensis.

WRKY transcription factor participates in plant growth and development and response to biotic and abiotic stresses. Arachis duranensis, a turfgrass, has high drought tolerance, yet little is known about AdWRKYs response to drought stress in A. duranensis. In this study, RNA-seq identified five AdWRKYs, including AdWRKY18, AdWRKY40, AdWRKY42, AdWRKY56, and AdWRKY64, which were upregulated under drought stress. Orthologous relationships between AdWRKYs and Arabidopsis WRKY were determined to predict the regulatory networks of the five AdWRKYs based on AtWRKYs. Additionally, protein-protein interactions were predicted using differentially expressed proteins from RNA-seq. The quantitative real-time PCR (qRT-PCR) results showed that AdWRKY40 was upregulated, while AdWRKY42, AdWRKY56, and AdWRKY64 were downregulated at different time-points under drought stress. The predicted regulatory networks showed that AdWRKY40 activates COR47, RD21, and RD29A expression under drought stress. Besides, AdWRKY56 regulated CesA8 under drought stress. Aradu.YIQ80 (NAC019) interacted with AdWRKY40, AdWRKY42, AdWRKY56, and AdWRKY64, while Aradu.Z5H58 (NAC055) interacted with AdWRKY42 and AdWRKY64 under drought stress. This study used Arabidopsis to assess AdWRKYs function and regulatory networks, providing a basis for understanding drought tolerance in A. duranensis.

Genome-scale transcriptomic insights into the gene co-expression network of seed abortion in triploid Siraitia grosvenorii.

BACKGROUND: Siraitia grosvenorii (Swingle) C. Jeffrey, also known as Luohanguo or monk fruit, is a famous traditional Chinese medicine ingredient with important medicinal value and broad development prospects. Diploid S. grosvenorii has too many seeds, which will increase the utilization cost of active ingredients. Thus, studying the molecular mechanism of seed abortion in triploid S. grosvenorii, identifying the abortion-related genes, and regulating their expression will be a new direction to obtain seedless S. grosvenorii. Herein, we examined the submicroscopic structure of triploid S. grosvenorii seeds during abortion. RESULTS: Upon measuring the endogenous hormone content, we found that abscisic acid (ABA) and trans-zeatin (ZR) levels were significantly downregulated after days 15 and 20 of flowering. RNA sequencing of triploid seeds at different developmental stages was performed to identify key genes regulating abortion in triploid S. grosvenorii seeds. Multiple genes with differential expression between adjacent stages were identified; seven genes were differentially expressed across all stages. Weight gene co-expression network analysis revealed that the enhancement of monoterpene and terpene metabolic processes might lead to seed abortion by reducing the substrate flow to ABA and ZR. CONCLUSIONS: These findings provide insights into the gene-regulatory network of seed abortion in triploid S. grosvenorii from different perspectives, thereby facilitating the innovation of the breeding technology of S. grosvenorii.

OsTTG1, a WD40 repeat gene, regulates anthocyanin biosynthesis in rice.

Anthocyanins play an important role in the growth of plants, and are beneficial to human health. In plants, the MYB-bHLH-WD40 (MBW) complex activates the genes for anthocyanin biosynthesis. However, in rice, the WD40 regulators remain to be conclusively identified. Here, a crucial anthocyanin biosynthesis gene was fine mapped to a 43.4-kb genomic region on chromosome 2, and a WD40 gene OsTTG1 (Oryza sativa TRANSPARENT TESTA GLABRA1) was identified as ideal candidate gene. Subsequently, a homozygous mutant (osttg1) generated by CRISPR/Cas9 showed significantly decreased anthocyanin accumulation in various rice organs. OsTTG1 was highly expressed in various rice tissues after germination, and it was affected by light and temperature. OsTTG1 protein was localized to the nucleus, and can physically interact with Kala4, OsC1, OsDFR and Rc. Furthermore, a total of 59 hub transcription factor genes might affect rice anthocyanin biosynthesis, and LOC_Os01g28680 and LOC_Os02g32430 could have functional redundancy with OsTTG1. Phylogenetic analysis indicated that directional selection has driven the evolutionary divergence of the indica and japonica OsTTG1 alleles. Our results suggest that OsTTG1 is a vital regulator of anthocyanin biosynthesis, and an important gene resource for the genetic engineering of anthocyanin biosynthesis in rice and other plants.

Sugarcane/peanut intercropping system improves physicochemical properties by changing N and P cycling and organic matter turnover in root zone soil.

BACKGROUND: The sugarcane/peanut intercropping system is a specific and efficient cropping pattern in South China. Intercropping systems change the bacterial diversity of soils and decrease disease rates. It can not only utilized light, heat, water and land resources efficiently, but also increased yield and economic benefits of farmers. METHODS: We determined soil nutrients, enzymes and microbes in sugarcane/peanut intercropping system, and analyzed relevance of the soil physicochemical properties and the genes involved in N and P cycling and organic matter turnover by metagenome sequencing. RESULTS: The results showed that sugarcane/peanut intercropping significantly boosted the content of total nitrogen, available phosphorus, total potassium, organic matter, pH value and bacteria and enhanced the activity of acid phosphatase compared to monocropping. Especially the content of available nitrogen, available phosphorus and organic matter increased significantly by 20.1%, 65.3% and 56.0% in root zone soil of IP2 treatment than monocropping treatment. The content of available potassium and microbial biomass carbon, as well as the activity of catalase, sucrase and protease, significantly decreased in intercropping root zone soil. Intercropping resulted in a significant increase by 7.8%, 16.2% and 23.0% in IS, IP1 and IP2, respectively, of the acid phosphatase content relative to MS. Metagenomic analysis showed that the pathways involved in carbohydrate and amino acid metabolism were dominant and more abundant in intercropping than in monocropping. Moreover, the relative abundances of genes related to N cycling (glnA, GLUD1_2, nirK), P cycling (phoR, phoB) and organic matter turnover (PRDX2_4) were higher in the intercropping soil than in the monocropping soil. The relative abundance of GLUD1_2 and phoR were 25.5% and 13.8% higher in the IP2 treatment respectively,and bgIX was higher in IS treatment compared to the monocropping treatment. Genes that were significantly related to phosphorus metabolism and nitrogen metabolism (TREH, katE, gudB) were more abundant in intercropping than in monocropping. CONCLUSION: The results of this study indicate that the intercropping system changed the numbers of microbes as well as enzymes activities, and subsequently regulate genes involved in N cycling, P cycling and organic matter turnover. Finally, it leads to the increase of nutrients in root zone soil and improved the soil environment.

Sugarcane/peanut intercropping system improves the soil quality and increases the abundance of beneficial microbes.

Sugarcane/peanut intercropping is a highly efficient planting pattern in South China. However, the effects of sugarcane/peanut intercropping on soil quality need to be clarified. This study characterized the soil microbial community and the soil quality in sugarcane/peanut intercropping systems by the Illumina MiSeq platform. The results showed that the intercropping sugarcane (IS) system significantly increased the total N (TN), available N (AN), available P (AP), pH value, and acid phosphatase activity (ACP), but it had little effect on the total P (TP), total K (TK), available K (AK), organic matter (OM), urease activity, protease activity, catalase activity, and sucrase activity, compared with those in monocropping sugarcane (MS) and monocropping peanut (MP) systems. Both intercropping peanut (IP) and IS soils contained more bacteria and fungi than soils in the MP and MS fields, and the microbes identified were mainly Chloroflexi and Acidobacteria, respectively. Intercropping significantly increased the number of unique microbes in IS soils (68 genera), compared with the numbers in the IP (14), MS (17), and MP (16) systems. The redundancy analysis revealed that the abundances of culturable Acidobacteriaceae subgroup 1, nonculturable DA111, and culturable Acidobacteria were positively correlated with the measured soil quality in the intercropping system. Furthermore, the sugarcane/peanut intercropping significantly increased the economic benefit by 87.84% and 36.38%, as compared with that of the MP and MS, respectively. These results suggest that peanut and sugarcane intercropping increases the available N and P content by increasing the abundance of rhizospheric microbes, especially Acidobacteriaceae subgroup 1, DA111, and Acidobacteria.

Cassava/peanut intercropping improves soil quality via rhizospheric microbes increased available nitrogen contents.

BACKGROUND: Intercropping, an essential cultivation pattern in modern agricultural systems, increases crop yields and soil quality. Cassava and peanut intercropping systems exhibit advantages in solar utilization and cadmium absorption, etc. However, the inner mechanisms need to be elucidated. In this study, Illumina MiSeq platform was used to reveal the rhizospheric microbes and soil quality in cassava/peanut intercropping systems, and the results provided a reference for the application of this method in studying other intercropping systems. RESULTS: Both intercropping cassava/peanut (IP) and intercropping peanut/cassava (IC) systems significantly increased available N, available K, pH value, and urease activity, comparing with that in monocropping cassava (MC) and monocropping peanut (MP) system. However, there were few effects on the total N, total P, total K, available P, organic matter, protease activity, catalase activity, sucrase activity, and acid phosphatase activity. Both IP and MP soils contained more bacteria and fungi than those in the IC and MC soils, which were mainly made of Proteobacteria and Actinobacteria. Intercropping remarkably increased the number of Nitrospirae in IP and IC soils comparing those in MC and MP soils. Redundancy analysis (RDA) revealed that the abundances of DA101, Pilimelia, and Ramlibacter were positively correlated to the soil quality. These results suggest that intercropping enhances the available nitrogen content of soil through increasing the quantity of rhizospheric microbes, especially that of DA101 and Pilimelia. CONCLUSIONS: The cassava/peanut intercropping system improves soil quality through increasing the available nitrogen content and abundance of DA101, Pilimelia, and Ramlibacter in the soil.

Integrated mRNA and small RNA sequencing reveals microRNA regulatory network associated with internode elongation in sugarcane (Saccharum officinarum L.).

BACKGROUND: Internode elongation is one of the most important traits in sugarcane because of its relation to crop productivity. Understanding the microRNA (miRNA) and mRNA expression profiles related to sugarcane internode elongation would help develop molecular improvement strategies but they are not yet well-investigated. To identify genes and miRNAs involved in internode elongation, the cDNA and small RNA libraries from the pre-elongation stage (EI), early elongation stage (EII) and rapid elongation stage (EIII) were sequenced and their expression were studied. RESULTS: Based on the sequencing results, 499,495,518 reads and 80,745 unigenes were identified from stem internodes of sugarcane. The comparisons of EI vs. EII, EI vs. EIII, and EII vs. EIII identified 493, 5035 and 3041 differentially expressed genes, respectively. Further analysis revealed that the differentially expressed genes were enriched in the GO terms oxidoreductase activity and tetrapyrrole binding. KEGG pathway annotation showed significant enrichment in "zeatin biosynthesis", "nitrogen metabolism" and "plant hormone signal transduction", which might be participating in internode elongation. miRNA identification showed 241 known miRNAs and 245 novel candidate miRNAs. By pairwise comparison, 11, 42 and 26 differentially expressed miRNAs were identified from EI and EII, EI and EIII, and EII and EIII comparisons, respectively. The target prediction revealed that the genes involved in "zeatin biosynthesis", "nitrogen metabolism" and "plant hormone signal transduction" pathways are targets of the miRNAs. We found that the known miRNAs miR2592-y, miR1520-x, miR390-x, miR5658-x, miR6169-x and miR8154-x were likely regulators of genes with internode elongation in sugarcane. CONCLUSIONS: The results of this study provided a global view of mRNA and miRNA regulation during sugarcane internode elongation. A genetic network of miRNA-mRNA was identified with miRNA-mediated gene expression as a mechanism in sugarcane internode elongation. Such evidence will be valuable for further investigations of the molecular regulatory mechanisms underpinning sugarcane growth and development.

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.

Identification of candidate genes for gelatinization temperature, gel consistency and pericarp color by GWAS in rice based on SLAF-sequencing.

Rice is an important cereal in the world. The study of the genetic basis of important agronomic traits in rice landraces and identification of genes will facilitate the breed improvement. Gelatinization temperature (GT), gel consistency (GC) and pericarp color (PC) are important indices of rice cooking and eating quality evaluation and potential nutritional importance, which attract wide attentions in the application of genetic and breeding. To dissect the genetic basis of GT, GC and PC, a total of 419 rice landraces core germplasm collections consisting of 330 indica lines, 78 japonica lines and 11 uncertain varieties were planted, collected, then GT, GC, PC were measured for two years, and sequenced using specific-locus amplified fragment sequencing (SLAF-seq) technology. In this study, 261,385,070 clean reads and 56,768 polymorphic SLAF tags were obtained, which a total of 211,818 single nucleotide polymorphisms (SNPs) were discovered. With 208,993 SNPs meeting the criterion of minor allele frequency (MAF) > 0.05 and integrity> 0.5, the phylogenetic tree and population structure analysis were performed for all 419 rice landraces, and the whole panel mainly separated into six subpopulations based on population structure analysis. Genome-wide association study (GWAS) was carried out for the whole panel, indica subpanel and japonica subpanel with subset SNPs respectively. One quantitative trait locus (QTL) on chromosome 6 for GT was detected in the whole panel and indica subpanel, and one QTL associated with GC was located on chromosome 6 in the whole panel and indica subpanel. For the PC trait, 8 QTLs were detected in the whole panel on chromosome 1, 3, 4, 7, 8, 10 and 11, and 7 QTLs in the indica subpanel on chromosome 3, 4, 7, 8, 10 and 11. For the three traits, no QTL was detected in japonica subpanel, probably because of the polymorphism repartition between the subpanel, or small population size of japonica subpanel. This paper provides new gene resources and insights into the molecular mechanisms of important agricultural trait of rice phenotypic variation and genetic improvement of rice quality variety breeding.

QTL Mapping by Whole Genome Re-sequencing and Analysis of Candidate Genes for Nitrogen Use Efficiency in Rice.

Nitrogen is a major nutritional element in rice production. However, excessive application of nitrogen fertilizer has caused severe environmental pollution. Therefore, development of rice varieties with improved nitrogen use efficiency (NUE) is urgent for sustainable agriculture. In this study, bulked segregant analysis (BSA) combined with whole genome re-sequencing (WGS) technology was applied to finely map quantitative trait loci (QTL) for NUE. A key QTL, designated as qNUE6 was identified on chromosome 6 and further validated by Insertion/Deletion (InDel) marker-based substitutional mapping in recombinants from F2 population (NIL-13B4 × GH998). Forty-four genes were identified in this 266.5-kb region. According to detection and annotation analysis of variation sites, 39 genes with large-effect single-nucleotide polymorphisms (SNPs) and large-effect InDels were selected as candidates and their expression levels were analyzed by qRT-PCR. Significant differences in the expression levels of LOC_Os06g15370 (peptide transporter PTR2) and LOC_Os06g15420 (asparagine synthetase) were observed between two parents (Y11 and GH998). Phylogenetic analysis in Arabidopsis thaliana identified two closely related homologs, AT1G68570 (AtNPF3.1) and AT5G65010 (ASN2), which share 72.3 and 87.5% amino acid similarity with LOC_Os06g15370 and LOC_Os06g15420, respectively. Taken together, our results suggested that qNUE6 is a possible candidate gene for NUE in rice. The fine mapping and candidate gene analysis of qNUE6 provide the basis of molecular breeding for genetic improvement of rice varieties with high NUE, and lay the foundation for further cloning and functional analysis.

Allergen composition analysis and allergenicity assessment of Chinese peanut cultivars.

Peanut (Arachis hypogaea) is among the eight major food allergens in the world. Several attempts have been made to decrease or eliminate the allergenicity of peanut. Systemic screening of thousands of peanut cultivars may identify peanut with low allergenicity. In this study, the allergen compositions of 53 Chinese peanut cultivars were characterized, and their allergenicity to sera IgE of Chinese patients and in a mouse model was assessed. Contents of total protein and allergens were quantified by SDS-PAGE and densitometry analysis on gel. Although the contents of allergens broadly varied among cultivars, they were related to one another. The IgE binding capacity of cultivars was tested by ELISA, and their allergenicity was further evaluated in a mouse model by oral sensitization. Results showed that the allergenicity of peanut was affected by allergen composition rather than a single allergen. Peanut cultivars with low allergenicity may contain more Ara h 3/4 (24 kDa), Ara h 2 and less Ara h 3/4 (43, 38, and 36 kDa), Ara h 6. Screening based on allergen composition would facilitate the identification of low-allergenic peanut.

Molecular profiling of genetic variability in domesticated groundnut (Arachis hypogaea L.) based on ISJ, URP, and DAMD markers.

To detect DNA polymorphisms in the peanut, we screened 26 polymorphic primers using intron-exon splice junction (ISJ), universal rice primer (URP), and directed amplification of minisatellite region DNA (DAMD) techniques. Amplification of genomic DNA of 16 peanut accessions yielded 121 ISJ, 50 URP, and 25 DAMD fragments, of which 34, 25 and 16 were polymorphic, respectively. The range of polymorphism was 10.0-62.5%, averaging 27.7%, for ISJ; 20-80%, averaging 49.5%, for URP; and 28.6-50.0%, averaging 36.3%, for DAMD. In comparisons of multiplex ratio, average polymorphism information content, and marker index, the URP markers were relatively more efficient than ISJ and DAMD markers. Clustering results remained more or less the same with ISJ and URP markers. To the best of our knowledge, this is the first report on the study of the genetic diversity of the peanut using ISJ, URP, and DAMD markers.

Molecular characterization of high plant species using PCR with primers designed from consensus branch point signal sequences.

A novel method is introduced for producing molecular markers in plants using single 15- to 18-mer PCR primers designed from the short conserved consensus branch point signal sequences and standard agarose gel electrophoresis. This method was tested on cultivated peanut and verified to give good fingerprinting results in other plant species (mango, banana, and longan). These single primers, designed from relatively conserved branch point signal sequences within gene introns, should be universal across other plant species. The method is rapid, simple, and efficient, and it requires no sequence information of the plant genome of interest. It could be used in conjunction with, or as a substitute for, conventional RAPD or ISSR techniques for applications including genetic diversity analysis, phylogenetic tree construction, and quantitative trait locus mapping. This technique provides a new way to develop molecular markers for assessing genetic diversity of germplasm in diverse species based on conserved branch point signal sequences.

Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (Arachis hypogaea 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.