Dong-Hong Min

Genetic Diversity, Population Structure and Linkage Disequilibrium in Elite Chinese Winter Wheat Investigated with SSR Markers

To ascertain genetic diversity, population structure and linkage disequilibrium (LD) among a representative collection of Chinese winter wheat cultivars and lines, 90 winter wheat accessions were analyzed with 269 SSR markers distributed throughout the wheat genome. A total of 1,358 alleles were detected, with 2 to 10 alleles per locus and a mean genetic richness of 5.05. The average genetic diversity index was 0.60, with values ranging from 0.05 to 0.86. Of the three genomes of wheat, ANOVA revealed that the B genome had the highest genetic diversity (0.63) and the D genome the lowest (0.56); significant differences were observed between these two genomes (P<0.01). The 90 Chinese winter wheat accessions could be divided into three subgroups based on STRUCTURE, UPGMA cluster and principal coordinate analyses. The population structure derived from STRUCTURE clustering was positively correlated to some extent with geographic eco-type. LD analysis revealed that there was a shorter LD decay distance in Chinese winter wheat compared with other wheat germplasm collections. The maximum LD decay distance, estimated by curvilinear regression, was 17.4 cM (r2>0.1), with a whole genome LD decay distance of approximately 2.2 cM (r2>0.1, P<0.001). Evidence from genetic diversity analyses suggest that wheat germplasm from other countries should be introduced into Chinese winter wheat and distant hybridization should be adopted to create new wheat germplasm with increased genetic diversity. The results of this study should provide valuable information for future association mapping using this Chinese winter wheat collection.

Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)

Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M2 mutants in a common wheat cultivar ‘Jinmai 47’. Numerous phenotypes with altered morphological and agronomic traits were observed from the M2 and M3 lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.

AtTGA4, a bZIP transcription factor, confers drought resistance by enhancing nitrate transport and assimilation in Arabidopsis thaliana

To cope with environmental stress caused by global climate change and excessive nitrogen application, it is important to improve water and nitrogen use efficiencies in crop plants. It has been reported that higher nitrogen uptake could alleviate the damaging impact of drought stress. However, there is scant evidence to explain how nitrogen uptake affects drought resistance. In this study we observed that bZIP transcription factor AtTGA4 (TGACG motif-binding factor 4) was induced by both drought and low nitrogen stresses, and that overexpression of AtTGA4 simultaneously improved drought resistance and reduced nitrogen starvation in Arabidopsis. Following drought stress there were higher nitrogen and proline contents in transgenic AtTGA4 plants than in wild type controls, and activity of the key enzyme nitrite reductase (NIR) involved in nitrate assimilation processes was also higher. Expressions of the high-affinity nitrate transporter genes NRT2.1 and NRT2.2 and nitrate reductase genes NIA1 and NIA2 in transgenic plants were all higher than in wild type indicating that higher levels of nitrate transport and assimilation activity contributed to enhanced drought resistance of AtTGA4 transgenic plants. Thus genetic transformation with AtTGA4 may provide a new approach to simultaneously improve crop tolerance to drought and low nitrogen stresses.

G-protein β subunit AGB1 positively regulates salt stress tolerance in Arabidopsis

Abstract The heterotrimeric GTP-binding proteins (G-proteins) in eukaryotes consisted of α, β and γ subunits and are important in molecular signaling by interacting with G-protein-coupled receptors (GPCR), on which to transduce signaling into the cytoplast through appropriate downstream effectors. However, downstream effectors regulated by the G-proteins in plants are currently not well defined. In this study, the transcripts of AGB1, a G protein β subunit gene in Arabidopsis were found to be down-regulated by cold and heat, but up-regulated by high salt stress treatment. AGB1 mutant (agb1-2) was more sensitive to high salt stress than wild-type (WT). Compared with WT, the cotyledon greening rates, fresh weight, root length, seedling germination rates and survival rates decreased more rapidly in agb1-2 along with increasing concentrations of NaCl in normal (MS) medium. Physiological characteristic analysis showed that compared to WT, the contents of chlorophyll, relative proline accumulation and peroxidase (POD) were reduced, whereas the malonaldehyde (MDA) content and concentration ratio of Na+/K+ were increased in agb1-2 under salt stress condition. Further studies on the expression of several stress inducible genes associated with above physiological processes were investigated, and the results revealed that the expressions of genes related to proline biosynthesis, oxidative stress response, Na+ homeostasis, stress- and ABA-responses were lower in agb1-2 than in WT, suggesting that those genes are possible downstream genes of AGB1 and that their changed expression plays an important role in determining phenotypic and physiologic traits in agb1-2. Taken together, these findings indicate that AGB1 positively regulates salt tolerance in Arabidopsis through its modulation of genes transcription related to proline biosynthesis, oxidative stress, ion homeostasis, stress- and ABA-responses.

The NAC-like transcription factor SiNAC110 in foxtail millet (Setaria italica L.) confers tolerance to drought and high salt stress through an ABA independent signaling pathway

Abstract Foxtail millet (Setaria italica (L.) P. Beauv) is a naturally stress tolerant crop. Compared to other gramineous crops, it has relatively stronger drought and lower nutrition stress tolerance traits. To date, the scope of functional genomics research in foxtail millet (S. italic L.) has been quite limited. NAC (NAM, ATAF1/2 and CUC2)-like transcription factors are known to be involved in various biological processes, including abiotic stress responses. In our previous foxtail millet (S. italic L.) RNA seq analysis, we found that the expression of a NAC-like transcription factor, S1NAC110 could be induced by drought stress; additionally, other references have reported that SiNAC110 expression could be induced by abiotic stress. So, we here selected SiNAC110 for further characterization and functional analysis. First, the predicted SiNAC110 protein encoded indicated SiNAC110 has a conserved NAM (no apical meristem) domain between the 11–139 amino acid positions. Phylogenetic analysis then indicated that SiNAC110 belongs to subfamily III of the NAC gene family. Subcellular localization analysis revealed that the SiNAC110-GFP fusion protein was localized to the nucleus in Arabidopsis protoplasts. Gene expression profiling analysis indicated that expression of SiNAC110 was induced by dehydration, high salinity and other abiotic stresses. Gene functional analysis using SiNAC110 overexpressed Arabidopsis plants indicated that, under drought and high salt stress conditions, the seed germination rate, root length, root surface area, fresh weight, and dry weight of the SiNAC110 overexpressed lines were significantly higher than the wild type (WT), suggesting that the SiNAC110 overex-pressed lines had enhanced tolerance to drought and high salt stresses. However, overexpression of S1NAC110 did not affect the sensitivity of SiNAC110 overexpressed lines to abscisic acid (ABA) treatment. Expression analysis of genes involved in proline synthesis, Na+/K+ transport, drought responses, and aqueous transport proteins were higher in the SiNAC110 overexpressed lines than in the WT, whereas expression of ABA-dependent pathway genes did not change. These results indicated that overexpression of SiNAC110 conferred tolerance to drought and high salt stresses, likely through influencing the regulation of proline biosynthesis, ion homeostasis and osmotic balance. Therefore, SiNAC110 appears to function in the ABA-independent abiotic stress response pathway in plants.

Genome-Wide Analysis of the RAV Family in Soybean and Functional Identification of GmRAV-03 Involvement in Salt and Drought Stresses and Exogenous ABA Treatment

Transcription factors play vital roles in plant growth and in plant responses to abiotic stresses. The RAV transcription factors contain a B3 DNA binding domain and/or an APETALA2 (AP2) DNA binding domain. Although genome-wide analyses of RAV family genes have been performed in several species, little is known about the family in soybean (Glycine max L.). In this study, a total of 13 RAV genes, named as GmRAVs, were identified in the soybean genome. We predicted and analyzed the amino acid compositions, phylogenetic relationships, and folding states of conserved domain sequences of soybean RAV transcription factors. These soybean RAV transcription factors were phylogenetically clustered into three classes based on their amino acid sequences. Subcellular localization analysis revealed that the soybean RAV proteins were located in the nucleus. The expression patterns of 13 RAV genes were analyzed by quantitative real-time PCR. Under drought stresses, the RAV genes expressed diversely, up- or down-regulated. Following NaCl treatments, all RAV genes were down-regulated excepting GmRAV-03 which was up-regulated. Under abscisic acid (ABA) treatment, the expression of all of the soybean RAV genes increased dramatically. These results suggested that the soybean RAV genes may be involved in diverse signaling pathways and may be responsive to abiotic stresses and exogenous ABA. Further analysis indicated that GmRAV-03 could increase the transgenic lines resistance to high salt and drought and result in the transgenic plants insensitive to exogenous ABA. This present study provides valuable information for understanding the classification and putative functions of the RAV transcription factors in soybean.