Limiao Chen

Transcript Profile of the Response of Two Soybean Genotypes to Potassium Deficiency

The macronutrient potassium (K) is essential to plant growth and development. Crop yield potential is often affected by lack of soluble K. The molecular regulation mechanism of physiological and biochemical responses to K starvation in soybean roots and shoots is not fully understood. In the present study, two soybean varieties were subjected to low-K stress conditions: a low-K-tolerant variety (You06-71) and a low-K-sensitive variety (HengChun04-11). Eight libraries were generated for analysis: 2 genotypes ×2 tissues (roots and shoots) ×2 time periods [short term (0.5 to 12 h) and long term (3 to 12 d)]. RNA derived from the roots and shoots of these two varieties across two periods (short term and long term) were sequenced and the transcriptomes were compared using high-throughput tag-sequencing. To this end, a large number of clean tags (tags used for analysis after removal of dirty tags) corresponding to distinct tags (all types of clean tags) were identified in eight libraries (L1, You06-71-root short term; L2, HengChun04-11-root short term; L3, You06-71-shoot short term; L4, HengChun04-11-shoot short term; L5, You06-71-root long term; L6, HengChun04-11-root long term; L7, You06-71-shoot long term; L8, HengChun04-11-shoot long term). All clean tags were mapped to the available soybean (Glycine max) transcript database (http://www.soybase.org). Many genes showed substantial differences in expression across the libraries. In total, 5,440 transcripts involved in 118 KEGG pathways were either up- or down-regulated. Fifteen genes were randomly selected and their expression levels were confirmed using quantitative RT-PCR. Our results provide preliminary information on the molecular mechanism of potassium absorption and transport under low-K stress conditions in different soybean tissues.

Genetic analysis and molecular mapping of resistance gene to Phakopsora pachyrhizi in soybean germplasm SX6907

Key messageIn this study,Rpp6907,a novel resistance gene/allele toPhakopsora pachyrhiziin soybean, was mapped in a 111.9-kb region, including three NBS-LRR type predicted genes, on chromosome 18.AbstractSoybean rust caused by Phakopsora pachyrhizi Sydow has been reported in numerous soybean-growing regions worldwide. The development of rust-resistant varieties is the most economical and environmentally safe method to control the disease. The Chinese soybean germplasm SX6907 is resistant to P. pachyrhizi and exhibits immune reaction compared with the known Rpp genes. These characteristics suggest that SX6907 may carry at least one novel Rpp gene/allele. Three F2 populations from the crosses of SX6907 (resistant) and Tianlong 1, Zhongdou40, and Pudou11 (susceptible) were used to map the Rpp gene. Three resistance responses (immune, red-brown, and tan-colored lesion) were observed from the F2 individuals. The segregation follows a ratio of 1(resistance):2(heterozygous):1(susceptible), indicating that the resistance in SX6907 is controlled by a single incomplete dominant gene (designated as Rpp6907). Results showed that Rpp6907 was mapped on soybean chromosome 18 (molecular linkage group G, MLG G) flanked by simple sequence repeat (SSR) markers SSR24 and SSR40 at a distance of 111.9 kb. Among the ten genes marked within this 111.9-kb region between the two markers, three genes (Glyma18g51930, Glyma18g51950, and Glyma18g51960) are nucleotide-binding site and leucine-rich repeat-type genes. These genes may be involved in recognizing the presence of pathogens and ultimately conferring resistance. Based on resistance spectrum analysis and mapping results, we inferred that Rpp6907 is a novel gene different from Rpp1 in PI 200492, PI 561356, PI 587880A, PI 587886, and PI 594538A, or a new Rpp1-b allele.

RNA-Seq Analysis of Differential Gene Expression Responding to Different Rhizobium Strains in Soybean (Glycine max) Roots

The root nodule symbiosis (RNS) between legume plants and rhizobia is the most efficient and productive source of nitrogen fixation, and has critical importance in agriculture and mesology. Soybean (Glycine max), one of the most important legume crops in the world, establishes a nitrogen-fixing symbiosis with different types of rhizobia, and the efficiency of symbiotic nitrogen fixation in soybean greatly depends on the symbiotic host-specificity. Although, it has been reported that rhizobia use surface polysaccharides, secretion proteins of the type-three secretion systems and nod factors to modulate host range, the host control of nodulation specificity remains poorly understood. In this report, the soybean roots of two symbiotic systems (Bradyrhizobium japonicum strain 113-2-soybean and Sinorhizobium fredii USDA205-soybean)with notable different nodulation phenotypes and the control were studied at five different post-inoculation time points (0.5, 7–24 h, 5, 16, and 21 day) by RNA-seq (Quantification). The results of qPCR analysis of 11 randomly-selected genes agreed with transcriptional profile data for 136 out of 165 (82.42%) data points and quality assessment showed that the sequencing library is of quality and reliable. Three comparisons (control vs. 113-2, control vs. USDA205 and USDA205 vs. 113-2) were made and the differentially expressed genes (DEGs) between them were analyzed. The number of DEGs at 16 days post-inoculation (dpi) was the highest in the three comparisons, and most of the DEGs in USDA205 vs. 113-2 were found at 16 dpi and 21 dpi. 44 go function terms in USDA205 vs. 113-2 were analyzed to evaluate the potential functions of the DEGs, and 10 important KEGG pathway enrichment terms were analyzed in the three comparisons. Some important genes induced in response to different strains (113-2 and USDA205) were identified and analyzed, and these genes primarily encoded soybean resistance proteins, NF-related proteins, nodulins and immunity defense proteins, as well as proteins involving flavonoids/flavone/flavonol biosynthesis and plant-pathogen interaction. Besides, 189 candidate genes are largely expressed in roots and\or nodules. The DEGs uncovered in this study provides molecular candidates for better understanding the mechanisms of symbiotic host-specificity and explaining the different symbiotic effects between soybean roots inoculated with different strains (113-2 and USDA205).

Genome-Wide Survey of the Soybean GATA Transcription Factor Gene Family and Expression Analysis under Low Nitrogen Stress

GATA transcription factors are transcriptional regulatory proteins that contain a characteristic type-IV zinc finger DNA-binding domain and recognize the conserved GATA motif in the promoter sequence of target genes. Previous studies demonstrated that plant GATA factors possess critical functions in developmental control and responses to the environment. To date, the GATA factors in soybean (Glycine max) have yet to be characterized. Thus, this study identified 64 putative GATA factors from the entire soybean genomic sequence. The chromosomal distributions, gene structures, duplication patterns, phylogenetic tree, tissue expression patterns, and response to low nitrogen stress of the 64 GATA factors in soybean were analyzed to further investigate the functions of these factors. Results indicated that segmental duplication predominantly contributed to the expansion of the GATA factor gene family in soybean. These GATA proteins were phylogenetically clustered into four distinct subfamilies, wherein their gene structure and motif compositions were considerably conserved. A comparative phylogenetic analysis of the GATA factor zinc finger domain sequences in soybean, Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa) revealed four major classes. The GATA factors in soybean exhibited expression diversity among different tissues; some of these factors showed tissue-specific expression patterns. Numerous GATA factors displayed upregulation or downregulation in soybean leaf in response to low nitrogen stress, and two GATA factors GATA44 and GATA58 were likely to be involved in the regulation of nitrogen metabolism in soybean. Overexpression of GmGATA44 complemented the reduced chlorophyll phenotype of the Arabidopsis ortholog AtGATA21 mutant, implying that GmGATA44 played an important role in modulating chlorophyll biosynthesis. Overall, our study provides useful information for the further analysis of the biological functions of GATA factors in soybean and other crops.

Search for Nodulation and Nodule Development-Related Cystatin Genes in the Genome of Soybean (Glycine max)

Nodulation, nodule development and senescence directly affects nitrogen fixation efficiency, and previous studies have shown that inhibition of some cysteine proteases delay nodule senescence, so their nature inhibitors, cystatin genes, are very important in nodulation, nodule development, and senescence. Although several cystatins are actively transcribed in soybean nodules, their exact roles and functional diversities in legume have not been well explored in genome-wide survey studies. In this report, we performed a genome-wide survey of cystatin family genes to explore their relationship to nodulation and nodule development in soybean and identified 20 cystatin genes that encode peptides with 97–245 amino acid residues, different isoelectric points (pI) and structure characteristics, and various putative plant regulatory elements in 3000 bp putative promoter fragments upstream of the 20 soybean cystatins in response to different abiotic/biotic stresses, hormone signals, and symbiosis signals. The expression profiles of these cystatin genes in soybean symbiosis with rhizobium strain Bradyrhizobium japonicum strain 113-2 revealed that 7 cystatin family genes play different roles in nodulation as well as nodule development and senescence. However, these genes were not root nodule symbiosis (RNS)—specific and did not encode special clade cystatin protein with structures related to nodulation and nodule development. Besides, only two of these soybean cystatins were not upregulated in symbiosis after ABA treatment. The functional analysis showed that a candidate gene Glyma.15G227500 (GmCYS16) was likely to play a positive role in soybean nodulation. Besides, evolutionary relationships analysis divided the cystatin genes from Arabidopsis thaliana, Nicotiana tabacum, rice, barley and four legume plants into three groups. Interestingly, Group A cystatins are special in legume plants, but only include one of the above-mentioned 7 cystatin genes related to nodulation and nodule development. Overall, our results provide useful information or clues for our understanding of the functional diversity of legume cystatin family proteins in soybean nodulation and nodule development and for finding nodule-specific cysteine proteases in soybean.

Identification and Comparative Analysis of CBS Domain-Containing Proteins in Soybean (Glycine max) and the Primary Function of GmCBS21 in Enhanced Tolerance to Low Nitrogen Stress

Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain–containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield.

Genome-Wide Identification and Classification of Soybean C2H2 Zinc Finger Proteins and Their Expression Analysis in Legume-Rhizobium Symbiosis

Root nodule symbiosis (RNS) is one of the most productive and economical systems for nitrogen fixation, and previous studies have shown that several nodule-specific C2H2-zinc finger proteins (ZFPs) play important roles in symbiosis establishment and nodule function. However, C2H2-ZFPs are the most widespread ZFPs in eukaryotes, and a great variation of structure and function exist among the family members. It remains largely unclear whether or not special types of C2H2-ZF genes participate in symbiosis, especially in soybean. In the present study, we performed a genome-wide survey of soybean C2H2-ZF genes, and 321 soybean C2H2-ZF genes were identified and classified into 11 clearly distinguishable subsets (Gm-t1-SF, Gm-t2-SF, Gm-1i-Q-SF, Gm-1i-M-SF, Gm-1i-Z-SF, Gm-1i-D-SF, Gm-2i-Q-SF, Gm-2i-M-SF, Gm-2i-Mix-SF, Gm-3i-SF, and Gm-4i-SF) based on the arrangements, numbers, and types of C2H2-ZF domains. Phylogenetic and gene ontology analyses were carried out to assess the conserved sequence and GO function among these subsets, and the results showed that the classification of soybean C2H2-ZFPs was reasonable. The expression profile of soybean C2H2-ZFPs in multiple tissues showed that nearly half of soybean C2H2-ZFPs within different subsets had expressions in nodules, including a clustering branch consisting of 11 Gm-1i-Q-SF genes specifically expressed in symbiotic-relative tissues. RNA-Seq was used to identify symbiosis-related soybean C2H2-ZFPs, and the expression pattern of the soybean C2H2-ZFPs in roots and nodules at different development stages showed that soybean C2H2-ZFPs mainly played roles in nodule development or nodule function rather than nodulation signal transduction, and nearly half of these genes had high expressions and/or different expression patterns during soybean nodule development, especially for the six clustering branches of genes consisting of different subsets of C2H2-ZFPs. Furthermore, the selected symbiosis-related soybean C2H2-ZFPs might function in legume-rhizobium symbiosis through regulating or interacting with other key proteins. Taken together, our findings provided useful information for the study on classification and conservative function of C2H2-ZFPs, and offered solid evidence for investigation of rhizobium symbiosis-related C2H2-ZFPs in soybean or other legumes.

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Combining QTL and candidate gene analysis with phenotypic model to unravel the relationship between lodging and related traits in soybean

Lodging is one of the major influencing factors of yield and quality in soybean [Glycine max (L.) Merr.] and other crops. To dissect the genetic basis of lodging in soybean, a recombinant inbred line population consisting of 165 lines was used to evaluate lodging percentage and eight related traits (branch number, internode length, number of nodes, plant height, stem diameter, stem strength, root length, and root weight) in three environments. Regression analysis indicated that plant height and root weight, which explain more than 55% of the variation in lodging percentage, might be the key factors influencing lodging in soybean. Nine consensus quantitative trait locus (QTLs) of lodging percentage were detected in one to three environments. Of which, eight consensus QTLs were colocated with 16 consensus QTLs of lodging-related traits by meta-analysis. In addition, seven candidate genes with the biological functions of shoot branching, root development, internode elongation, and lignin biosynthesis were identified on four pleiotropic QTL regions (oq.13-1, oq.13-2, oq.19-2, and oq.19-3) for lodging percentage and related traits. These findings showed that the consensus QTLs of lodging percentage might result from the pleiotropic QTLs affecting the lodging-related traits. Soybean lodging is determined by the cumulative effect of many traits/processes of growth and development. The combination of MAS, statistical model, and phenotypic selection will provide a powerful breeding strategy for lodging resistance in soybean.

Molecular Cloning and Bioinformatics Analysis of K+Transporter Gene (GmKT12) from Soybean (Glycine max[L．] Merri)

GmKT12 predicted as a potassium transporter is of great importance in absorbing nutrients,signal transduction in plant growth.At present,there are few reports about the gene.In this paper,one low K tolerant soybean line You 06-71 and one low K intolerant line Hengchun 04-11 were used as plant materials.In order to research the expression level of GmKT12 in low potassium stress conditions,RNA was extracted and tested by Real-time PCR in eight periods.The results showed that there were highly significant differences between two lines due to their was diversity of amino acid sequence and protein structure.Homology and Bioinformatic analysis after cloning the target sequence from the two lines showed that comparing with GmKT12,there were 56 homologous genes with more than 30 percent similarity,GmKT12 and Glyma18g18822 were nearest in the phylogenetic tree.The protein encoded by GmKT12 was soluble transmembrane protein which contained 11–12 membrane structural domains,and possessed multiple phosphorylation sites.These indicated that the protein might take part in plant signal transduction and regulate potassium ions transportation into or out of cells.In conclusion,GmKT12 might play a pivotal role in potassium absorption in soybean,on which the research is important.