Characterization of Pingliang xiaoheidou (ZDD 11047), a soybean variety with resistance to soybean cyst nematode Heterodera glycines.

KEY MESSAGE: A novel QTL (qSCN-PL10) for SCN resistance and related candidate genes were identified in the soybean variety Pingliang xiaoheidou, and plant basal immunity seems to contribute to the SCN resistance. Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most devastating soybean pests worldwide. The development of host plant resistance represents an effective strategy to control SCN. However, owing to the lack of diversity of resistance genes in soybean varieties, further investigation is necessary to identify new SCN resistance genes. By analyzing the resistance phenotypes of soybean variety Pingliang xiaoheidou (Pingliang, ZDD 11047), we found that it exhibited the different resistance phenotypes from PI 88788 and Peking varieties. Because Pingliang variety contains the Rhg1-a (low copy) haplotype and lacks the resistant Rhg4 haplotype, novel quantitative trait locus might account for their SCN resistance. After sequencing parental lines (Magellan and Pingliang) and 200 F2:3 progenies, a high-density genetic map was constructed using the specific length amplified fragment sequencing method and qSCN-PL10 was identified as a novel locus for SCN resistance. Candidate genes were predicted by RNA sequencing (RNA-seq) in the qSCN-PL10 locus region. The RNA-seq analysis performed also indicated that plant basal immunity plays an important role in the resistance of Pingliang to SCN. These results lay a foundation for the use of marker-assisted breeding to enhance the resistance to SCN.

RNA-Seq analysis of nodule development at five different developmental stages of soybean (Glycine max) inoculated with Bradyrhizobium japonicum strain 113-2.

Nodule development directly affects nitrogen fixation efficiency during soybean growth. Although abundant genome-based information related to nodule development has been released and some studies have reported the molecular mechanisms that regulate nodule development, information on the way nodule genes operate in nodule development at different developmental stages of soybean is limited. In this report, notably different nodulation phenotypes in soybean roots inoculated with Bradyrhizobium japonicum strain 113-2 at five developmental stages (branching stage, flowering stage, fruiting stage, pod stage and harvest stage) were shown, and the expression of nodule genes at these five stages was assessed quantitatively using RNA-Seq. Ten comparisons were made between these developmental periods, and their differentially expressed genes were analysed. Some important genes were identified, primarily encoding symbiotic nitrogen fixation-related proteins, cysteine proteases, cystatins and cysteine-rich proteins, as well as proteins involving plant-pathogen interactions. There were no significant shifts in the distribution of most GO functional annotation terms and KEGG pathway enrichment terms between these five development stages. A cystatin Glyma18g12240 was firstly identified from our RNA-seq, and was likely to promote nodulation and delay nodule senescence. This study provides molecular material for further investigations into the mechanisms of nitrogen fixation at different soybean developmental stages.

Genome-wide transcriptional analysis of two soybean genotypes under dehydration and rehydration conditions.

BACKGROUND: Soybean is an important crop that provides valuable proteins and oils for human use. Because soybean growth and development is extremely sensitive to water deficit, quality and crop yields are severely impacted by drought stress. In the face of limited water resources, drought-responsive genes are therefore of interest. Identification and analysis of dehydration- and rehydration-inducible differentially expressed genes (DEGs) would not only aid elucidation of molecular mechanisms of stress response, but also enable improvement of crop stress tolerance via gene transfer. Using Digital Gene Expression Tag profiling (DGE), a new technique based on Illumina sequencing, we analyzed expression profiles between two soybean genotypes to identify drought-responsive genes. RESULTS: Two soybean genotypes - drought-tolerant Jindou21 and drought-sensitive Zhongdou33 - were subjected to dehydration and rehydration conditions. For analysis of DEGs under dehydration conditions, 20 cDNA libraries were generated from roots and leaves at two different time points under well-watered and dehydration conditions. We also generated eight libraries for analysis under rehydration conditions. Sequencing of the 28 libraries produced 25,000-33,000 unambiguous tags, which were mapped to reference sequences for annotation of expressed genes. Many genes exhibited significant expression differences among the libraries. DEGs in the drought-tolerant genotype were identified by comparison of DEGs among treatments and genotypes. In Jindou21, 518 and 614 genes were differentially expressed under dehydration in leaves and roots, respectively, with 24 identified both in leaves and roots. The main functional categories enriched in these DEGs were metabolic process, response to stresses, plant hormone signal transduction, protein processing, and plant-pathogen interaction pathway; the associated genes primarily encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significantly expressed (|log2 ratio| ≥ 8) genes - Glyma15g03920, Glyma05g02470, Glyma15g15010, Glyma05g09070, Glyma06g35630, Glyma08g12590, and Glyma11g16000 - are more likely to determine drought stress tolerance. The expression patterns of eight randomly-selected genes were confirmed by quantitative RT-PCR; the results of QRT-PCR analysis agreed with transcriptional profile data for 96 out of 128 (75%) data points. CONCLUSIONS: Many soybean genes were differentially expressed between drought-tolerant and drought-sensitive genotypes. Based on GO functional annotation and pathway enrichment analysis, some of these genes encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significant DEGs are candidates for improving soybean drought tolerance. These findings will be helpful for analysis and elucidation of molecular mechanisms of drought tolerance; they also provide a basis for cultivating new varieties of drought-tolerant soybean.

Identification of genes associated with nitrogen-use efficiency by genome-wide transcriptional analysis of two soybean genotypes.

BACKGROUND: Soybean is a valuable crop that provides protein and oil. Soybean requires a large amount of nitrogen (N) to accumulate high levels of N in the seed. The yield and protein content of soybean seeds are directly affected by the N-use efficiency (NUE) of the plant, and improvements in NUE will improve yields and quality of soybean products. Genetic engineering is one of the approaches to improve NUE, but at present, it is hampered by the lack of information on genes associated with NUE. Solexa sequencing is a new method for estimating gene expression in the transcription level. Here, the expression profiles were analyzed between two soybean varieties in N-limited conditions to identify genes related to NUE. RESULTS: Two soybean genotypes were grown under N-limited conditions; a low-N-tolerant variety (No.116) and a low-N-sensitive variety (No.84-70). The shoots and roots of soybeans were used for sequencing. 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) responses] and compared the transcriptomes by high-throughput tag-sequencing analysis. 5,739,999, 5,846,807, 5,731,901, 5,970,775, 5,476,878, 5,900,343, 5,930,716, and 5,862,642 clean tags were obtained for the eight libraries: L1, 116-shoot short-term; L2 84-70-shoot short-term; L3 116-shoot long-term; L4 84-70-shoot long-term; L5 116-root short-term; L6 84-70-root short-term; L7 116-root long-term;L8 84-70-root long-term; these corresponded to 224,154, 162,415, 191,994, 181,792, 204,639, 206,998, 233,839 and 257,077 distinct tags, respectively. The clean tags were mapped to the reference sequences for annotation of expressed genes. Many genes showed substantial differences in expression among the libraries. In total, 3,231 genes involved in twenty-two metabolic and signal transduction pathways were up- or down-regulated. Twenty-four genes were randomly selected and confirmed their expression patterns by quantitative RT-PCR; Twenty-one of the twenty-four genes showed expression patterns consistent with the Digital Gene Expression (DGE) data. CONCLUSIONS: A number of soybean genes were differentially expressed between the low-N-tolerant and low-N-sensitive varieties under N-limited conditions. Some of these genes may be candidates for improving NUE. These findings will help to provide a detailed understanding of NUE mechanisms, and also provide a basis for breeding soybean varieties that are tolerant to low-N conditions.