Global analysis of seed transcriptomes reveals a novel PLATZ regulator for seed size and weight control in soybean.

Seed size and weight are important factors that influence soybean yield. Combining the weighted gene co-expression network analysis (WGCNA) of 45 soybean accessions and gene dynamic changes in seeds at seven developmental stages, we identified candidate genes that may control the seed size/weight. Among these, a PLATZ-type regulator overlapping with 10 seed weight QTLs was further investigated. This zinc-finger transcriptional regulator, named as GmPLATZ, is required for the promotion of seed size and weight in soybean. The GmPLATZ may exert its functions through direct binding to the promoters and activation of the expression of cyclin genes and GmGA20OX for cell proliferation. Overexpression of the GmGA20OX enhanced seed size/weight in soybean. We further found that the GmPLATZ binds to a 32-bp sequence containing a core palindromic element AATGCGCATT. Spacing of the flanking sequences beyond the core element facilitated GmPLATZ binding. An elite haplotype Hap3 was also identified to have higher promoter activity and correlated with higher gene expression and higher seed weight. Orthologues of the GmPLATZ from rice and Arabidopsis play similar roles in seeds. Our study reveals a novel module of GmPLATZ-GmGA20OX/cyclins in regulating seed size and weight and provides valuable targets for breeding of crops with desirable agronomic traits.

Genome-Wide Association Study of Partial Resistance to P. sojae in Wild Soybeans from Heilongjiang Province, China.

Phytophthora root rot (PRR) is a destructive disease of soybeans (Glycine max (L.) Merr) caused by Phytophthora sojae (P. sojae). The most effective way to prevent the disease is growing resistant or tolerant varieties. Partial resistance provides a more durable resistance against the pathogen compared to complete resistance. Wild soybean (Glycine soja Sieb. & Zucc.) seems to be an extraordinarily important gene pool for soybean improvement due to its high level of genetic variation. In this study, 242 wild soybean germplasms originating from different regions of Heilongjiang province were used to identify resistance genes to P. sojae race 1 using a genome-wide association study (GWAS). A total of nine significant SNPs were detected, repeatedly associated with P. sojae resistance and located on chromosomes 1, 10, 12, 15, 17, 19 and 20. Among them, seven favorable allelic variations associated with P. sojae resistance were evaluated by a t-test. Eight candidate genes were predicted to explore the mechanistic hypotheses of partial resistance, including Glysoja.19G051583, which encodes an LRR receptor-like serine/threonine protein kinase protein, Glysoja.19G051581, which encodes a receptor-like cytosolic serine/threonine protein kinase protein. These findings will provide additional insights into the genetic architecture of P. sojae resistance in a large sample of wild soybeans and P. sojae-resistant breeding through marker-assisted selection.

A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis.

Soybean (Glycine max) production is severely affected in unfavorable environments. Identification of the regulatory factors conferring stress tolerance would facilitate soybean breeding. In this study, through coexpression network analysis of salt-tolerant wild soybeans, together with molecular and genetic approaches, we revealed a previously unidentified function of a class B heat shock factor, HSFB2b, in soybean salt stress response. We showed that HSFB2b improves salt tolerance through the promotion of flavonoid accumulation by activating one subset of flavonoid biosynthesis-related genes and by inhibiting the repressor gene GmNAC2 to release another subset of genes in the flavonoid biosynthesis pathway. Moreover, four promoter haplotypes of HSFB2b were identified from wild and cultivated soybeans. Promoter haplotype II from salt-tolerant wild soybean Y20, with high promoter activity under salt stress, is probably selected for during domestication. Another promoter haplotype, III, from salt-tolerant wild soybean Y55, had the highest promoter activity under salt stress, had a low distribution frequency and may be subjected to the next wave of selection. Together, our results revealed the mechanism of HSFB2b in soybean salt stress tolerance. Its promoter variations were identified, and the haplotype with high activity may be adopted for breeding better soybean cultivars that are adapted to stress conditions.

Cloning of the soybean E2 ubiquitin-conjugating enzyme GmUBC1 and its expression in Arabidopsis thaliana.

Plant E2 Ubiquitin-conjugating enzymes regulate various biological pathways such as stress resistance, growth and development. Reports on its functions are more frequent in Arabidopsis thaliana, but relatively rare in soybean (Glycine max), which is one of the most important economic crops. In this study, a gene Glyma.12G161200, which may be related to the soybean cotyledon folding mutant, was cloned from soybean "Nanong 94-16". Analysis of its sequence suggested that it encodes an E2 ubiquitin binding enzyme, so it was named as GmUBC1. Its coding region is 462 bp in length, which encodes a protein of 153 amino acids with a predicted molecular mass of 17.25 kDa and an isoelectric point of 6.74. The expression pattern of GmUBC1 in different tissues of soybean and its response patterns to different stresses and hormone treatments were analyzed by real-time PCR. The results showed that the gene was expressed at the highest level in mutant seeds at 40 days after flowering. Moreover, the expression of the GmUBC1 gene was down-regulated by the treatments of PEG, cold, JA and ABA, respectively. Subcellular localization analysis of GmUBC1 revealed that the protein was expressed in the whole cell. When GmUBC1 was ectopically expressed in Arabidopsis, the 1000-grain weight and total amino acid content of some transgenic lines were found to be significantly increased. Collectively, heterologous overexpression of GmUBC1 can regulate seed weights and amino acid contents, which may provide genetic resources for soybean quality improvement.

Functional analysis of flower development related gene GsLFY from Glycine soja.

LEAFY/FLORICAULA (LFY/FLO) is a family of plant-specific transcription factors, which plays an important role(s) in the regulation of floral organ formation and development. So far, LFY regulation on floral development in wild soybean has not been reported in the literature. In this study, the LFY gene, GsLFY, has been isolated from Glycine soja, and characterized with molecular and transgenic techniques. The cDNA for GsLFY gene is 1224 bp in length and contains an open reading frame encoding a polypeptide of 407 amino acids. Quantitative RT-PCR analysis shows that GsLFY is prominently expressed in various tissues, including roots, flowers and seeds. Among the four floral organs, GsLFY is expressed highly in sepals and stamens while weakly in the petals and carpels. Yeast two-hybrid experiments show that GsLFY possesses transactivation activity while transient expression analysis with Arabidopsis thaliana protoplasts shows that GsLFY protein is localized in the nucleus, supporting the notion that GsLFY is a transcription factor. The GsLFY transgenic tobacco plants flower about 29 days earlier than the wild-type plants, thereby providing a potential rationale for developing new soybean varieties with altered flowering time.

[Mapping and cloning of low phosphorus tolerance genes in soybeans].

Soybean is a major source of edible oil and phytoprotein. Low phosphorus available in soil is an important factor limiting the current soybean production. Effective ways to solve the problem include identification of germplasms and genes tolerant to low-phosphorus stress, and cultivation of soybean varieties with high phosphorus efficiency. Recently many researches have been carrying out investigations to map and clone genes related to phosphorus efficiency in soybeans. However, due to the complexity of the soybean genome and little knowledge of functional genes, it has been difficult to understand the mechanism of soybean tolerance to low phosphorus. Although quantitative trait locus (QTL) mapping related to low phosphorus tolerance has made some progress, it remains elusive to obtain accurate candidate genes for molecular breeding applications, due to the limited accuracy of QTL. Even for the cloned soybean low phosphorus tolerance genes, the molecular mechanisms are largely unknown, further limiting the application to breeding. In this review, we summarize the progresses on mapping, cloning and functional characterization of soybean low phosphorus tolerance genes.

[Transgenic technology and soybean quality improvement].

Soybean is an important source of edible oil, protein and protein diet. The breeding process of high quality soybean can be accelerated via employment of transgenic technology, by which the key genes for soybean quality traits could be directly manipulated. Thus, various soybean varieties could be bred to fulfill different needs for specific consumers. Here, we reviewed the contribution of transgenic technology to improvement of soybean qualities in recent years. We also introduce some newly developed safe transgenic technologies and hope this information could relieve some concerns on the GM food.

GmRFP1 encodes a previously unknown RING-type E3 ubiquitin ligase in Soybean (Glycine max).

RING-finger proteins with E3 ubiquitin ligase activity play important roles in the regulation of plant growth and development. In this study, a cDNA clone encoding a novel RING-finger protein, designated as GmRFP1, was isolated and characterized from soybean. GmRFP1 was an intronless gene encoding a predicted protein product of 392 amino acid residues with a molecular mass of ~43 kDa. The protein contained a RING-H2 motif and an N-terminal transmembrane domain. The transcript was observed in all detected organs and was up-regulated by abscisic acid (ABA) and salt stress, but down-regulated by cold and drought treatments. We further expressed and purified both wild type and mutant version of GmRFP1 in E. coli. In vitro assays showed that the purified GmRFP1 induced the formation of polyubiquitin chains while mutation within the RING-finger region abolished the ubiquitination activity. These findings suggest that GmRFP1 is a previously unknown E3 ubiquitin ligase in soybean and that the RING domain is required for its activity. It may play unappreciated roles in ABA signaling and stress responses via mediating the ubiquitination and degradation of target proteins through the ubiquitin-proteasome pathway.

[Cloning and characterization of an aldehyde dehydrogenase gene GmALDH3-1 from soybean].

To research the mechanism of soybean reproductive development, we identified a number of flower development related genes in soybean by microarray hybridization. A gene predominately expressed in soybean flowers was chosen for further analysis. Through bioinformatic and RT-PCR approaches, the full-length gene was cloned from soybean flowers. The results of BLAST searching indicated that this gene encoded for an aldehyde dehydrogenase and was named as GmALDH3-1. GmALDH3-1 contains a complete open reading frame of 1485 bp in length, which encodes for a peptide of 494 amino acids. The product encoded by GmALDH3-1 shows 83% similarity and 68% identity with Populus tomentosa PtALDH3, respectively, and 39% and 59% with human ALDH3B. Phylogenetic analysis shows that GmALDH3-1 and other ALDH3 subfamily members are grouped into the same branch and GmALDH3-1 is close to PtALDH3 and Arabidopsis AtALDH3F1. Real-time RT-PCR analysis demonstrated that the highest expression level of GmALDH3-1 occurred in flowers, but the expression of this gene was almost undetectable in leaves and roots. We further analyzed GmALDH3-1 expression during the course of seed development based on publicly available microarray data and found that GmALDH3-1 was highly expressed in the seed endothelium, epidermis, outer integument, and hilum.

[Mapping QTLs related to oil content of soybeans].

The soybean Recombinant Inbred Lines(RIL), including 133 lines, from the cross Wan82-178 x Tongshan-baopihuangdoujia were used as experimental materials in this study. Based on the linkage map constructed with Single Sequence Repeat(SSR) markers using this RIL population, the software Cartgrapher(V.2.0) and the composite interval mapping were employed to identify quantitative traits loci(QTL) associated with oil content of soybean in 2004 and 2005. It was found that the results of mapping QTL for the oil content were similar for these two years. They were both mapped near satt331 on linkage group wt-11, and they could be used to explain 13.95% and 15.01% of the total variation of the oil content, respectively. In addition, the software QTL Mapper 1.6 was applied to detect QTLs related to oil con-tent in two years. The result indicated that the QTL related to oil content was still mapped near satt331 on linkage group wt-11.

[Mapping insect resistance QTLs of soybean with RIL population].

A recombinant inbred line (RIL) population, NJRIKY, which was derived from the cross Kefeng 1 xNannong 1138-2, was used to constructed the genetic linkage map. Larval weight and pupae weight of cotton worm [Prodenia litura (L.) Fabricius] were examined and used as indicators of resistance. Based on the linkage map constructed with SSR markers of this RIL population, one QTL responsible for larval weight was mapped on linkage group G20-O and the position was 31.91 cM. The QTL's additive effect was 0.0408 and explained 11.74 of the total variation of the larval weight. Two QTLs associated with pupae weight were mapped on linkage group G8-D1b+W and G17-L and the positions were 14.71 cM and 0.01 cM, respectively. The QTLs' additive effects were -0.0139 and 0.0103 ,which explained 11.30 and 6.36 of the total variation of larval weight, respectively.

[Crude extraction and activity assay of CEL I].

CEL I, extracted from celery, is the first known eukaryotic nuclease that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. It is a key enzyme for TILLING research. Here we reported a crude extraction method and activity assay of CEL I. Incision at mismatches of single nucleotide suggested that CEL I can effectively detect DNA at G-->A base substitution and the result can be obtained from an ABI377 Sequencer. Therefore, the extracted enzyme can be used in TILLING.

[Genetic analysis and RAPD marker of the genes for brachytic stem trait in soybean].

Three crosses between NG94-156 (brachytic stem) and three varieties (normal stem) were made, and F2 segregative population and two recombined inbred line populations(F(7:8)) were obtained. Genetic analysis indicated that the brachytic stem of NG94-156 was controlled by two duplicate recessive genes. In searching for RAPD marker linked to the genes controlling brachytic stem, 260 RAPD primers were applied to screen four parents of three combinations and RIL. Polymorphic bands revealed by the primer S-506 exhibited the best repeatability among all primers. Linkage analysis indicated the genetic distance between S-506(1600) and brachytic stem gene was 6.94 cM.

The evolution of soybean mosaic virus: An updated analysis by obtaining 18 new genomic sequences of Chinese strains/isolates.

Soybean mosaic virus (SMV) is widely recognized as a highly damaging pathogen of soybean, and various strains/isolates have been reported to date. However, the pathogenic differences and phylogenetic relationships of these SMV strains/isolates have not been extensively studied. In the present work, by first obtaining 18 new genomic sequences of Chinese SMV strains/isolates and further compiling these with available data, we have explored the evolution of SMV from multiple aspects. First, as in other potyviruses, recombination has occurred frequently during SMV evolution, and a total of 32 independent events were detected. Second, using a maximum-likelihood method and removing recombinant fragments, a phylogeny covering 83 SMV sequences sampled from all over the world was reconstructed and the results showed four separate SMV clades, with clade I and II recovered for the first time. Third, the population structure analysis of SMV revealed significant genetic differentiations between China and two other countries (Korea and U.S.A.). Fourth, certain SMV-encoded genes, such as P1, HC-Pro and P3, exhibited higher non-synonymous substitution rate (dN) than synonymous substitution rate (dS), indicating that positive selection has influenced these genes. Finally, four Chinese SMV strains/isolates were selected for inoculation of both USA and Chinese differential soybean cultivars, and their pathogenic phenotypes were significantly different from that of the American strains. Overall, these findings have further broadened our understanding on SMV evolution, which would assist researchers to better deal with this harmful virus.

[Mapping of five genes resistant to SMV strains in soybean].

Soybean mosaic virus (SMV) is one of the most prevalent pathogens that impact the soybean world widely. Previous reports showed that most of the resistances were controlled by one pair of dominant genes. In this study, a soybean RIL population NJRIKY derived from Kefeng 1 x Nannong 1138-2 was used to study the inheritance of resistance to five SMV strains (Sa,Sc-8, Sc-9, N1, and N3) and mapping of resistant genes. Kefeng 1 is resistant to all five SMV strains while Nannong 1138-2 is susceptible to all five SMV strains. The parents and RIL populations were planted in green house and five SMV strains were inoculated on different populations. The results showed that each ratio of the number of resistant families to that of susceptible families was consistent with 1:1 for the five strains. This indicated that the resistance to each of the five strains was controlled by one dominant gene, respectively. RFLP and SSR markers were used to analyze the RIL population, Mapmaker/Exp 3.0b was used to study the linkage between markers and the resistant genes. Through linkage analysis, Rsa was found linked Rn1, Rn3 and Rsc9 with 21.4 cM, 23.5 cM and 35.3 cM, Rsc8 was found to be linked only Rn1 with 35.8 cM. Multi locus analysis showed that the order and intervals of the five resistance genes were Rsc8-35.8 cM-Rn1-10.3 cM-Rn3-21.5 cM- Rsa-35.8 cM-Rsc9. According to the result of RFLP and SSR analysis, a genetic map was constructed which consisted of 256 markers that covered 3050.9 cM and converged into 22 linkage groups. The five resistance genes were mapped on the linkage group N8-D1b + W. The RFLP markers A691T, K4771, LC5T were found linked to the resistant genes Rn1 and Rn3 with distances of 15.04 cM, 17.82 cM, 15.37 cM, 16.14 cM, 17.82 cM, 16.58 cM.

[Mapping the trait controlled by two duplicate genes in the DH or RIL population].

While there is linkage between molecular marker and trait controlled by two duplicate genes in the DH or RIL population, the recombination rate (RR) between molecular marker and one gene controlling the above trait may be estimated by the maximum likelihood method. Moreover, the standard deviation of RR was also obtained in this paper. Finally, the results from Monte Carlo simulation with 3000 replications showed that the unbiasedness of RR for various sample size and RR was good, and the variation of the estimated value of RR decreased with the increase of sample size or RR.

[Mapping QTLs of soybean root weight with RIL population NJRIKY].

The recombinant inbred line (RIL) population, NJRIKY with 184 families derived from a cross Kefeng No.1 x Nannong1138-2 was used in mapping QTLs of root weight of soybean. Based on the linkage map constructed by Wang, with the software Cartographer V. 1.21 of the composite interval mapping procedure, three QTLs of root weight were mapped on N3-B1 and N6-C2 linkage group. The left telomere distance of rw1 was 66.31cM on N3-B1 linkage group, and those of rw2 and rw3 were 169.91cM and 179.71 cM, respectively, on N6-C2 linkage group. The former was located in A520T approximately ACCCAGO5 and the latter were overlapped with OPW13 and ACGCATO6, respectively. Their LOD values were 10.34,4.01 and 3.15, respectively. The QTLs of the root weight explained 26.3%,9.2% and 6.8% of the total variation, and their additive effects were -0.514,-0.303 and -0.260, respectively.

Proteomic analysis of seed germination under salt stress in soybeans.

Soybean (Glycine max (L.) Merrill) is a salt-sensitive crop, and its production is severely affected by saline soils. Therefore, the response of soybean seeds to salt stress during germination was investigated at both physiological and proteomic levels. The salt-tolerant cultivar Lee68 and salt-sensitive cultivar N2899 were exposed to 100 mmol/L NaCl until radicle protrusion from the seed coat. In both cultivars, the final germination percentage was not affected by salt, but the mean germination times of Lee68 and N2899 were delayed by 0.3 and 1.0 d, respectively, compared with controls. In response to salt stress, the abscisic acid content increased, and gibberellic acid (GA1+3) and isopentenyladenosine decreased. Indole-3-acetic acid increased in Lee68, but remained unchanged in N2899. The proteins extracted from germinated seeds were separated using two-dimensional gel electrophoresis (2-DE), followed by Coomassie brilliant blue G-250 staining. About 350 protein spots from 2-DE gels of pH range 3 to 10 and 650 spots from gels of pH range 4 to 7 were reproducibly resolved, of which 18 protein spots showed changes in abundance as a result of salt stress in both cultivars. After matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of the differentially expressed proteins, the peptide mass fingerprint was searched against the soybean UniGene database and nine proteins were successfully identified. Ferritin and 20S proteasome subunit ß-6 were up-regulated in both cultivars. Glyceraldehyde 3-phosphate dehydrogenase, glutathione S-transferase (GST) 9, GST 10, and seed maturation protein PM36 were down-regulated in Lee68 by salt, but still remained at a certain level. However, these proteins were present in lower levels in control N2899 and were up-regulated under salt stress. The results indicate that these proteins might have important roles in defense mechanisms against salt stress during soybean seed germination.

[Culture of soybean somatic embryo line and stability of microsatellite DNA].

We cultured soybean immature cotyledons to induce somatic embryos and set up soybean somatic embryo lines by culturing the induced somatic embryos in liquid medium on shaker. Regenerated plants of normal fertility were easily obtained with the cultures of various ages by culturing the somatic embryos on differentiation media. DNAs were isolated from the embryogenic cultures after 5, 9, 15 or 17 months' suspension and from 42 plants regenerated from somatic embryos of various culturing ages. 102 SSR markers covering soybean genome almost evenly were chosen to check variation of microsatellite DNA during suspension culture and differentiation. Among the eight DNA samples of soybean somatic embryos of various ages and 42 DNA samples of regenerated plants, there was no any variation of the major bands of the 102 SSR markers during one year's subculturing and differentiation. There were only six weaker subsidiary bands of five SSR markers among the 102 SSR markers added in four of the fifty DNA samples. Two of them happened to the same regenerated plant differentiated from the 9-month embryogenic cultures. Three happened to the two DNA samples from somatic embryos irregular in morphology of the 5-month embryogenic cultures. The last subsidiary band variation happened to a DNA sample of the 17-month embryogenic cultures. The results show that stable microsatellites were maintained during the suspension culture and differentiation while we made the cultures highly embryogenic potential and easy to regenerate.