The identification of presence/absence variants associated with the apparent differences of growth period structures between cultivated and wild soybeans

The cultivated soybean（Glycine max（L.） Merr.） was distinguished from its wild progenitor Glycine soja Sieb.＆ Zucc.in growth period structure,by a shorter vegetative phase（V）,a prolonged reproductive phase（R） and hence a larger R/V ratio.However,the genetic basis of the domestication of soybean from wild materials is unclear.Here,a panel of 123 cultivated and 97 wild accessions were genotyped using a set of 24 presence/absence variants（PAVs） while at the same time the materials were phenotyped with respect to flowering and maturity times at two trial sites located at very different latitudes.The major result of this study showed that variation at PAVs is informative for assessing patterns of genetic diversity in Glycine spp.The genotyping was largely consistent with the taxonomic status,although a few accessions were intermediate between the two major clades identified.Allelic diversity was much higher in the wild germplasm than in the cultivated materials.A significant domestication signal was detected at 11 of the PAVs at 0.01 level.In particular,this study has provided information for revealing the genetic basis of photoperiodism which was a prominent feature for the domestication of soybean.A significant marker-trait association with R/V ratio was detected at 14 of the PAVs,but stripping out population structure reduced this to three.These results will provide markers information for further finding of R/V related genes that can help to understand the domestication process and introgress novel genes in wild soybean to broaden the genetic base of modern soybean cultivars.

Wild soybean(Glycine soja)transcription factor GsWRKY40 plays positive roles in plant salt tolerance

Wild soybean(Glycine soja),a relative of cultivated soybean,shows high adaptability to adverse environmental conditions.We identified and characterized a wild soybean transcription factor gene,GsWRKY40,that promotes plant salt stress.GsWRKY40 was highly expressed in wild soybean roots and was up-regulated by salt treatment.GsWRKY40 was localized in nucleus and demonstrated DNA-binding activities but without transcriptional activation.Mutation and overexpression of GsWRKY40 altered salt tolerance of Arabidopsis plants.To understand the molecular mechanism of GsWRKY40 in regulating plant salt resistance,we screened a cDNA library and identified a GsWRKY40 interacting protein GsbHLH92 by using yeast two-hybrid approach.The physical interaction of GsWRKY40 and GsbHLH92 was confirmed by co-immunoprecipitation(co-IP),GST pull-down,and bimolecular fluorescence complementation(BiFC)techniques.Intriguingly,co-overexpression of GsWRKY40 and GsbHLH92 resulted in higher salt tolerance and lower ROS levels than overexpression of GsWRKY40 or GsbHLH92 in composite soybean plants,suggesting that GsWRKY40 and GsbHLH92 may synergistically regulate plant salt resistance through inhibiting ROS production.qRT-PCR data indicated that the expression level of GmSPOD1 gene encoding peroxidase was cooperatively regulated by GsWRKY40 and GsbHLH92,which was confirmed by using a dual luciferase report system and yeast one-hybrid experiment.Our study reveals a pathway that GsWRKY40 and GsbHLH92 collaboratively up-regulate plant salt resistance through impeding GmSPOD1 expression and reducing ROS levels,providing a novel perspective on the regulatory mechanisms underlying plant tolerance to abiotic stresses.

Screening for Glyphosate Resistant Wild Soybean(Glycine soja) and Study on Its Physiological Mechanisms of Resistance

[Objective] This study aimed to preliminarily determine the physiological mechanism of glyphosate resistance produced in wild soybean（Glycine soja） and further provide a basis for the breeding of glyphosate-resistant soybean. [Method] First,a screening for glyphosate resistant varieties among sixty-seven wild soybean materials was done in a field trial; subsequently, physiological indexes of the screened resistant variety ZYD0685 and the sensitive variety ZYD0790 were studied. [Result]At the glyphosate dose of 1.23 kg a.i/hm2, glyphoaste resistance varied greatly among different wild soybean materials, with the highest survival rate of 87% and83% occurring in ZYD0685 and ZYD2405, respectively, and that of another seven accessions ranged from 2.7% to 38%, and all the remaining fifty-eight soybean materials died. After treatment with glyphoaste at different doses, there were no significant differences in chlorophyll content and shikimate content in the resistant ZYD0685, but there was an evident increase in the activity of gultathione-S-transferases（GSTs）; while in the sensitive ZYD0790, the content of shikimic acid increased significantly, and chlorophyll content decreased significantly, and GSTs activity revealed a slight change. [Conclusion] Therefore, lowering the amount of accumulated shikimic acid is the major physiological response to glyphosate in wild soybean.

Identification of Endophytic Bacillus BHL3501 from Wild Soybean and Its Inhibitory Effect against Soybean Cyst Nematode (Heterodera glycines Ichinohe)

[ Objective] The paper was to identify endophytic bacillus BHL3501 from wild soybean and its inhibitory effect against soybean cyst nematode. [ Meth- od] Through morphological characteristic, physiological and biochemical characteristics and 16S rDNA analysis, a strain of endophytic bacterium BHL3501 with ne- maticidal activity isolated and screened from the root of wild soybean was identified, the effects of BHL3501 metabolite solutions with different dilution multiples on egg hatching and activity of 2rd instar juvenile of soybean cyst nematode were also studied. [ Result ] BHL3501 strain was preliminarily identified to be Bacillus sp.. The relative inhibitory rate of its original fermentation broth on egg hatching of soybean cyst nematode after 24 h was 98.3% ; the relative inhibitory rate under 10- time diluted solution treatment was 79.5%, which had significant difference with sterile water control. The corrected mortality rate of 24 instar juvenile was 93.1% after treated by original fermentation broth for 24 h, the treatments of all diluted solutions had significant difference with sterile water control. [ Conclusion ] BHL3501 metabolites had strong inhibitory effect against egg hatching of soybean cyst nematode, which also had strong toxic effect on its 2nd instar juvenile.

Research Progress of Wild Soybean Germplasms and Utilization

Wild soybean （Glycine soja） is the wild relative species of cultivated soy- bean, which is of high protein content, strong tolerance and high propagation coeffi- cient. The diverse growing conditions of wild soybean in China contribute to numer- ous phenotypes of wild soybean, which also reflect the evolution process through long-term natural and artificial selection. The research progress of wild soybean germpfasms was summarized in resource status and innovative utilization in order to provide information for research of wild soybean germplasms.

Sexual compatibility of transgenic soybean and different wild soybean populations

The introduction of genetically modified(GM) soybean into farming systems raises great concern that transgenes from GM soybean may flow to endemic wild soybean via pollen. This may increase the weediness of transgenic soybean by increasing the fitness of hybrids under certain conditions and threaten the genetic diversity of wild soybean populations. Although pollen-mediated gene flow between GM crops and wild relatives is dependent on many factors, the sexual compatibility(SC)determined by their genetic backgrounds is the conclusive factor. The considerable genetic variation among wild soybean populations may cause compatibility differences between different wild and cultivated soybeans. Thus, an evaluation of the SC between transgenic soybean and different wild soybeans is essential for assessing the environmental consequences of cultivated soybean–wild soybean transgene flow. The podding and seed sets were assessed after artificial hybridization using transgenic glyphosate-resistant soybean as the paternal parent and 18 wild soybean populations as the maternal parents. Then, the average number of filled seeds produced in 200 flowers(AFS) was calculated for each wild soybean under natural self-pollination as well as under artificial crossing with transgenic soybean. Finally, the index of cross-SC was calculated(ICSC) as the ratio of the AFS of wild soybean artificially crossed with transgenic soybean and the AFS of naturally self-pollinated wild soybean. The results demonstrated that after self-pollination and crossing with transgenic soybean, the average podding rates of 18 wild soybean populations ranged within 96.50–99.50% and 4.92–18.03%, and the average filled seed numbers per pod varied from 1.70 to 2.69 and 0.20 to 0.48, respectively. The results showed that approximately 89% of wild soybeans displayed either medium or higher than medium SC with transgenic soybean(ICSC>1.0%). This implied the high possibility of gene flow via pollen from transgenic soybean to wild soybean.

Wild Soybean Germplasm Resources Conservation and Use in Shandong Province

Glycine soja or wild soybean is the wild ancestor of the soybean, being an important source for property improvement and variety breeding. With abundant wild soybeans, their habitat is undermining, and wild soybeans are facing increasing- ly threatened in Shandong if measures are not taken yet. Besides, wild soybean germplasm is far from further exploiting or using. The research analyzed status quo and problems of wild soybean germplasm posed countermeasures or suggestions in crop germplasm conservation and use. resources conservation and use and pro- order to provide references for agricultural crop germplasm conservation and use.

Gene flow from transgenic roundup-ready soybean to wild soybean

A study was conducted in the field of the Institute of Vegetable Crops, Jiangsu province from July 2000 to August 2003. The transgenic roundup-ready soybean was sown in the middle of the field in a circular manner for 5 circles, with the distance of 3 m, from one circle to another. Then the wild soybean was planted in plots as the rays of the circles in 8 directions (N, E, W, S, NE, NW, SE and SW), spaced every 5 m until 50 m. Each plot comprised 25 plants. In the second year, the wild soybean seeds from the first year were planted in the field together with the original wild soybean as check. Before flowering time, high concentrations of roundups (about 4-5 times of the normal dose) were sprayed on the plants and the surviving plants were identified. The leaves were taken to the lab for DNA extraction to determine the unique DNA for roundup-ready soybean (CTAB method). About 2% of the plants survived, but some leaves were yellow. One plant of wild soybean was found to have the roundup-ready gene from the original roundup-ready soybean. The other surviving wild soybeans should also had some fragments of the roundup tolerance gene. However, the DNA bands were not very clear in the PCR map.

Linkage and association mapping of wild soybean(Glycine soja)seeds germinating under salt stress

Salinity threatens soybean germination,growth and production.The germination stage is a key period in the life of soybean.Wild soybean contains many genes related to stress resistance that are valuable resources for the genetic improvement of soybean.To identify the genetic loci of wild soybean that are active during seed germination under salt stress,two populations,a soybean interspecific hybrid population comprising 142 lines and a natural population comprising 121 wild soybean accessions,were screened for three germination-related traits in this study.By using single-nucleotide polymorphism(SNP)markers with three salt tolerance indices,25 quantitative trait loci(QTLs),21 significant SNPs(–log_(10)(P)≥4.0)and 24 potential SNPs(3.5<–log_(10)(P)<4.0)were detected by linkage mapping and a genome-wide association study(GWAS)in two environments.The key genetic region was identified based on these SNPs and QTLs.According to the gene functional annotations of the W05 genome and salt-induced gene expression qRT-PCR analysis,GsAKR1 was selected as a candidate gene that responded to salt stress at the germination stage in the wild soybean.These results could contribute to determining the genetic networks of salt tolerance in wild soybean and will be helpful for molecular marker-assisted selection in the breeding of salt-tolerant soybean.

Influences of Temperature Regime on Germination of Seed of Wild Soybean(Glycine soja)

As an important plant species with high protein contents,wild soybean(Glycine soja),has drawn much attention and appeared to be useful for the genetic improvement of soybean germplasms.Since temperature is one of the numerous environmental factors affecting the germination of most plants,an experimental study was carried out to determine the effect of temperature on germination of wild soybean(G.soja)seeds.Germination test was conducted by setting up thirty-six constant and alternating temperature regimes,ranging from 5 to 40 ℃(16 h night/8 h day).Responses in germination rate to these temperature regimes were then used to construct a quadratic response surface,giving estimated germination rates with confidence intervals at P ≤ 0.05.The results showed that germination capacity was significantly greater while exposed to constant temperatures of 25 ℃,and under the alternating temperature regime the optimum temperature occurred at the 20/25,25/25,25/30 ℃ regime(16 h/8 h)with the amplitude widened from 0 to 5 ℃.Together with regional monthly climate data,these results could be used to improve and promote the cultivation of wild soybean(G.soja),making it available to develop the location-specific optimum seeding time and to apply weed-control treatments.

Evaluation and Utilization Methods of Wild Soybean (Glycine Soja)Germplasms

Soybean is one of the world's most important crops,which provids a large amount of protein and fat for the people.For such an important crop,it's breeding still depends upon a relatively narrow base of genetic resources(Delannay et al.,1983,Lohnes & Bernard,1991,Zhang,1987),so itrestricts the improvement of soybean yield.quality and resistance.Wild soybeans are of great interest as a potential genetic resource(Hadley & Hymowitz,1973),therefore,studing and using them to create new germplasms have been becoming an important program in soybean breeding.

Comparative Transcriptome Profiling of Glycine soja Roots Under Salinity and Alkalinity Stresses Using RNA-seq

Saline-alkaline stress can dramatically inhibit plant growth and limit crop production. Wild soybean （Glycine soja） is a crop that adapts well to such environmental stresses. In this study, RNA-sequencing technology was used to analyze the transcriptome profles of G. soja roots subjected to 50 mmol·L^-1 NaHCO3 and 150 mmol·L^-1 NaCl treatments. Totally, 2 125 differentially-expressed genes （DEGs） after NaCl treatment and 1 839 DEGs after NaHCO3 treatment were identifed. The top 14 DEGs revealed by RNA-seq were analyzed using qRT-PCR （quantitative real-time polymerase chain reaction）. Gene ontology （GO） annotation showed that most of DEGs under salt and alkali stresses were enriched in ＂metabolic process＂, ＂catalytic activity＂ and ＂binding＂ terms. To search for transcription factors （TFs） among DEGs, the data were screened against TF database PlantTFDB, and it was found that fve TF families, Apetala2/ethylene-responsive element binding proteins （AP2-EREBP）, V-myb avian myeloblastosis viral oncogene homolog （MYB）, WRKYGQK and Zinc fnger motif （WRKY）, NAM, ATAF1/2, CUC1/2 （NAC） and Cys2/His2 （C2H2） were involved in salt stress response. Other fve TF families, NAC, WRKY, MYB, AP2-EREBP and bZIP were involved in response to alkali stress. These two stress treatments shared NAC, WRKY, AP2-EREBP and MYB, and the only two different TFs were bZIP and C2H2. Forty-eight MYB TFs were differentially expressed under salt and alkali stresses, and most of them were up-regulated. This study provided useful information for further investigation of DEGs and TFs in response to saline and alkaline stresses and helped in understanding the molecular basis of the response of G. soja to saline and alkaline stresses.

Circadian Rhythm of Root＇s Apical Meristem Mitosis Cells of Soybean

The circadian rhythms of apical meristem mitosis cells of the two types of soybean were investigated： wild soybeans - Glycine max （L.） Merr. （G. hispida Max.）, G. soja Sieb., and Zucc （G, ussuriensis Reg. and Maack）; and the hybrids of their homozygous forms （F6）. Representatives of the initial kinds and interspecific hybrids for reproduction were grown up in comparable conditions. Seeds of a soybean were grown up at 24℃ in Petri cups in thermostat in darkness during winter. For research of mitotic meristem cages activity the tips of roots were fixed temporal by acetoalcohol （fractional fixing） from 1 p.m. till 12 a.m., further up to 12 p.m. each 60 minutes. Acetokarmin was used for preparing the coloring. For researching of mitotic indexes, there have been analyzed from 6 up to 10 thousand cells at each stage （during each moment of time： 01 hour, 02, 03 06 ... 12, 13 ... 18 hours and further）. Within a day （day ＋ night） in cells of root apical meristem, three mitosis ＂waves＂ in each of the investigated forms with an absolute starting point of rest at 12 p.m., with precisely expressed further periodicity were found out. Each of the investigated forms misses in character of wave processes, on a degree of increase and recession of mitosis waves, on the level of mitotic indexes. Maximal mitotic activity at all the investigated forms is noted at 6 and 12 a.m. and 6 p.m.

Identification of MicroRNAs in Wild Soybean (Glycine soja)

MicroRNAs （miRNAs） play important roles in post-transcriptional gene silencing by directing target mRNA cleavage or translational inhibition. Currently, hundreds of miRNAs have been identified in plants, but no report has been published of wild soybean （Glycine soja Sieb）. We constructed a small-RNA library consisting of 2 880 sequences with high quality, in which 1 347 were 19-24 nt in length. By utilizing the miRNA, Rfam and domesticated soybean expressed sequence tag database, we have analyzed and predicted the secondary structure of these small RNAs. As a result, t5 conserved miRNA candidates belonging to eight different families and nine novel miRNA candidates comprising eight families were identified in wild soybean seedlings. All these miRNA candidates were validated by northern blot and the novel candidates expressed in a tissue-specific manner. Furthermore, putative target genes were predicted for novel miRNA candidates and two of them were verified by 5＇-rapid amplification of cDNA ends experiments. These results provided useful information for miRNA research in wild soybean and plants.

Sampling strategy for wild soybean(Glycine soja)populations based on their genetic diversity and fine-scale spatial genetic structure

A total of 892 individuals sampled from a wild soybean population in a natural reserve near the Yellow River estuary located in Kenli of Shandong Province(China)were investigated.Seventeen SSR(simple sequence repeat)primer pairs from cultivated soybeans were used to estimate the genetic diversity of the population and its variation pattern versus changes of the sample size(sub-samples),in addition to investigating the fine-scale spatial genetic structure within the population.The results showed relatively high genetic diversity of the population with the mean value of allele number(A)being 2.88,expected heterozygosity(He)0.431,Shannon diversity index(I)0.699,and percentage of poly-morphic loci(P)100%.Sub-samples of different sizes(ten groups)were randomly drawn from the population and their genetic diversity was calculated by computer simulation.The regression model of the four diversity indexes with the change of sample sizes was computed.As a result,27-52 individuals can reach 95%of total genetic variability of the population.Spatial autocorrelation analysis revealed that the genetic patch size of this wild soybean population is about 18 m.The study provided a scientific basis for the sampling strategy of wild soybean populations.