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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Estimating genetic diversity and sampling strategy for a wild soybean (Glycine soja) population based on different molecular markers

Genetic diversity is the basic and most important component of biodiversity. It is essential for the effective conservation and utilization of genetic resources to accurately estimate genetic diversity of the targeted species and populations. This paper reports analyses of genetic diversity of a wild soy-bean population using three molecular marker tech-nologies (AFLP, ISSR and SSR), and computer simulation studies of randomly selected subsets with different sample size (5―90 individuals) drawn 50 times from a total of 100 wild soybean individuals. The variation patterns of genetic diversity indices, including expected heterozygosity (He), Shannon diversity index (I), and percentage of polymorphic loci (P), were analyzed to evaluate changes of genetic diversity associated with the increase of individuals in each subset. The results demonstrated that (1) val-ues of genetic diversity indices of the same wild soybean population were considerably different when estimated by different molecular marker techniques; (2) genetic diversity indices obtained from subsets with different sample sizes also diverged considera-bly; (3) P values were relatively more reliable for comparing genetic diversity detected by different molecular marker techniques; and (4) different diver-sity indices reached 90% of the total genetic diversity of the soybean population quite differently in terms of the sample size (number of individuals) analyzed. When using the P value as a determinator, 30―40individuals could capture over 90% of the total ge-netic diversity of the wild soybean population. Results from this study provide a strong scientific basis for estimating genetic diversity and for strategic conser-vation of plant species.

Establishment of a Core Collection for the Chinese annual wild soybean (Glycine Soja)

With more than 6000 accessions collected from across China, the Chinese National Crop Gene Bank (CNCGB) holds the largest collection of annual wild soybean (Glycine Soja) germplasm in the world. To facilitate the management and utilization of these germplasm collections, it is important to establish a Core Collection. This study compares five sampling strategies, namely random, constant, proportional, logarithmic and genetic diversity-based, to establish a Core Collection for the annual wild soybean germplasm. Among the strategies evaluated, the genetic di- versity-based was found to be the simplest and most efficient. Using hierarchical classification and cluster analysis, in the genetic diversity diversity-based strategy, 652 accessions, accounting for 10.65% of the total 6172 accessions, were se- lected out to represent the total accessions. The established Core Collection has the following features: (i) the 21 de- scriptors observed in the entire collection were compared by the Core Collection, all 18 quality characters of entire collec- tion were preserved by core collection, and coincidence rate of average was 98.4%; (ii) the variant of 13 descriptors of the two collections was very similar, with the coincidence index being 0.96; (iii) the coincidence rate of genetic diversity be- tween the two collections was 81.38% DNA alleles; (iv) the geographic distribution pattern of core collection was the same as the entire collection; (v) molecular marker analysis by 20 SSR primer pairs on 299 accessions showed that the Core Collection covered 83.64% of the entire collection. It thus is concluded that the established Core Collection is rep- resentative and will be a valuable entry point for better evaluation and more efficient utilization of the genetic re- sources available in the annual wild soybean germplasm bank.

寒地野生大豆种质资源游离氨基酸组分分析

旨在对采集自黑龙江省各地区的寒地野生大豆种质资源游离氨基酸组分进行深入分析。以153份寒地野生大豆为试材,利用全自动氨基酸分析仪进行了17种游离氨基酸含量的测定,并对测定结果进行了系统分析比较。结果表明:17种游离氨基酸中,甲硫氨酸变异幅度最大,变异系数为0.469,总游离氨基酸含量为2.069 mg/g,其中精氨酸含量最高,为0.894 mg/g。17种游离氨基酸含量与总游离氨基酸含量之间均为正相关,大部分游离氨基酸之间表现为正相关,仅天冬氨酸与苯丙氨酸以及丝氨酸与精氨酸之间表现为显著负相关。主成分分析提取的前5个主成分累计贡献率达85.15%,第1、2主成分贡献率分别为26.47%和20.55%。以第1、2主成分作图可知,北部地区资源和中南部地区资源之间差异较大,东部地区和西部地区资源之间差异较小。该研究明确了寒地野生大豆中17种游离氨基酸含量变异情况和组分之间的相关性,明确了不同来源地对游离氨基酸含量和组分的影响,为大豆育种应用提供了重要的基础数据。

喷施水杨酸对野生大豆草甘膦耐性的影响

为进一步提高前期筛选到的高耐草甘膦野生大豆材料的抗性,本研究以喷施清水为对照,先后对野生大豆材料进行外源喷施水杨酸(SA)和草甘膦处理,测定过氧化氢(H_(2)O_(2))、超氧阴离子(O_(2)^(-))、莽草酸含量、脂质过氧化水平(丙二醛含量)、叶绿素含量、抗氧化酶活性及5-稀醇式丙酮-莽草酸-3-磷酸合成酶(EPSPS)基因表达等指标。结果表明,与仅喷施草甘膦阳性对照相比,喷施0.5 mmol·L^(-1) SA的2个野生大豆材料H_(2)O_(2)、O_(2)^(-)、莽草酸和丙二醛含量在草甘膦处理后整体显著降低,叶绿素含量、过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性整体显著升高,EPSPS基因表达整体显著上调。上述结果表明,外源喷施SA可提高野生大豆植株过氧化物清除酶活性,进一步提高植株对草甘膦的耐性水平。本研究为提高栽培大豆草甘膦耐性,降低草甘膦对大豆植株损伤提供了参考。

野生大豆内生真菌YD09鉴定及促生耐盐特性研究

为从野生大豆中筛选具有促生抗逆的内生菌,利用组织分离法从野生大豆根中分离内生真菌,根据菌株形态和ITS序列系统发育树对菌株进行鉴定,研究菌株自身耐盐特性、胞内和胞外产物对大豆幼苗生长及耐盐性的影响。结果显示:分离得到1株内生真菌,基于菌株形态学和rRNA基因ITS序列系统发育分析,鉴定为交链孢霉菌(Alternaria alternata),命名为YD09。菌株YD09耐NaCl浓度可达30%。菌株YD09作用于大豆幼苗,具有良好的促生效果,且可使幼苗耐NaCl浓度达到200 mmol·L^(-1)。菌株YD09胞内产物对大豆幼苗具有明显的促生效果,胞外产物能提高大豆幼苗对盐胁迫的抗性。综上,菌株YD09具有较好的促生、耐盐能力,能够缓解盐胁迫对大豆幼苗的伤害,可作为农业生产上应用的新菌种资源。

野生大豆功能基因的研究进展

野生大豆(Glycine soja Sieb.&Zucc.)是栽培大豆(Glycine max [L.] Merr.)的近缘祖先种。在大豆驯化的过程中,栽培大豆丢失了大量的基因或等位变异,导致栽培大豆的遗传多样性降低,这严重限制了栽培大豆品种选育和改良的有效性与丰富性。我国野生大豆种质资源丰富,蕴藏着许多高蛋白含量、抗病虫、耐干旱、耐盐碱等方面的潜力基因,挖掘潜力基因并利用分子设计育种技术应用到现代的栽培大豆品种中,能够有效地拓宽栽培大豆的遗传多样性。本文综述了野生大豆的分布规律和形态特征、近年来在野生大豆中发掘的重要功能基因或位点,包括百粒重、开花期和成熟期、蛋白质和油分含量、抗病、抗虫、耐盐碱、耐干旱等重要农艺性状基因,并讨论这些重要基因或位点在未来栽培大豆育种中的应用潜力,以期为育种家培育和改良大豆新品种提供一种新的育种思路和策略。