基于大豆胞囊线虫病抗性候选基因的SNP位点遗传变异分析

以我国363份栽培和野生大豆资源为材料, 对大豆胞囊线虫抗性候选基因(rhg1和Rhg4)的SNP位点(8个)进行遗传变异分析, 以期阐明野生和栽培大豆间遗传多样性及连锁不平衡水平差异。结果表明, 与野生大豆相比, 代表我国栽培大豆总体资源多样性的微核心种质及其补充材料的连锁不平衡水平较高(R²值为0.216)。在栽培大豆群体内, 基因内和基因间分别有100％和16.6％的SNP位点对连锁不平衡显著, 形成两个基因特异的连锁不平衡区间(Block)。在所有供试材料中共检测到单倍型46个, 野生大豆的单倍型数目(27)少于栽培大豆(31), 但单倍型多样性(0.916)稍高于栽培大豆(0.816)。单倍型大多数(67.4％)为群体所特有(31个), 其中15个为野生大豆特有单倍型。野生大豆的两个主要优势单倍型(Hap_10和Hap_11)在栽培大豆中的发生频率也明显下降, 推测野生大豆向栽培大豆进化过程中, 一方面形成了新的单倍型, 另一方面因为瓶颈效应部分单倍型的频率降低甚至消失。     

Nucleotide sequences encoding glycinin B4 polypeptide of the cultivated soybean Glycine max and its presumed wild-type ancestor Glycine soja in connection with the origin of cultivated soybean

Nucleotide sequences of cDNAs encoding soybean glycinin B4 polypeptide were compared in three soybean cultivars and two plant introductions of wild soybean Glycine soja. Only two nucleotide substitutions were found in three cultivars G. max, as compared with G. max and G. soja having nucleotide sequences which contain four nucleotide substitutions. These data serve as additional evidence, at molecular level, indicating the origin of G. max from G. soja. On the other hand, the time period required for four nucleotide substitutions' accumulation, as calculated from parameters of molecular evolution of 11S globulins, is much longer than the term having passed after soybean domestication.     

Proteomic and genomic characterization of Kunitz trypsin inhibitors in wild and cultivated soybean genotypes

In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.     

大豆优异种质资源的利用与创新

黑龙江省农科院利用已获得的具有野生大豆(含半野生大豆)多花荚、多分枝、高蛋白等有益性状基因的优异种间杂交新种质(G.max×G.soja)与栽培大豆(G.max)回交,一方面继续拓宽大豆育种遗传基础,另一方面改善现有种间杂交种质的农艺性状和品质,并提高产量水平,已选育出外贸制纳豆、豆芽、制酱及青瓤黑豆等特用大豆品种(系).实践表明,利用野生大豆及种间杂交新种质,对拓宽大豆育种遗传基础,提高大豆育成品种水平,尤其对特用大豆品种(系)的选育是有效可行的.     

Studies on rDNA ITS1 Regions of Soybean and Its Wild Relatives

The rDNA internal transcribed spacer 1 (ITS1) regions of wild soybean (Glycine soja), semiwild soybean (G. gracillis), perennial wild soybean (G. tomentella, G. tabacina) and two accessions of cultivated soybean (G. max) were amplified by PCR and cloned. The copy number in soybean genome was about 2 × 103. Sequence analysis showed that the G/C content of G. soja (61.40%), G. gracillis (61.40%), G. max (61.40%), G. tabacina (58.11%) and G. tomentella (59.01%) were very similar to that of Phaseolus radiatus (59.81%), and the G/C content of G. tabacina was the lowest one in all known ITS1 re- gions of plants. Maxium-homology analysis proved that the ITS1 sequence of soybean was the most homologous to its counterpart of P. radiatus. It was implied that the homology of ITS1 regions of relative species were related with the genetic relationships among these species. Sequence analysis disclosed that there were two conserved sequences (GACCCGC- GAA) and (GCGCCAAGGAA) in all sequenced ITS1 regions of plants.     

Investigation of genetically modified soybean biosafety in the center of the origin and diversity of Russian Far East

Wild soybean (Glycine soja Sieb. & Zucc.) is the nearest relative of a soybean crop (Glycine max (L.) Merr.). Study of population genetic structure of wild-growing relatives ofgenetically modified (GM) plants in the centers of their origin is one of the main procedures before introduction of GM crops in these areas. We have studied genetic variability of nine wild growing soya populations of Primorye Territory using RAPD analysis. The level of G. soja genetic variability was considerably higher than that of G. max. We have analyzed phylogenetic relationships in the genus Glycine subgenus Soja using RAPD markers. Our data confirm validity of allocation G. gracilis in a rank of a species.     

Importance of genetic characterization and conservation of plant genetic resources: The breeding system and genetic diversity of wild soybean (Glycine soja)

Abstract Plant genetic resources play an important role in the improvement of cultivated plants. To characterize and evaluate the ecological and reproductive features of wild soybean ( Glycine soja Sieb. et Zucc.), which is the most probable ancestor of cultivated soybean ( G. max (L) Merr.), the breeding system and genetic diversity of G. soja were investigated. The extent of natural cross-pollination of G. soja was estimated in four populations along the Omono River in Akita Prefecture, Japan by examining allozyme variation. Although it has been previously believed that G. soja is autogamous, as is cultivated soybean, the mean multilocus outcrossing rate ( t m) estimate was 13%. These values are much higher than the outcrossing rate previously reported for both G. soja and G. max . Frequent visits by honeybees and carpenter bees to flowers were also observed, which supported this conjecture. Furthermore, to evaluate the genetic variation of G. soja as a genetic resource, the genetic structure of 447 populations over Japan were analyzed. Wild soybean populations had a higher degree of variation of isozyme loci. The G ST coefficient of gene differentian values among the sites within the district were particularly high, revealing that the isozyme genotype was greatly different among site populations and homogeneous within the sites. The genetic differentiation among nine districts was observed in the allele frequencies of a few loci, indicating that geographic isolation in the wild soybean population was effectively created through the distance between the districts. The difference in the allele frequency among the districts may be produced under genetic drift. Finally, the importance of the preservation of natural plant populations and the habitats of wild progenitors (i.e. the in situ conservation of plant genetic resources) was emphasized.     

野生大豆rbcS基因的克隆及结构分析

核酮糖１,５二磷酸羧化酶（Ｒｕｂｉｓｃｏ,Ｅ．Ｃ．４．１．１．３９）是光合碳代谢中的关键酶,也是植物中研究最为广泛深入的一种酶。高等植物的Ｒｕｂｉｓｃｏ大、小亚基分别由叶绿体和核基因组编码。迄今已有几十种光合生物的Ｒｕｂｉｓｃｏ大、小亚基的基因（ｒｂｃＬ、ｒｂｃＳ）结构得到阐明［１］。在高等植物中ｒｂｃＳ基因由多基因家族编码,结构较为复杂,但它同时又是一种相对保守的基因,且同一物种内各ｒｂｃＳ基因成员是协同进化的,因此ｒｂｃＳ基因适合于植物分子进化及系统分类的研究［２］。我国是栽培大豆（Ｇｌｙｃ…     

Cloning and Structure Analysis of rbcS Gene from Wild Soybean

About 1 kb fragment of rbcS (ribulose 1, 5-bisphosphate carboxylase small subunit) gene in wild soybean (Glycine soja, Ji 50017) was amplified from total DNA by PCR assay. Sequence analysis of the fragment indicated that 1089 bp sequenced included the whole coding region for Rubisco small snbunit. The rbcS gene in wild soybean encoded a precursor composed of a transit peptide of 55 amino acids and a mature protein of 123 amino acids. There were two introns found in the rbcS gene as other dicotyledonous species previously sequenced. Comparison of DNA sequences showed high degree homology of rbcS genes between wild soybean and cultivated soybean (Glycine max var. wayne). Some changes of amino acids emerged from the diverse nucleotides did not affect the function of the small subunit. These results may contribute some basic data in molecular biology to study the origin and evolution of soybean.     

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.     

Resistance of Glycine species and various cultivated legumes to the soybean aphid (Homoptera: Aphididae)

The soybean aphid, Aphis glycines Matsumura, is a new pest of soybean, Glycine max (L.) Merr., in North America. It has become widespread on soybean in North America since it was first identified in the Midwest in 2000. Species of Rhamnus L. (buckthorn) are the primary hosts of A. glycines, and soybean is known as a secondary host. There is limited information about the secondary host range of A. glycines. Aphid colonization on various legume hosts was compared in choice experiments. Aphid colonization occurred on species in the genus Glycine Wild. No colonization occurred on Lablab purpureus (L.) Sweet, Lens culinaris Medik, Phaseolus vulgaris L., Pisum sativum L., or species of Vicia L. and Vigna Savi. Colonization was limited or aphids were transient on species of Medicago L., Phaseolus L., and Trifolium L. There were significant differences in aphid colonization among Medicago truncatula accessions with numbers ranging from 7 to 97 aphids per plant. Six Glycine soja Sieb. & Zucc. accessions were as resistant as G. max accessions to A. glycines; these may represent novel sources of A. glycines resistance not found in G. max. Antibiosis was found to play a large role in the expression of resistance in three of the G. soja accessions. Results of this study indicated that G. max and G. soja were the best secondary hosts of A. glycines; however, its secondary host range may include other leguminous species. Therefore, A. glycines did not seem to have a highly restricted monophagous secondary host range.     

盐生野大豆的异黄酮积累及其生态学意义

以自然生长在盐碱地上的野大豆(Glycine soja)和不耐盐的栽培大豆(G. max)为材料,测定了它们在不同盐度条件下叶片、根部和种子的异黄酮含量,并测定了它们叶片的L-苯丙氨酸含量和苯丙氨酸裂解酶(PAL)活性,还测定了它们根部的结瘤量和固氮酶活性。通过两者比较,分析了它们的大豆异黄酮代谢和盐渍环境的关系。结果表明:盐渍处理不抑制盐生野大豆PAL酶的活性,其大豆异黄酮大量积累;相反,盐渍处理明显抑制栽培大豆PAL酶活性,其大豆异黄酮含量减少,而大豆异黄酮合成前体L-苯丙氨酸积累。结果还显示:在盐渍条件下,盐生野大豆根部异黄酮积累的同时,其根瘤结瘤量较多,且固氮酶活性也较高;而栽培大豆随着其根部异黄酮的减少,其根瘤结瘤量大大减少,且固氮活性大大下降。野大豆和栽培大豆的这些差别说明:盐生野大豆积累大豆异黄酮有其生态学意义,这很可能是野大豆通过异黄酮次生代谢途径适应盐渍环境的一种重要机制。     

大豆种质资源SRAP分子标记中的引物筛选

以113个大豆栽培品种和20个野生品种为材料,从288对引物组合中筛选出12对多态性丰富、条带清晰、可重复性好的SRAP引物组合。用筛选出的12对引物组合对大豆品种进行PCR扩增,获得了带型丰富和清晰可辨的DNA的PAGE指纹图谱;共扩增出251条谱带,其中多态性条带220条,多态性谱带比率为87.6%,平均每个引物扩增出18.3条谱带。结果显示,所筛选出的12对引物组合可以有效的应用于大豆种质资源的SRAP分析。     

Multigene family for Bowman-Birk type proteinase inhibitors of wild soja and soybean: the presence of two BBI-A genes and pseudogenes

Genes for Bowman-Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.     

Differential sensitivity to chloride and sodium ions in seedlings of Glycine max and G. soja under NaCl stress

High Na+ and Cl- concentrations in soil cause hyperionic and hyperosmotic stress effects, the consequence of which can be plant demise. Ion-specific stress effects of Na+ and Cl- on seedlings of cultivated (Glycine max (L.) Merr) and wild soybean (Glycine soja Sieb. Et Zucc.) were evaluated and compared in isoosmotic solutions of Cl-, Na+ and NaCl. Results showed that under NaCl stress, Cl- was more toxic than Na+ to seedlings of G. max. Injury of six G. max cultivars, including 'Jackson' (salt sensitive) and 'Lee 68' (salt tolerant), was positively correlated with the content of Cl- in the leaves, and negatively with that in the roots. In subsequent research, seedlings of two G. max cultivars (salt-tolerant Nannong 1138-2, and salt-sensitive Zhongzihuangdou-yi) and two G. soja populations (BB52 and N23232) were subjected to isoosmotic solutions of 150mM Na+, Cl- and NaCl, respectively. G. max cv. Nannong 1138-2 and Zhongzihuangdou-yi were damaged much more heavily in the solution of Cl- than in that of Na+. Their Leaves were found to be more sensitive to Cl- than to Na+, and salt tolerance of these two G. max cultivars was mainly due to successful withholding of Cl- in the roots and stems to decrease its content in the leaves. The reverse response to isoosmotic stress of 150 mM Na+ and Cl- was shown in G. soja populations of BB52 and N23232; their leaves were not as susceptible to toxicity of Cl- as that of Na+. Salt tolerance of BB52 and N23232 was mainly due to successful withholding of Na+ in the roots and stems to decrease its content in the leaves. These results indicate that G. soja have advantages over G. max in those traits associated with the mechanism of Cl-tolerance, such as its withholding in roots and vacuoles of leaves. It is possible to use G. soja to improve the salt tolerance of G. max.     

Superoxide Dismutase (SOD)Zymogram Patterns and Their Geographical Distribution of Wild (Glycine soja) and Cultivated Soybean (G. max) in China

The purpose of this study was to examine the superoxide dismutase (SOD) zymogram patterns, their frequency and geographical distribution of wild (Glycine soja) and cultivated soybean (G. max) in China. Seeds of 226 wild soybean germplasms and 104 cultivated soybean cultivars (land races) were collected from all provinces and autonomous regions in China except Taiwan, Xinjiang and Qinghai provinces About 50 embryos per wild soybean germplasm and I0 embryos per cultivated soybean cultivars were used for test. Vertical polyacrylamide gel electrophoresis and a stainning system modified after Luo (1984)were used. The Japanese GS- 930 Scanner was used in gel-plate scanning. In program scanning the maximum and minimum absorption wavelength were 700 and 550 nm respectively. The results showed that: 1. Six zymogram patterns were found in soybean (Fig. 1, 2). Wild soybean displayed five patterns (Ⅰ, Ⅱ, Ⅳ Ⅴ, Ⅵ), while the cultivated soybean displayed only two patterns (Ⅱ, Ⅲ). 2. Fourty six percent of wild germplasms gave an 7-band zymogram (Table Ⅰ) (pattern Ⅰ), fourty nine percent had a 6th and 7th band with faster mobility (pattern Ⅱ), about two percent produced a 6-band zymogram which lacked the SODc4 band (pattern Ⅳ), about two percent had a 5-band pattern which lacked the SODc,c4 bands (pattern Ⅴ), and only one germptasm displayed a 5-band zymogram which lacked SODb2b3 bands (pattern Ⅵ). 3. More than ninty eight percent of cultivated cultivars belonged to pattern Ⅱ, only about two percent belonged to pattern Ⅲ. 4. The geographical distribution of frequency of pattern Ⅱ between wild and cultivated soybean was most close in 36–51º N area. The difference of zymograms between G. soja and G. max, and the problems of the origional area and evolution of soybean were discussed.     

Diversity of chloroplast DNA SSRs in wild and cultivated soybeans: evidence for multiple origins of cultivated soybean

Soybean [ Glycine max (L.) Merr.] is one of the major crops in the world and was domesticated from a wild progenitor, Glycine soja Sieb. & Zucc., in East Asia. In order to address the questions concerning the evolution and maternal lineage of soybean, we surveyed the variation in chloroplast DNA simple sequence repeats (cpSSR) of 326 wild and cultivated soybean accessions that were collected from various Asian countries. Twenty-three variants were detected at six cpSSRs in the accessions tested. All of the variants were found in wild soybean, whereas only 14 variants existed in the cultigen. Combining the variants at the six cpSSRs gave 52 haplotypes in the former and eight haplotypes in the latter. Both analyses indicated a considerably higher genetic diversity in the wild soybean. Around 75% of the cultivated accessions tested possessed a common haplotype (no. 49), which was detected in only seven wild accessions, six from southern Japan and one from southern China. The predominant haplotype in the cultigen may therefore have originated from a rare haplotype of the wild soybean that is presently distributed in the southern areas of Japan and China. The remaining seven haplotypes in the cultigen were distributed regionally, and except for three rare haplotypes, largely overlapped with the distributions of wild accessions with the same respective haplotypes. Our results strongly suggest that the cultivated soybeans with different cpDNA haplotypes originated independently in different regions from different wild gene pools and/or hybrid swarms between cultivated and wild forms.     

大豆炸荚发生规律及分子遗传基础

炸荚是野生大豆繁衍后代的一种原始自然属性,同时也是栽培大豆减产的主要原因之一,因此对其发生规律和分子遗传基础的研究具有重要的理论意义和潜在的育种应用价值。文章在剖析抗炸荚大豆荚部细胞学微观组织结构特征的基础上,总结了大豆炸荚的发生规律和大豆炸荚表型性状的鉴定指标与方法,介绍了抗炸荚种质鉴定与抗炸荚品种选育概况,同时详细阐述了大豆抗炸荚性状的分子遗传基础研究进展,最后对大豆抗炸荚性的研究与应用进行了展望。     

Genetic divergence between North American ancestral soybean lines and introductions with resistance to soybean cyst nematode revealed by chloroplast haplotype

Domesticated soybean [Glycine max (L.) Merr.] is a major crop with an established ancestral relationship to wild soybean (Glycine soja Sieb. & Zucc.) native to Asia. Soybean genetic diversity can be assessed at different levels by identification of polymorphic alleles at genetic loci, in either the plastid or nuclear genomes. The objective of this study was to evaluate genetic diversity based on chloroplast haplotypes for soybean genotypes present in the USDA germplasm resource collection. Shared chloroplast haplotypes represent broad groups of genetic relatedness. Previous work categorized three-quarters of the cultivated soybeans from Asia into a single haplotype group. Our results confirmed the close relationship of North American soybean ancestors and G. max plant introductions previously identified as representing potential sources of soybean genetic variation with the finding that these genotypes belonged to a single chloroplast haplotype group. Genetic diversity was identified in soybean genotypes determined to have a high density of single nucleotide polymorphisms and in a screen of accessions with resistance to soybean cyst nematode. Characterization of soybean plant introduction lines into chloroplast haplotype group may be an important initial step in evaluating the appropriate use of particular soybean genotypes.