Inheritance and Gene Mapping of Resistance to Soybean Mosaic Virus Strain SC14 in Soybean

Soybean mosaic virus (SMV) is one of the most broadly distributed diseases worldwide. It causes severe yield loss and seed quality deficiency in soybean (Glycine max (L.) Merr.). SMV Strain SC14 isolated from Shanxi Province, China, was a newly identified virulent strain and can infect Kefeng No. 1, a source with wide spectrum resistance. In the present study, soybean accessions, PI96983, Qihuang No. 1 and Qihuang No. 22 were identified to be resistant (R) and Nannong 1138‐2, Pixianchadou susceptible (S) to SC14. Segregation analysis of PI96983 x Nannong 1138‐2 indicated that a single dominant gene (designated as R_SC14) controlled the resistance to SC14 at both V₂ and R₁ developmental stages. The same results were obtained for the crosses of Qihuang No. 1 × Nannong 1138‐2 and Qihuang No. 22 × Nannong 1138‐2 as in PI96983 × Nannong 1138‐2 at V₂ stage, but at R₁ stage, the F₁ performed as necrosis (a susceptible symptom other than mosaic), F₂ segregated in a ratio of 1R:2N:1S, and the progenies of necrotic (N) F₂ individuals segregated also in R, N and S. It indicated that a single gene (designated as R_SC14Q, to be different from that of PI96983) controlled the resistance to SC14, its dominance was the same as in PI96983 × Nannong 1138‐2 (without symptoms) at V₂ stage and not the same at R₁ stage. The tightly linked co‐dominant simple sequence repeat (SSR) marker Satt334 indicated that all the heterozygous bands were completely corresponding to the necrotic F₂ individuals, or all the necrotic F₂ individuals were heterozygotes. It was inferred that necrosis might be due to the interaction among SMV strains, resistance genes, genetic background of the resistance genes, and plant development stage. Furthermore, the bulked segregant analysis (BSA) of SSR markers was conducted to map the resistance genes. In F2of PI96983 × Nannong 1138‐2, five SSR markers, Sat_297, Sat_234, Sat_154, Sct_033 and Sat_120, were found closely linked to RSC14, with genetic distances of 14.5 cM, 11.3cM, 4.3cM,3.2cM and 6cM, respectively. In F₂ of Qihuang No. 1 × Nannong 1138‐2, three SSR markers, Sat_234, Satt334 and Sct_033, tightly linked to R_SC14Q with genetic distances of 7.2 cM, 1.4 cM and 2.8 cM, respectively. Based on the integrated joint map by Cregan et al. (1999), both RScMand R_SC14Q were located between Sat_234 and Sct_033 on linkage with group F of soybean, with their distances from Sct_033 at the same side being 3.2 cM and 2.8 cM, respectively. Therefore, RSC14and R_SC14Q might be on a same locus. The obtained information provides a basic knowledge for marker‐assisted selection of the resistance gene in soybean breeding programs and fine mapping and map‐based cloning of the resistance gene. (Managing editor: Li‐Hui Zhao)     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mutagenesis of Embryogenic Cultures of Soybean and Detecting Polymorphisms Using RAPD Markers

Embryogenic suspension cultures of soybean (Glycine max L. cv. Iroquois) were subjected to mutagenesis using varying concentrations (1, 3, 10, and 30 mM) of ethyl methanesulfonate (EMS). Depending on the concentration of EMS used, the mean survival rate of embryogenic cultures decreased from 74 % (1 mM EMS) to 43 % after 30 mM EMS treatment. Random amplified polymorphic DNA (RAPD) analysis was used to determine whether induction of genetic variability in embryogenic cultures in response to the different EMS treatments may result in identification of polymorphic markers. Two of 35 ‘core’ primers tested revealed polymorphisms. One of the primers, OPO-01/1150, revealed polymorphism in tissue treated with 10 mM EMS, while the other primer, OPO-05/1200, revealed polymorphism in tissue treated with either 1 or 30 mM EMS. These results suggest that RAPD markers are useful in detecting mutations in embryogenic cultures of soybean. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

用PCR法检测献血员单个核细胞中的CMV—DNA

107份自愿献血新鲜血标本和22份库存血标本分别用PCR检测单个核细胞中巨细胞病毒DNA（CMV－DNA）和用ELISA检测其血浆中CMV－IgG。结果CMV－DNA阳性率达80．4％和77．3％,CMV－IgC阳性纺为65．9％。其中CMV－IgG阳性者,基本上都携带CMV－DNA;CMV－IgG阴性者,亦有部分携带CMV－DNA。因此认为献血员血液中CMV高带毒率应引起临床有关部门的高度重视;PCR是筛选无传播CMV危险性血液制品的最可靠方法。     

Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers

The use of genetically diverse resistance sources is important in breeding for durable disease resistance. Detection and evaluation of resistance genes by conventional inheritance experiments, however, often require laborious screening and genetic testing. In the present study, a marker-assisted screening for resistance sources was initiated in soybean [Glycine max (L.) Merr] using one DNA microsatellite and two RFLP markers tightly linked to a soybean mosaic virus (SMV) resistance gene (Rsv1). The three marker loci were used to screen 67 diverse soybean cultivars, breeding lines, and plant introductions. Five variants were found at the microsatellite locus (HSP176L), and the two RFLP loci (pA186 and pK644a) near Rsv1 show a remarkably higher level of restriction polymorphism than Rsv1-independent RFLP loci. Several specific variants at the three marker loci were found to be correlated with virus resistance, among which HSP176L-2 can be detected by PCR, thus may be useful for germplasm screening. The grouping of the 67 accessions according to their multilocus marker variants agrees with the available pedigree information. When all, or most, of the cultivars within a given group with the same Rsv1-linked marker variant are resistant, their SMV resistance is most likely conferred by Rsv1. These putatively Rsv1-carrying groups contain a total of 38 SMV-resistant lines including six differential cultivars that are known to carry Rsv1. The remaining seven resistant accessions (Columbia, Holladay, Peking, Virginia, FFR-471, PI 507403, and PI 556949) do not carry resistance marker variants, and at least some of them could be sources of resistance genes independent of Rsv1.     

The Decrease of Extracted Apoplast Protein in Soybean Root Tip by Aluminium Treatment

Aluminium effect on the mobility of apoplast protein in root tips was studied. Two-day seedlings of soybean (Glycine max. (L.) Merr. cv. Tsurunoko) were treated with 50 μM AlCl₃ for 2 h. Using infiltration method, the apoplast protein in root tips was extracted with 20 or 100 mM MgCl₂. When 20 mM MgCl₂ was used to collect weakly bound protein to apoplast, the amount of protein extracted was reduced to be about 20 % compared with that of control and the band of 97 kDa disappeared in SDS-PAGE gel. However, the 97 kDa protein could be extracted by 100 mM MgCl₂, which were used for extraction of more tightly bound protein to apoplast, and the amount was estimated to be the same as that of control. When the protein was further developed in two-dimensional electrophoresis, three spots were found between pI 6.4 and 6.5. This is the first report of an Al effect on the mobility of apoplast protein. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

纵纹腹小鸮DNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列 (GTG) 5,(GT) 8,(CAC) 5和人源小卫星 33 1 5作引物 ,扩增纵纹腹小的基因组DNA ,产生多态性DNA片段 ,回收了 8个表现个体特异性的片段。当用小的基因组总DNA探针与它们杂交时 ,其中 2个表现阳性 ,说明PCR方法扩增出的高变异产物含有重复序列。用含重复序列的个体特异性PCR产物作探针 ,与无关个体小基因组DNA的HaeⅢ酶切产物进行DNA印迹 ,获得了变异性较高的DNA指纹图谱。且通过对京白鸡家系分析表明 ,用小基因组DNA的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传。因此 ,高变异的PCR产物可以有效地用作DNA指纹探针。     

Differential Expression of Peroxidase Isoenzymes in Soybean Roots Treated with the Benzothiadiazole

The protection compound benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) was applied to soybean roots or leaves in a dose of 20 cm³ of a solution containing 25 μg g⁻¹ of the active ingredient. Electrophoretic profiles of chitinase and superoxide dismutase were not altered by the product. Increased activity of two root anionic peroxidases and three differential isoforms of these enzymes were observed in plants with roots treated by BTH, which can be used as biochemical markers of the BTH effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

大豆GmABI4基因克隆及表达分析

该研究基于大豆基因组数据库,根据拟南芥ABI4蛋白的氨基酸序列,经比对分析,获得了大豆中的2个GmABI4基因,分别命名为GmABI4-1(GenBank登录号为XM_014766551.1)和GmABI4-2(GenBank登录号为NM_001249003)。TMHMM软件和系统进化转录分析表明,这2个基因编码的蛋白均不具有信号肽,二级结构主要以无规则卷曲和延伸链为主;进化树分析表明,大豆GmABI4和野生大豆亲缘关系较近。荧光定量PCR分析表明,GmABI4-1与GmABI4-2基因在大豆种子与豆荚中的表达量均高于根、茎、叶、花等其他组织,推测可能与调控大豆种子生命活动相关。     

Soybean aphid resistance genes in the soybean cultivars Dowling and Jackson map to linkage group M

The soybean aphid [Aphis glycines Matsumura] is an important pest of soybean [Glycine max (L.) Merr.] in North America. Single dominant genes in the cultivars ‘Dowling’ and ‘Jackson’ control resistance to the soybean aphid. The gene in Dowling was named Rag1, and the genetic relationship between Rag1 and the gene in Jackson is not known. The objectives of this study were to map the locations of Rag1 and the Jackson gene onto the soybean genetic map. Segregation of aphid resistance and simple sequence repeat (SSR) markers in F _2:3 populations developed from crosses between Dowling and the two susceptible soybean cultivars ‘Loda’ and ‘Williams 82’, and between Jackson and Loda, were analyzed. Both Rag1 and the Jackson gene segregated 1:2:1 in the F _2:3 populations and mapped to soybean linkage group M between the markers Satt435 and Satt463. Rag1 mapped 4.2 cM from Satt435 and 7.9 cM from Satt463. The Jackson gene mapped 2.1 cM from Satt435 and 8.2 cM from Satt463. Further tests to determine genetic allelism between Rag1 and the Jackson gene are in progress. The SSR markers flanking these resistance genes are being used in marker-assisted selection for aphid resistance in soybean breeding programs. Trade and manufacturers’ names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究.结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果.基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略.同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法.     

Dynamics of Seed Protein Biosynthesis in Two Soybean Genotypes Differing in Drought Susceptibility

The dynamics of seed storage protein biosynthesis was studied under field conditions during two vegetative seasons. Two soybean (Glycine max L. Merr.) genotypes were examined: BOSA (drought tolerant) and L 121 (drought susceptible). Seed samples were taken from plants at three stages of seed maturation (50 and 70 d after flowering, and at full maturity). The earlier synthesis of the -subunit of the 7S protein occurred in the drought susceptible cultivar. We have not found such differences in the synthesis of the - and -subunits of the 7S protein. Our results did not confirm significant genotypic differences in protein composition of the mature seeds between the cultivars studied, but have pointed out to the differences in the dynamics of protein biosynthesis during seed maturation and desiccation.     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究。结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果。基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略。同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法。     

Identification of QTLs Underlying Water-Logging Tolerance in Soybean

Soil water-logging can cause severe damage to soybean [Glycine max (L.) Merr.] and results in significant yield reduction. The objective of this study was to identify quantitative trait loci (QTL) that condition water-logging tolerance (WLT) in soybean. Two populations with 103 and 67 F_6:11 recombinant inbred lines (RILs) from A5403 × Archer (Population 1) and P9641 × Archer (Population 2), respectively, were used as the mapping populations. The populations were evaluated for WLT in manually flooded fields in 2001, 2002, and 2003. Significant variation was observed for WLT among the lines in the two populations. No transgressive tolerant segregants were observed in either population. Broad-sense heritability of WLT for populations 1 and 2 were 0.59 and 0.43, respectively. The tolerant and sensitive RILs from each population were selected to create a tolerant bulk and a sensitive bulk, respectively. The two bulks and the parents of each population were tested with 912 simple sequence repeat (SSR) markers to select candidate regions on the linkage map that were associated with WLT. Markers from the candidate regions were used to genotype the RILs in both populations. Both single marker analysis (SMA) and composite interval mapping (CIM) were used to identify QTL for WLT. Seventeen markers in Population 1 and 15 markers in Population 2 were significantly (p <0.0001) associated with WLT in SMA. Many of these markers were linked to Rps genes or QTL conferring resistance to Phytophthora sojae Kaufmann and Gerdemann. Five markers, Satt599 on linkage group (LG) A1, Satt160, Satt269, and Satt252 on LG F, and Satt485 on LG N, were significant (p <0.0001) for WLT in both populations. With CIM, a WLT QTL was found close to the marker Satt385 on LG A1 in Population 1 in 2003. This QTL explained 10% of the phenotypic variation and the allele that increased WLT came from Archer. In Population 2 in 2002, a WLT QTL was located near the marker Satt269 on LG F. This QTL explained 16% of the phenotypic variation and the allele that increased WLT also came from Archer.     

Influence of Media Components and pH on Somatic Embryo Induction in Three Genotypes of Soybean

The influence of media components on the initiation of somatic embryogenesis in three genotypes of soybean was investigated. The following genotypes were used: Iroquois, Macon, and Savoy. Media modifications included sucrose concentration, type and concentration of auxin at two pH levels, and pH level independently. Immature cotyledons were used as the source of explant. Cotyledons were placed on a medium containing MS salts, B5 vitamins, sucrose, and auxin. Gelrite (0.2%) was used as the solidifying agent. Sucrose concentrations of 1, 2, 3, 4.5, or 6% were used. The auxins used included 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) with each at concentrations of 45.2, 90.4, 135.7, 180.9, and 226.2 M. The pH of each the media was adjusted to either 5.7 or 7.0 with 1 N NaOH. In an additional experiment, the effect of the two pH levels, 5.7 and 7.0, was investigated independently. Overall, the frequency of somatic embryogenesis significantly varied among the different genotypes used in this study, with Iroquois showing the highest response. Frequency of somatic embryogenesis also varied in response to the different treatments used, including sucrose and auxin. The highest initiation (91.7%) and mean number of somatic embryos per responding explant (14.9) of Iroquois was observed in a medium containing 2% sucrose. The highest initiation (97.1%) and mean number of somatic embryos per responding explant (19.5) was observed in Iroquois on 135.7 M 2,4-D and Savoy on 135.7 M 2,4-D, respectively, for the auxin by pH level experiment. No significant differences were observed among the two pH treatments used.     

Fine Mapping of Xa2, a Bacterial Blight Resistance Gene in Rice

The rice bacterial blight resistance gene, Xa2, confers resistance to T7147 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. It is located on the long arm of chromosome 4. Here, we report the fine mapping of Xa2 by genetic recombination analysis with simple sequence repeat (SSR) markers according to the genome sequence. Two F₂ populations are constructed to localize Xa2. In a primary analysis with 136 random F₂ plants of Zhenzhuai/IRBB2, it was found that Xa2 was located in approximately 20 cM region. To accurately determine the locus of Xa2, 120 new SSR markers were developed in this region by screening the sequence. Twelve new SSR markers were successfully used in genetic recombination analysis in IR24/IRBB2 population, while 20 in ZZA/IRBB2 population. We found that the nearest SSR markers to Xa2 are HZR950-5 and HZR970-4, which cover approximately 190-kb region. The sequence analysis of this 190-kb region revealed the presence of a homologous sequence of leucine rich repeat (LRR)-kinase. These results are very useful for transferring or pyramiding Xa2 by molecular marker-assistant selection in rice breeding programs and for cloning Xa2 by map-based cloning in combination with a long-range PCR strategy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

GmHSFA1基因克隆及其过量表达提高转基因大豆的耐热性

热激转录因子在调节植物对逆境胁迫应答和热激蛋白基因表达方面起重要作用。采用生物信息学和比较基因组学方法结合RACE技术从大豆基因组中克隆到一个新的热激转录因子基因GmHsfA1, 其cDNA全长1 781 bp, 包含1个1 533 bp的开放阅读框, 编码含有510个氨基酸残基的蛋白质(GenBank登录号为AY458843)。与其他转录因子的分子结构相似,GmHSFA1也含有4个典型的结构功能域—DNA结合域、寡聚域、核定位信号和C端激活域。BLAST分析表明, GmHSFA1与其同源性最高的番茄热激转录因子LpHSFA1之间的氨基酸序列相似性为52.46%。RT-PCR、Northern和遗传转化结果显示: 1)GmHsfA1在大豆的不同组织中呈现组成型表达模式; 2)常温下转基因大豆植株的GmHsfA1表达水平明显高于非转基因对照; 3)GmHsfA1的过量表达激活了转基因大豆植株中热激蛋白基因GmHsp22在非诱导条件下的转录, 并加强了高温胁迫下另2个热激蛋白基因GmHsp23和GmHsp70的表达; 4)转GmHsfA1大豆植株的耐热温度(达52℃)明显高于非转基因植株。上述结果说明, GmHsfA1的过量表达激活或促进其下游3个热激蛋白基因的转录或表达, 明显提高了转基因大豆植株的耐热能力。