Marker-assisted breeding for transgressive seed protein content in soybean [Glycine max (L.) Merr].

KEY MESSAGE: After two cycles of marker-assisted breeding on three loci, lines with transgressive segregation of 8.22-9.32 % protein content were developed based on four original soybean parents with 35.35-44.83 % protein content. Marker-assisted breeding has been an innovative approach in conventional breeding, which is to be further demonstrated, especially for quantitative traits. A study on continuous transgressive breeding for seed protein content (SPC) in soybean using marker-assisted procedures is reported here. The SPC of the recombinant inbred line (RIL) population XG varied in 38.04-47.54 % under five environments with P 1 of 35.35 %, P 2 of 44.34 % and total heritability of 89.11 %. A transgressive segregant XG30 with SPC 45.53 % was selected for further improvement. The linkage mapping of XG showed its genetic constitution composed of five additive QTL (32.16 % of phenotypic variation or PV) and two pairs of epistatic QTL (2.96 % PV) using 400 SSR markers with the remnant heritability 53.99 % attributed to the undetected collective of minor QTL. Another transgressive segregant WT133 with SPC 48.39 % was selected from the RIL population WT (44.83 % SPC for both parents). XG30 and WT133 were genotyped on the three major additive QTL (Prot-08-1, Prot-14-1 and Prot-19-2) as A 2 A 2 B 2 B 2 L 1 L 1 and A 1 A 1 B 1 B 1 L 2 L 2 , respectively. From WT133×XG30, surprising transgressive progenies were obtained, among which the recombinants with all three positive alleles A 2 _B 2 _L 2 _ performed the highest SPC, especially that of Prot-08-1. The five F 2-derived superior families showed their means higher than the high parent value in F 2:3 and F 2:4 and more transgressive effect in F 2:5:6, with the highest as high as 54.15 %, or 4.82 and 9.32 % more than WT133 and its original high parent, respectively. This study demonstrated the efficiency of marker-assisted procedure in breeding for transgressive segregation of quantitative trait.

Genome-wide identification and transcription analysis of soybean carotenoid oxygenase genes during abiotic stress treatments.

Carotenoid oxygenase is a key enzyme in carotenoid metabolism leading to the synthesis of two phytohormones, abscisic acid (ABA) and strigolactone, as well as norisoprenoids. Few studies have analyzed inter-relationship of the metabolic networks of these three substances. In this present paper, soybean carotenoid oxygenase genes were identified to reveal their phylogenetic relationships, and the transcriptional response of these genes to four abiotic stresses (NaCl, PEG, high and low temperature) and ABA treatment were investigated to characterize their potential roles in plant resistance. Positive selection was found in the branches of carotenoid cleavage dioxygenase (CCD1), CCD8 and NCED (9-cis-epoxycarotenoid oxygenase), indicating an adaptive evolution in these clades. In soybean eight carotenoid oxygenase genes were identified. The transcriptional responses of almost all of them under stress and ABA conditions were significantly altered when assessed by quantitative polymerase chain reaction. Notably, CCD1 and CCD4, previously known as the key genes in norisoprenoids metabolism, showed especially strong responses to the abiotic stresses and ABA treatment. Furthermore, transcription levels of CCD7 and CCD8, key genes for the strigolactone pathway, highly increased during ABA treatment providing further evidence that ABA is involved in regulating strigolactone metabolism. All of the carotenoid oxygenase genes in soybean are involved in plant abiotic stress physiology, and ABA is presumed to be a core regulatory substance. These findings provide some insights into the mechanisms that underlie the regulation of tolerance response to abiotic stresses in soybean.

[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.

Global Transcriptome Profiling of Enterobacter Strain NRS-1 in Response to Hydrogen Peroxide Stress Treatment.

Microbes are often subjected to oxidative stress in nature that badly affects their growth rate and viability. Although the response of microbes against oxidative stress has been characterized at the chemical, physiological, and molecular levels, the mechanism of gene-regulation network adaptations of bacteria in response to oxidative stress remains largely unknown. In this study, transcriptomic profiling of glyphosate-tolerant Enterobacter strain NRS-1 was analyzed under 9 mM H2O2 stress using RNA-seq and qRT-PCR. The lag period in the growth of NRS-1 was very short compared with wild-type strain under H2O2 treatment. A total of 113 genes are identified as differentially expressed genes (DEGs) under H2O2 that include 38 upregulated and 75 downregulated transcripts. But not any genes regulated by major oxidative regulons, viz., oxyR, soxR, rpoS, perR, ohrR, and σв, have been reported in DEGs, hence potentially reflecting that specific changes have occurred in NRS-1 for adaptation to oxidative stress. Based on the functions of the DEGs, six elements namely formate dehydrogenase, processes associated with iron ions, repair programs, multidrug resistance, antioxidant defense, and energy generation (mqo, sdhC) might have contributed for stress tolerance in NRS-1. These elements are proposed to form a molecular network explaining gene response of NRS-1 to stress, and ensure global cell protection and growth recovery of NRS-1. These findings enrich the view of gene regulation in bacteria in response to H2O2 oxidative stress.

Complex gene response of herbicide-resistant Enterobacter strain NRS-1 under different glyphosate stresses.

Knowledge of biological evolution and genetic mechanisms is gained by studying the adaptation of bacteria to survive in adverse environmental conditions. In this regard, transcriptomic profiling of a glyphosate-tolerant Enterobacter strain NRS-1 was studied under four different treatments to investigate the gene-regulatory system for glyphosate tolerance. A total of 83, 83, 60 and 74 genes were up-regulated and 108, 87, 178 and 117 genes down-regulated under 60-NPG, 110-NPG, NaCl (355 mM) and HCl (pH 4.46) stress treatments, respectively. Complex gene network was identified to be involved in regulating tolerance to glyphosate. This study revealed that NRS-1 has gained glyphosate tolerance at the cost of osmotic and acidic resistance. The 25 differentially expressed genes are reported to may have partly changed the function for providing resistance to glyphosate directly, among them genes metK, mtbK, fdnG and wzb that might detoxify/degrade the glyphosate. However, under 110-NPG condition, NRS-1 might have utilized economical and efficient ways by depressing its metabolism and activity to pass through this stress. Hence, the present study provides insights into the genes involved in glyphosate tolerance, which can be effectively utilized to engineer herbicide-resistant crop varieties after their proper validation to manage weed growth.

[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.

[Identification of drought tolerant germplasm and inheritance and QTL mapping of related root traits in soybean (Glycine max (L.) Merr.)].

Fifty nine accessions of soybean (Glycine max (L.) Merr.) selected from 301 ones in Huang-Huai-Hai and Middle-Lower Changjiang Valleys were tested in two years for their tolerance to drought by using the mean membership index value averaged over those of plant height,leave number,dry root weight and dry stem and leaf weight. Four most tolerant accessions (Rank 1) and two most sensitive ones (Rank 5) were identified. There existed very significant correlations between drought tolerance and relative values of dry root weight,total root length, and root volume (per plant dry weight basis), respectively,which could be used as root indicators of drought tolerance. The RIL population derived from Kefeng 1 x Nannong 1138-2 was used to analyze the inheritance of the three related root traits by using the segregation analysis of quantitative traits under the major gene plus polygene mixed inheritance model. The results showed that between the two parents (Rank 1 x Rank 4), the relative values of dry root weight,total root length and root volume were respectively controlled by two major genes (linked together for the latter two traits, recombination value being 4.30% and 1.93%, respectively) plus polygenes with their major gene heritability values of 62.26%-91.81% and polygene heritability values 2.99%-24.75%, indicating that the major genes,especially the one with larger effect,accounted for a major part of the genetic variation between the two parents. It was identified that five, three, and five QTLs located on N6-C2, N8-D1b + W, N11-E, and N18-K linkage groups for relative dry root weight, total root length and root volume, respectively. Each of the traits appeared to have one locus (Dw1, R/1, and Rv1) with relatively large effect in comparison with their other loci, and those major ones were located near the same site of the same linkage group N6-C2. The results of segregation analysis and QTL mapping appeared pretty consistent with each other, which could be used as a demonstration of each other.

[Segregation analysis of genetic system of quantitative traits in plants].

Based on the traditional polygene inheritance model of quantitative traits, the mixed major gene and polygene inheritance model was raised and considered as the general model, while pure major gene or pure polygene inheritance model being only the specific case of the general model. From the proposed theory, the segregation analysis procedure was established for studying the genetic system of quantitative traits of plants. At present this procedure can be used to evaluate the genetic effects of individual major genes (up to 2-3 major genes) and the collective genetic effects of polygenes as well as their heritability values. The present paper introduces the process of the establishment of the procedure, main achievements and application results. An example was given to illustrate the steps, methods and effectiveness of the procedure.

[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.

[Analysis on the major gene and multigene mixed inheritance of wide compatibility gene in rice].

The intersubspecific hybrids between Indica and Japanica varieties are generally semi sterile, which limits the use of Indica-Japanica heterosis. Genetic study of wide compatibility gene (WCG) in rice helps to understand the mechanism of semi sterility and make it possible to overcome this phenomenon. In this study, the P1,P2,F1,B1,B2,and F2 of Indica-Japanica hybrid of 3037/02428 were analyzed by the major gene and minor gene mixed inheritance model. The research showed that Apart from single major gene, the WCG inheritance is also affected by minor genes. Not only major gene's effect on fertility but also minor gene's effect should be considered when utilize WCG to overcome semisterile between subspecies.

Identification of regulated genes conferring resistance to high concentrations of glyphosate in a new strain of Enterobacter.

Glyphosate is a widely used herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity. Most plants and microbes are sensitive to glyphosate. However, transgenic-resistant crops that contain a modified epsps obtained from the resistant microbes have been commercially successful and therefore, new resistance genes and their adaptive regulatory mechanisms are of great interest. In this study, a soil-borne, glyphosate-resistant bacterium was selected and identified as Enterobacter. The EPSPS in this strain was found to have been altered to a resistant one. A total of 42 differentially expressed genes (DEGs) in the glyphosate were screened using microarray techniques. Under treatment, argF, sdhA, ivbL, rrfA-H were downregulated, whereas the transcripts of speA, osmY, pflB, ahpC, fusA, deoA, uxaC, rpoD and a few ribosomal protein genes were upregulated. Data were verified by quantitative real-time PCR on selected genes. All transcriptional changes appeared to protect the bacteria from glyphosate and associated osmotic, acidic and oxidative stresses. Many DEGs may have the potential to confer resistance to glyphosate alone, and some may be closely related to the shikimate pathway, reflecting the complex gene interaction network for glyphosate resistance.

Cloning and comparative analysis of the gene encoding diacylglycerol acyltransferase from wild type and cultivated soybean.

Diacylglycerol acyltransferase (DGAT), as an important enzyme in triacylglycerol synthesis, catalyzes the final acylation of the Kennedy pathway. In the present study, the GmDGAT gene was cloned from Glycine max by using AtDGAT as a query to search against the soybean EST database and the rapid amplification of cDNA ends (RACE) method. Allelic genes were also isolated from 13 soybean accessions and the divergence of the deduced amino acid sequences were compared. The comparison reveals that although GmDGAT is a highly conserved protein, several differences of insertion/deletion were identified in the N-terminal region of the GmDGATs from various soybean accessions. In the C-terminal regions, a single amino acid mutation specific to both G. max and G. soja was also found. The GmDGAT genomic sequences were further cloned and the number and size of exons in the DGAT genomic sequence were very similar among different plant species, whereas the introns were more diverged. These results may have significance in elucidating the genetic diversity of the GmDGAT among the soybean subgenus.

[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.

The EIM algorithm in the joint segregation analysis of quantitative traits.

In this article, a new algorithm for obtaining the maximum likelihood estimators (MLEs) of parameters in the joint segregation analysis (JSA) of multiple generations of P1, F1, P2, F2 and F2:3 (MG5) for quantitative traits was set up. Firstly, owing to the fact that the component variance of the heterogeneous genotype in F2:3 included both the first-order genetic parameters (denoted by the means of distributions) and the second-order parameters, a simple closed form for the MLEs of the means of component distributions did not exist while the expectation and maximization (EM) algorithm was used. To simplify the estimation of parameters, the first partial derivative of the above variance on the mean in the sample log-likelihood function was omitted. However, this would be remedied by the iterated method. Then, variances of component distributions for segregating populations were partitioned into major-gene, polygenic and environmental variances so that the generally iterated formulae for estimating the means as well as polygenic and environmental variances of component distributions in the maximization step (M-step) of the EM algorithm were obtained. Therefore, the EM algorithm for estimating parameters in the JSA model for the MG5 was simplified. This is called the expectation and iterated maximization (EIM) algorithm. Finally, an example of the inheritance of the resistance of soybean to beanfly showed that the results of mixed inheritance analysis in this paper coincided with those in both Wang & Gai (2001) and Wei et al. (1989), so the EIM algorithm was appropriate.

Isolation and characterization of a full-length resistance gene homolog from soybean.

Using mixed resistance gene analogs as probes, a putative resistance gene (KR1) was isolated from soybean and characterized further. The KR1 protein consists of a Toll/interleukin receptor (TIR) domain, a nucleotide binding site (NBS) domain, an imperfect leucine-rich repeat (LRR) domain and two C-terminal transmembrane segments. Due to these features, KR1 represents a distinct member in the TIR-NBS-LRR class of resistance genes. Southern-blot analysis indicated that there were several KR1-related sequences within the soybean genome, and two polymorphic loci were mapped onto linkage group L. KR1 was induced by SA treatment and soybean mosaic virus (SMV) infection in the resistant line (Kefeng 1). An orthologue (NR1) and a homologue (NR2) of the KR1 gene were also identified in the SMV susceptible-line Nannong1138-2. Sequencing analysis revealed that NR2 was highly homologous to KR1 and NR1, but had a 21-bp deletion. Moreover, the NR1, NR2 transcription and the ratio of NR1/ NR2 was up-regulated by viral infection in Nannong1138-2. These results indicated the complexity of the regulatory mechanism in the plant responses to SMV infection.