Identification of soybean drought-tolerant genotypes and loci correlated with agronomic traits contributes new candidate genes for breeding

Plant Molecular Biology - Drought tolerance level of 136 soybean genotypes, the correlations among traits were evaluated, and several important drought-tolerant genotypes, traits, SNPs and genes...     

Mapping candidate genes for oleate biosynthesis and their association with unsaturated fatty acid seed content in soybean

The development of high-oleate soybean germplasm is hindered by the lack of knowledge of the genetic factors controlling oleate phenotypic variation. In the present study, several candidate genes implicated in oleate biosynthesis were mapped and their cosegregation with oleate, linoleate and linolenate quantitative trait loci (QTLs) was investigated. FAD2-2C, a previously described ω-6 desaturase isoform, was localized on linkage group E; whereas, a novel FAD2-2 isoform, designated as FAD2-2D, mapped on linkage group N. In addition, two isoforms were identified for the aminoalcoholphosphotransferase-encoding GmAAPT1 gene, denoted AAPT1a and AAPT1b. A database query suggested that only one functional copy of the FAD6 gene, encoding a plastid localized ω-6 desaturase, exists in the soybean genome. AAPT1a and FAD6 mapped on linkage group D1b, 23.40 cM apart. Linolenate QTLs with minor effects were identified near the FAD6 and AAPT1a markers in two segregating populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

黄酮6位羟基化酶催化机制的理论研究与应用

灯盏乙素发酵生产过程中,黄酮6位羟基化酶催化效率不足,导致产生至少约18%的副产物。本研究以2种黄酮6位羟基化酶CYP82D4与CYP706X为研究目标,通过分子动力学模拟与量子化学计算,对两种黄酮6位羟基化酶的催化机制进行解析。结果表明,CYP82D4与CYP706X在反应决速步的能垒几乎相同,应当具有相似的反应速率,而CYP82D4相对较小的底物结合能可能有利于产物释放,是其具有更高催化效率的直接原因。最后,基于对底物进出过程的研究,CYP82D4的L540A突变将催化效率提高了1.37倍,证明了理论计算指导黄酮6位羟基化酶改造优化的可行性。本研究揭示了黄酮6位羟化酶的催化机制,为对其进行改造优化以提高灯盏乙素的发酵生产效率提供了参考。     

矮秆大豆突变体异黄酮含量变化的研究

以大豆品种东农42及其两个矮秆突变体(HK8和HK11)为原料,采用Luminol-K₃Fe(CN)₆化学发光体系的流动注射化学发光法对茎、叶和种子中的异黄酮进行检测,同时与HPLC检测方法相比较。结果表明:该方法能较简便快速地检测异黄酮含量,相对标准偏差(RSD)为0.69%～2.30%,回收率为95.5%～104.5%。该实验准确地检测出三种大豆材料中异黄酮含量; 异黄酮含量与株高成正比; 种子的异黄酮含量比茎、叶的均高     

QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Background

Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results

The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions

A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.     

Polymorphisms of soybean isoflavone synthase and flavanone 3-hydroxylase genes are associated with soybean mosaic virus resistance

Soybean mosaic disease, caused by soybean mosaic virus (SMV), is one of the most devastating diseases that limit soybean production throughout the world. Soybean isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) genes catalyze the production of isoflavones and flavonoids, the increase of which is correlated with increased disease resistance. We have cloned, sequenced, and analyzed the IFS1, IFS2 and F3H genomic regions from 33 Chinese soybean accessions including 16 Glycine soja and 17 Glycine max. High nucleotide diversity and low extent of linkage disequilibrium (LD) in these three genes provided sufficient genetic resolution for association mapping. As a result, a set of single nucleotide polymorphisms (SNPs) with significant (P < 0.05) association to SMV strain SC-3 and SC-7 resistance were discovered in these genes. Among them, the SNP haplotype ‘TCACAACGA-TACA’ in IFS1 gene was found to be extremely significantly (P < 0.01) associated with SMV SC-3 resistance. After 7 days of SC-3 inoculation, the expression level of IFS1 gene in the two SC-3 resistance accessions that have this significant site continued to increased and reach to 30–160 folds high, while in the SC-3 susceptible accession which does not carry the significant site the expression level decreased to near zero. These polymorphisms were corresponding to the trait variance and thus can be considered as the candidate sites for functional molecular markers for future SMV resistance breeding.     

Expression of candidate genes for residual feed intake in Angus cattle

Residual feed intake (RFI) has been adopted in Australia for the purpose of genetic improvement in feed efficiency in beef cattle. RFI is the difference between the observed feed intake of an animal and the predicted feed intake based on its size and growth rate over a test period. Gene expression of eight candidate genes (AHSG, GHR, GSTM1, INHBA, PCDH19, S100A10, SERPINI2 and SOD3), previously identified as differentially expressed between divergent lines of high‐ and low‐RFI animals, was measured in an unselected population of 60 steers from the Angus Society Elite Progeny Test Program using quantitative real‐time PCR. Results showed that the levels of gene expression were significantly correlated with RFI. The genes explain around 33.2% of the phenotypic variance in RFI, and prediction equations using the expression data are reasonably accurate estimators of RFI. The association of these genes with economically important traits, such as other feed efficiency‐related traits and fat, growth and carcass traits, was investigated as well. The expression of these candidate genes was significantly correlated with feed conversion ratio and daily feed intake, which are highly associated with RFI, suggesting a functional role for these genes in modulating feed utilisation. The expression of these genes did not show any association with average daily gain, eye muscle area and carcass composition.     

Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase ( CHS ) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase ( F3'H ) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.     

Extension-growth responses and expression of flavonoid biosynthesis genes in the Arabidopsis hy4 mutant

The hy4 mutant of Arabidopsis thaliana(L.) Heynh. was previously shown to be impaired in the suppression of hypocotyl extension specifically by blue light. We report here that hy4 is altered in a range of blue-light-mediated extension-growth responses in various organs in seedlings and mature plants: it shows greater length of bolted stems, increased petiole extension and increased leaf width and area in blue light compared to the wild type. The hy4 mutant shows decreased cotyledon expansion in both red and blue light compared to the wild type. Anthocyanin formation and the expression of several flavonoid biosynthesis genes is stimulated by blue light in the wild type but to a much lower extent in hy4. The results indicate that the HY4 gene product is concerned with the perception of blue light in a range of extension-growth and gene-expression responses in Arabidopsis.Abbreviations DFR dihydroflavonol reductase - CHS chalcone synthase - CHI chalcone isomerase We thank the UK Agricultural and Food Research Council for supporting this work through the award of a research grant to G.I.J. We are grateful to Robert Brown for excellent technical assistance and Drs B.W. Shirley (Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, USA), C.D. Silflow (Department of Genetics and Cell Biology, University of Minnesota, St. Paul, USA) and I.E. Somssich (Department of Biochemistry, Max-Planck-Institut, Köln, Germany) for providing plasmid DNA.     

Expression of the human genes for steroid 21-hydroxylase and its C169R mutant in insect cells and functional analysis of the expression products

Steroid 21-hydroxylase (CYP21A2) is a key enzyme of glucocorticoid and mineralocorticoid biosynthesis in the adrenal cortex and belongs to the family of microsomal cytochrome P450. CYP21A2 deficiency is the most common cause of human congenital adrenal hyperplasia (CAH). Human CYP21A2 and its C169R mutant, observed in a patient with classic CAH, were expressed in Sf9 and Hi5 insect cells infected with recombinant baculoviruses. Functional CYP21A2 was produced to 28% of the total microsomal protein under optimal conditions. The C169R mutation did not affect the efficiency of CYP21A2 synthesis in insect cells, nor did it prevent CYP21A2 incorporation in membranes of the endoplasmic reticulum. Functional analysis in vitro showed that the mutant enzyme almost completely lacked the catalytic activity towards two substrates, progesterone and 17-hydroxyprogesterone.     

Expression of candidate pheromone receptor genes in vomeronasal neurons

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VON). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Recent approaches have provided new insights into the molecular biology of sensory transduction in the VNO. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of genes which are likely to encode mammalian pheromone receptors. The isolation of these receptors from the VNO might permit the analysis of the molecular events which translate the bindings of pheromones into innate stereotypic behaviors and help to elucidate the logic of pheromone perception in mammals.      

Using the candidate gene approach for detecting genes underlying seed oil concentration and yield in soybean

Increasing the oil concentration in soybean seeds has been given more attention in recent years because of demand for both edible oil and biodiesel production. Oil concentration in soybean is a complex quantitative trait regulated by many genes as well as environmental conditions. To identify genes governing seed oil concentration in soybean, 16 putative candidate genes of three important gene families (GPAT: acyl-CoA:sn-glycerol-3-phosphate acyltransferase, DGAT: acyl-CoA:diacylglycerol acyltransferase, and PDAT: phospholipid:diacylglycerol acyltransferase) involved in triacylglycerol (TAG) biosynthesis pathways were selected and their sequences retrieved from the soybean database (http://www.phytozome.net/soybean). Three sequence mutations were discovered in either coding or noncoding regions of three DGAT soybean isoforms when comparing the parents of a 203 recombinant inbreed line (RIL) population; OAC Wallace and OAC Glencoe. The RIL population was used to study the effects of these mutations on seed oil concentration and other important agronomic and seed composition traits, including seed yield and protein concentration across three field locations in Ontario, Canada, in 2009 and 2010. An insertion/deletion (indel) mutation in the GmDGAT2B gene in OAC Wallace was significantly associated with reduced seed oil concentration across three environments and reduced seed yield at Woodstock in 2010. A mutation in the 3′ untranslated (3′UTR) region of GmDGAT2C was associated with seed yield at Woodstock in 2009. A mutation in the intronic region of GmDGAR1B was associated with seed yield and protein concentration at Ottawa in 2010. The genes identified in this study had minor effects on either seed yield or oil concentration, which was in agreement with the quantitative nature of the traits. However, the novel gene-specific markers designed in the present study can be used in soybean breeding for marker-assisted selection aimed at increasing seed yield and oil concentration with no significant impact on seed protein concentration.     

Expression analysis, single nucleotide polymorphisms and combined genotypes in candidate genes and their associations with growth and carcass traits in Qinchuan cattle

The apolipoprotein E (ApoE) gene is an important component of plasma lipoprotein, and Fas apoptosis inhibitory molecule (FAIM) is a novel anti-apoptotic gene. In this study, we researched and discussed seven genes in eight different tissues in Qinchuan cattle by quantitative Real-time PCR. The result of analysis showed that ApoE and FAIM ₂ genes had a correlation with muscle and fat. PCR–RFLP was applied to analyze the genetic variations of the ApoE and FAIM ₂ genes and verify the effect on growth and carcass traits in a total of 365 Qinchuan cattles. The result of haplotype analysis showed that nine different haplotypes were identified among the four SNPs in ApoE and FAIM ₂ genes. The statistical analyses indicated that the four SNPs were significant association with growth and carcass traits (P < 0.05, N = 365); and the four SNPs were significant association between nine combined genotypes of candidate genes and growth and carcass traits. Taken together, our results provide the evidence that polymorphisms in candidate genes are associated with growth and carcass traits in Qinchuan cattle, and may be used as a possible candidate for marker-assisted selection and management in beef cattle breeding program.     

Expression of normal and mutant human pre-pro-insulins in Xenopus oocytes

Conveniently situated PstI sites were used to delete a major segment from the C-peptide coding region of a human pre-pro-insulin cDNA. The resultant mutant cDNA encoded a protein with the structure: pre-peptide B chain--Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-Arg-A chain. Normal and mutant human pre-pro-insulin cDNAs were used as templates for the synthesis of mRNA in a reaction catalysed by T7 RNA polymerase. The mRNAs were then microinjected into Xenopus oocytes to determine the effect of the deletion on the secretion of pro-insulin. When normal pre-pro-insulin mRNA was microinjected, pre-pro-insulin was processed to pro-insulin, which in turn was secreted into the media. When the mutant pre-pro-insulin mRNA was microinjected, however, mutant pro-insulin could be detected in the oocytes but at a much lower level than the normal pro-insulin. No mutant pro-insulin could be detected in the media. The stability of the mRNAs in the oocytes was investigated by microinjecting [32P]mRNA. 24 and 48 h after microinjection, the recovery of [33P]mRNA from the oocytes was 95 and 24% and 20 and 16% of that injected, for the normal and mutant mRNAs, respectively. In a cell-free translation system supplemented with dog pancreatic microsomal membranes, the pre-peptide was cleaved from the normal pre-pro-insulin but not from the mutant pre-pro-insulin. These results suggest that C-peptide plays an important role in the segregation of pro-insulin within and transport through the cellular secretory pathway.