Map-Based Cloning of the Gene Associated With the Soybean Maturity Locus E3

Photosensitivity plays an essential role in the response of plants to their changing environments throughout their life cycle. In soybean [Glycine max (L.) Merrill], several associations between photosensitivity and maturity loci are known, but only limited information at the molecular level is available. The FT3 locus is one of the quantitative trait loci (QTL) for flowering time that corresponds to the maturity locus E3. To identify the gene responsible for this QTL, a map-based cloning strategy was undertaken. One phytochrome A gene (GmPhyA3) was considered a strong candidate for the FT3 locus. Allelism tests and gene sequence comparisons showed that alleles of Misuzudaizu (FT3/FT3; JP28856) and Harosoy (E3/E3; PI548573) were identical. The GmPhyA3 alleles of Moshidou Gong 503 (ft3/ft3; JP27603) and L62-667 (e3/e3; PI547716) showed weak or complete loss of function, respectively. High red/far-red (R/FR) long-day conditions enhanced the effects of the E3/FT3 alleles in various genetic backgrounds. Moreover, a mutant line harboring the nonfunctional GmPhyA3 flowered earlier than the original Bay (E3/E3; PI553043) under similar conditions. These results suggest that the variation in phytochrome A may contribute to the complex systems of soybean flowering response and geographic adaptation.FLOWERING represents the transition from the vegetative to the reproductive phase in plants. Various external cues, such as photoperiod and temperature, are known to initiate plant flowering under the appropriate seasonal conditions. Of these cues, light is the most important, being received by several photoreceptors, including the red light (R) and the far-red light (FR)-absorbing phytochromes and the blue/UV-A absorbing cryptochromes and phototorpins (Chen et al. 2004).Phytochrome is the best characterized of these photoreceptors. All higher plant phytochromes are thought to exist as specific dimer combinations (Sharrock and Clack 2004), with each monomer being attached to a light-absorbing linear tetrapyrrole, phytochromobilin. The phytochrome apoproteins are synthesized within the cytosol and assemble autocatalytically with a chromophore to form the phytochrome holoproteins. The R-absorbing form (Pr) is thought to be inactive but is then converted to the active FR-absorbing form (Pfr) by R absorption. The absorption of light triggers the transfer of the phytochrome to the nucleus, where it regulates gene expression. In most plant species, the phytochrome apoproteins are encoded by a small gene family. Type I phytochrome is degraded in the light and is abundant in dark-grown seedlings, whereas type II phytochrome is relatively stable in the light (reviewed by Bae and Choi 2008). In Arabidopsis, five phytochromes (PhyA–E) have been characterized (Clack et al. 1994; Quail et al. 1995). PhyA is type I and is responsible for the very low fluence response and high irradiance response, whereas the other phytochromes are type II and are responsible for red-far/red reversible low fluence response (reviewed by Whitelam et al. 1998).It is well known that mutations in the phytochrome A gene affect the photoperiodic control of flowering. In Arabidopsis, a phyA mutant flowered later in either long-day or short-day conditions with a night break (Johnson et al. 1994; Reed et al. 1994). In rice, combinations of mutant alleles of phytochrome genes conferred various effects on the flowering phenotype. For example, the phyA phyB and phyA phyC double mutants grown under natural-day-length conditions showed earlier flowering phenotypes than wild-type plants (Takano et al. 2005). In pea, a long-day plant, loss- or gain-of-function phyA mutants displayed late or early flowering phenotypes, respectively (Weller et al. 1997, 2001). It is likely that photoperiodic response via phyA signaling is important for crop adaptation to a wide range of growing conditions.In soybean [Glycine max (L.) Merrill], several maturity loci, designated as E loci (Cober et al. 1996a), have been characterized by classical methods. These are E1 and E2 (Bernard 1971), E3 (Buzzell 1971), E4 (Buzzell and Voldeng 1980), E5 (McBlain and Bernard 1987), E6 (Bonato and Vello 1999), and E7 (Cober and Voldeng 2001). Of these, the E1, E3, and E4 loci have been suggested to be related to photoperiod sensitivity under various light conditions (Saidon et al. 1989; Cober et al. 1996b; Abe et al. 2003). In previous studies, using the same populations as in this study, three flowering-time quantitative trait loci (QTL)—FT1, FT2, and FT3 loci—were identified and considered to be identical with the maturity loci E1, E2, and E3, respectively (Yamanaka et al. 2001; Watanabe et al. 2004). Although many loci related to soybean flowering and maturity have been identified, and some candidate genes were recognized using near isogenic lines (NILs) (Tasma and Shoemaker 2003), most of the genes responsible for these loci have not yet been isolated except for the E4 gene. Liu et al. (2008) reported an association between phytochrome A and photoperiod sensitivity. A retrotransposon sequence inserted into the exon of the e4 allele conferred an early flowering phenotype under long-day conditions extended by incandescent lighting.A relationship between the E3 gene and some photoreceptor genes was suggested from different photosensitivity responses of various soybean NILs (Cober et al. 1996a). Cober and Voldeng (1996) also reported a linkage relationship between the E3 and Dt1 loci, which is related to a determinate or indeterminate growth habit phenotype. Additionally, Molnar et al. (2003) reported that Satt229, on linkage group (LG) L, was a proximal simple sequence repeat (SSR) marker to the E3 loci. According to the Soybean Genome Database (Shultz et al. 2006a,b, 2007; http://soybeangenome.siu.edu/) and the Legume Information System (LIS; http://www.comparative-legumes.org/), there are numerous QTL and >60 loci associated with various agronomic traits in the region between Dt1 and Satt373 (∼30–40 cM). This extremely large number of QTL may be the result of linkage between the Dt1 and E3 loci because both loci can affect many aspects of plant morphology. Among these QTL, several associations with the E3 gene have been reported (Mansur et al. 1996; Orf et al. 1999; Funatsuki et al. 2005; Kahn et al. 2008).To identify the genes responsible for the target QTL, fine mapping and map-based cloning strategies are necessary (Salvi and Tuberosa 2005). QTL analysis using intercross-derived populations, such as F₂ and recombinant inbred lines (RILs), have some limitations in genome resolution (10–30 cM) because of the simultaneous segregation of several loci affecting the same trait (Kearsey and Farquhar 1998). Additional strategies are therefore required to locate QTL more precisely. The use of NILs that differ at a single QTL is an effective approach for fine mapping and characterization of an individual locus (Salvi and Tuberosa 2005). However, the development of NILs through repeated backcrossing is time-consuming and laborious (Tuinstra et al. 1997). The use of a residual heterozygous line (RHL), as proposed by Yamanaka et al. (2004), and which is derived from RIL, is a powerful tool for precisely evaluating QTL (Haley et al. 1994). An RHL harbors a heterozygous region where the target QTL is located and a homozygous background in most other regions of the genome. Tuinstra et al. (1997) used a similar term, heterogeneous inbred family, for a selfed RHL population to identify the QTL associated with seed weight in sorghum.This RHL strategy has already been used to identify loci underlying resistance to pathogens in soybean (Njiti et al. 1998; Meksem et al. 1999; Triwitayakorn et al. 2005). After identification of the target loci, novel DNA markers tightly linked to the loci were developed using the amplified fragment length polymorphism (AFLP) method (Meksem et al. 2001a,b). Physical contigs, screened by sequence-characterized amplified region (SCAR) markers converted from these AFLP fragments, are ideal sources for identifying candidate genes for the target traits (Ruben et al. 2006).The aim of this study is to characterize the FT3 locus using a map-based cloning strategy and to confirm the gene responsible for the E3/FT3 locus by allelism tests through comparisons of gene sequences and photosensitivity of several alleles.     

Soybean Resistance Genes Specific for Different Pseudomonas Syringae Avirulence Genes Are Allelic, or Closely Linked, at the Rpg1 Locus

RPG1 and RPM1 are disease resistance genes in soybean and Arabidopsis, respectively, that confer resistance to Pseudomonas syringae strains expressing the avirulence gene avrB. RPM1 has recently been demonstrated to have a second specificity, also conferring resistance to P. syringae strains expressing avrRpm1. Here we show that alleles, or closely linked genes, exist at the RPG1 locus in soybean that are specific for either avrB or avrRpm1 and thus can distinguish between these two avirulence genes.     

Allelic Combinations of Soybean Maturity Loci E1, E2, E3 and E4 Result in Diversity of Maturity and Adaptation to Different Latitudes

Soybean cultivars are extremely diverse in time to flowering and maturation as a result of various photoperiod sensitivities. The underlying molecular genetic mechanism is not fully clear, however, four maturity loci E1, E2, E3 and E4 have been molecularly identified. In this report, cultivars were selected with various photoperiod sensitivities from different ecological zones, which covered almost all maturity groups (MG) from MG 000 to MG VIII and MG X adapted from latitude N 18° to N 53°. They were planted in the field under natural daylength condition (ND) in Beijing, China or in pots under different photoperiod treatments. Maturity-related traits were then investigated. The four E maturity loci were genotyped at the molecular level. Our results suggested that these four E genes have different impacts on maturity and their allelic variations and combinations determine the diversification of soybean maturity and adaptation to different latitudes. The genetic mechanisms underlying photoperiod sensitivity and adaptation in wild soybean seemed unique from those in cultivated soybean. The allelic combinations and functional molecular markers for the four E loci will significantly assist molecular breeding towards high productivity.     

Genetic Variations in the Human Cannabinoid Receptor Gene Are Associated with Happiness

Happiness has been viewed as a temporary emotional state (e.g., pleasure) and a relatively stable state of being happy (subjective happiness level). As previous studies demonstrated that individuals with high subjective happiness level rated their current affective states more positively when they experience positive events, these two aspects of happiness are interrelated. According to a recent neuroimaging study, the cytosine to thymine single-nucleotide polymorphism of the human cannabinoid receptor 1 gene is associated with sensitivity to positive emotional stimuli. Thus, we hypothesized that our genetic traits, such as the human cannabinoid receptor 1 genotypes, are closely related to the two aspects of happiness. In Experiment 1, 198 healthy volunteers were used to compare the subjective happiness level between cytosine allele carriers and thymine-thymine carriers of the human cannabinoid receptor 1 gene. In Experiment 2, we used positron emission tomography with 20 healthy participants to compare the brain responses to positive emotional stimuli of cytosine allele carriers to that of thymine-thymine carriers. Compared to thymine-thymine carriers, cytosine allele carriers have a higher subjective happiness level. Regression analysis indicated that the cytosine allele is significantly associated with subjective happiness level. The positive mood after watching a positive film was significantly higher for the cytosine allele carriers compared to the thymine-thymine carriers. Positive emotion-related brain region such as the medial prefrontal cortex was significantly activated when the cytosine allele carriers watched the positive film compared to the thymine-thymine carriers. Thus, the human cannabinoid receptor 1 genotypes are closely related to two aspects of happiness. Compared to thymine-thymine carriers, the cytosine allele carriers of the human cannabinoid receptor 1 gene, who are sensitive to positive emotional stimuli, exhibited greater magnitude positive emotions when they experienced positive events and had a higher subjective happiness level.     

Diurnal Expression Pattern,Allelic Variation,and Association Analysis Reveal Functional Features of the E1 Gene in Control of Photoperiodic Flowering in Soybean

Although four maturity genes, E1 to E4, in soybean have been successfully cloned, their functional mechanisms and the regulatory network of photoperiodic flowering remain to be elucidated. In this study, we investigated how the diurnal expression pattern of the E1 gene is related to photoperiodic length; and to what extent allelic variation in the B3-like domain of the E1 gene is associated with flowering time phenotype. The bimodal expression of the E1 gene peaked first at around 2 hours after dawn in long-day condition. The basal expression level of E1 was enhanced by the long light phase, and decreased by duration of dark. We identified a 5bp (3 SNP and 2-bp deletion) mutation, referred to an e1-b3a, which occurs in the middle of B3 domain of the E1 gene in the early flowering cultivar Yanhuang 3. Subcellular localization analysis showed that the putative truncated e1-b3a protein was predominately distributed in nuclei, indicating the distribution pattern of e1-b3a was similar to that of E1, but not to that of e1-as. Furthermore, genetic analysis demonstrated allelic variations at the E1 locus significantly underlay flowering time in three F₂ populations. Taken together, we can conclude the legume specific E1 gene confers some special features in photoperiodic control of flowering in soybean. Further characterization of the E1 gene will extend our understanding of the soybean flowering pathway in soybean.     

Interaction of Four Soybean Cultivars with Subsoiling and a Nematicide

Yields of four soybean, Glycine max, cultivars were increased with subsoiling under the row and application of the nematicide, DBCP i 1,2-dibromo-3-chloropropane) in Tiflon sandy loam heavily infested with the root-knot nematode Meloidogyne incognita. These cultivars represent four maturity groups: very early (V), "Essex'', early (VI), "Davis'': medium (VII), ''Ransom''; and late (VIII), '' Hutton ''. The average increase for the four cullivars was about the same for subsoiling or DBCP. When the treatmcnts were used together, the increase was greater than when either was used alone, but the effects were not additive. Increased yields were obtained with subsoiling and DBCP for the most nematode resistant cultivar, Hutton, as well as for the most susccptiblc, Davis. Subsoiling reduced root-knot galling in nonfumigated plots but did not affect it in fumigated plots. On 12 September, M. incognita larvae were most numerous at the 0- to 20 cm depth, intermediate at 20 to 33 cm depth and least numerous at 33 to 46 cm depth, Subsoiling did not affect larval populations at the three levels.     

32个柿主栽品种SSR图谱构建及遗传变异分析

以32份柿主栽品种为试验材料,利用SSR标记技术构建其指纹图谱并进行遗传多样性分析。从78对候选引物中筛选出20对多态性高、稳定性好的引物作为核心引物,构建柿主栽品种SSR指纹图谱。结果显示:(1)20对引物在32份材料中共扩增出183条多态性条带,每对引物扩增出3～20条不等,平均每对引物扩增出9.15条,多态性比率为81.3%。各个位点的杂合度在0.410 3～0.914 3之间,平均为0.702 7。(2)8对引物在12个品种上具有特征带型,采用5对引物进行组合鉴定即可将32个柿品种完全区分开。(3)依据SSR带型特征,对每对引物生成的不同带型直接编号,简化柿SSR带型记录方法,并利用每个品种的带型编号,建立其DNA指纹图谱,结果表明,核心引物组合法比特征谱带法更适用于构建中国柿主栽品种DNA指纹图谱。(4)根据系统聚类分析将32个柿主栽品种分为两大类,品种间的亲缘关系与地理来源具有一定的相关性。     

Characterization of Transcriptional Repressor Gene MSX1 Variations for Possible Associations with Congenital Heart Diseases

Background

The human heart consists of several cell types with distinct lineage origins. Interactions between these cardiac progenitors are very important for heart formation. The muscle segment homeobox gene family plays a key role in the cell morphogenesis and growth, controlled cellular proliferation, differentiation, and apoptosis, but the relationships between the genetic abnormalities and CHD phenotypes still remain largely unknown. The aim of this work was to evaluate variations in MSX1 and MSX2 for their possible associations with CHD.

Methods

We sequenced the MSX1 and MSX2 genes for 300 Chinese Han CHD patients and 400 normal controls and identified the variations. The statistical analyses were conducted using Chi-Square Tests as implemented in SPSS (version 19.0). The Hardy-Weinberg equilibrium test of the population was carried out using the online software OEGE.

Results

Six variations rs4647952, rs2048152, rs4242182, rs61739543, rs111542301 and rs3087539 were identified in the MSX2 gene, but the genetic heterozygosity of those SNPs was very low. In contrast, the genetic heterozygosity of two variations rs3821949 near the 5’UTR and rs12532 within 3’UTR of the MSX1 gene was considerably high. Statistical analyses showed that rs3821949 and rs12532 were associated with the risk of CHD (specifically VSD).

Conclusions

The SNPs rs3821949 and rs12532 in the MSX1 gene were associated with CHD in Chinese Han populations.     

Identification of Soybean Genes Involved in Circadian Clock Mechanism and Photoperiodic Control of Flowering Time by In Silico Analyses

Glycine max is a photoperiodic short-day plant and the practical consequence of the response is latitude and sowing period limitations to commercial crops. Genetic and physiological studies using the model plants Arabidopsis thaliana and rice （Oryza sativa） have uncovered several genes and genetic pathways controlling the process, however information about the corresponding pathways in legumes is scarce. Data mining prediction methodologies, including multiple sequence alignment, phylogeneUc analysis, bioinformaUcs expression and sequence motif pattern identification, were used to identify soybean genes involved in day length perception and photoperiodic flowering induction. We have investigated approximately 330 000 sequences from open-access databases and have identified all bona fide central oscillator genes and circadian photoreceptors from A. thaliana in soybean sequence databases. We propose a working model for the photoperiodic control of flowering time in G. max, based on the identified key components. These results demonstrate the power of comparative genomics between model systems and crop species to elucidate the several aspects of plant physiology and metabolism.     

大豆开花基因GmCO和GmFT的克隆及表达

为了研究大豆光周期反应是否受开花基因CO(CONSTANS)和FT(FLOWERING LOCUS T)调控,采用同源序列法从大豆中分离了CO和FT的同源物GmCO和GmFT.GmCO和GmFT分别编码151和109个氨基酸,与水稻和拟南芥中相关蛋白的氨基酸序列同源性达到70%以上.通过RT-PCR分析GmCO和GmFT在短日照(short daylength,SD)、自然光照(natural light,NL)和长日照(long daylength,LD)处理大豆不同发育阶段叶片中的表达发现,GmCO在LD处理大豆早期发育的叶片中高丰度表达,GmFT在SD和NL处理大豆开花时期的叶片中高丰度表达.上述结果表明,GmCO和GmFT的表达与大豆开花时间及光照长度密切相关,且GmCO抑制GmFT的表达.     

Sequence Variations in Human Immunodeficiency Virus Type 1 Nef Are Associated with Different Stages of Disease

nef alleles derived from a large number of individuals infected with human immunodeficiency virus type 1 (HIV-1) were analyzed to investigate the frequency of disrupted nef genes and to elucidate whether specific amino acid substitutions in Nef are associated with different stages of disease. We confirm that deletions or gross abnormalities in nef are rarely present. However, a comparison of Nef consensus sequences derived from 41 long-term nonprogressors and from 50 individuals with progressive HIV-1 infection revealed that specific variations are associated with different stages of infection. Five amino acid variations in Nef (T15, N51, H102, L170, and E182) were more frequently observed among nonprogressors, while nine features (an additional N-terminal PxxP motif, A15, R39, T51, T157, C163, N169, Q170, and M182) were more frequently found in progressors. Strong correlations between the frequency of these variations in Nef and both the CD4(+)-cell count and the viral load were observed. Moreover, analysis of sequential samples obtained from two progressors revealed that several variations in Nef, which were more commonly observed in patients with low CD4(+)-T-cell counts, were detected only during or after progression to immunodeficiency. Our results indicate that sequence variations in Nef are associated with different stages of HIV-1 infection and suggest a link between nef gene function and the immune status of the infected individual.     

Polymorphic Sites at the Immunoregulatory CTLA-4 Gene Are Associated with Chronic Chagas Disease and Its Clinical Manifestations

Background

Chagas disease affects approximately 10 million people mainly in Latin America. The immune regulation by the host seems to be an essential factor for disease evolution, and immune system inhibitory molecules such as CTLA-4 and PD-1 favor the maintenance of peripheral tolerance. Considering that polymorphisms at the immunoregulatory CTLA-4 and PDCD1 genes may alter their inhibitory function, we investigated the association of alleles, genotypes and haplotypes of polymorphic sites observed at the CTLA-4 and PDCD1 genes with different clinical manifestations of chronic Chagas disease (indeterminate, cardiac, digestive and mixed).

Methods

The polymorphisms at the CTLA-4 (-1722T/C, -318C/T and +49A/G) and PDCD1 (PD-1.3G/A) genes were typed using TaqMan methodology in 277 chronic Chagas disease patients classified into four groups, according to clinical characteristics, and 326 non-infected controls.

Results

Our results showed that CTLA-4 -1722CC genotype (22%), -1722C allele (27%) and CTLA-4 TCG (8.6%), TCA (26%) and CCA (15%) haplotypes were strongly associated with the indeterminate form, while the CTLA-4 -318CT genotype (82%) and CTLA-4 -318T allele (47%) were found mainly in patients with the mixed form of the disease. The CTLA-4 TCG haplotype (10.2%) was associated with the digestive form. On the other hand, the PD-1.3G/A polymorphism was not associated with chronic Chagas disease and its clinical manifestations.

Conclusions

Here, we showed that alleles, genotypes and haplotypes reported to increase the expression of the regulatory molecule CTLA-4 were associated with the indeterminate form of the disease. Taken together, our data support the idea that polymorphic sites at immunoregulatory genes may influence the development of Chagas disease variants.     

大豆耐盐机理及相关基因分子标记

大豆耐盐涉及多种生理代谢途径.耐盐大豆能够通过Cl-排除、控制Na 的吸收和转运、合成渗透调节物质、改变细胞膜膜脂组分及相关酶类的活性等多种形式来适应盐胁迫;野生大豆群体具有盐腺,从形态结构上适应盐逆境;大豆-根瘤菌共生体在盐胁迫下通过互作来提高整体的耐盐性.分子生物学技术应用于大豆耐盐研究,已获得了一些与耐盐相关基因连锁的分子标记.广泛搜集筛选大豆栽培种和野生种资源,利用分子生物学技术和基因工程提高大豆耐盐性,将成为未来大豆耐盐研究的主要内容.     

大豆耐盐机理及相关基因分子标记

大豆耐盐涉及多种生理代谢途径。耐盐大豆能够通过Cl-排除、控制Na+的吸收和转运、合成渗透调节物质、改变细胞膜膜脂组分及相关酶类的活性等多种形式来适应盐胁迫;野生大豆群体具有盐腺,从形态结构上适应盐逆境;大豆-根瘤菌共生体在盐胁迫下通过互作来提高整体的耐盐性。分子生物学技术应用于大豆耐盐研究,已获得了一些与耐盐相关基因连锁的分子标记。广泛搜集筛选大豆栽培种和野生种资源,利用分子生物学技术和基因工程提高大豆耐盐性,将成为未来大豆耐盐研究的主要内容。