GS6, A Member of the GRAS Gene Family,Negatively Regulates Grain Size in Rice

Grain size is an important yield-related trait in rice. Intensive artificial selection for grain size during domestication is evidenced by the larger grains of most of today’s cultivars compared with t...     

Stimulation of RecA-mediated cleavage of phage phi 80 cI repressor by deoxydinucleotides

The presence of either deoxyguanylyl-(3'----5')-deoxyguanosine (d(G-G] or deoxyadenylyl-(3'----5')-deoxyguanosine (d(A-G] greatly stimulates cleavage of the phage phi 80 cI repressor mediated by the Escherichia coli RecA protein in vitro. No other deoxydinucleoside monophosphate or riboguanylyl-(3'----5')-guanosine (r(G-G] affects the cleavage reaction. Neither the cleavage site of the phi 80 cI repressor nor the requirement for single-stranded DNA and ATP for cleavage is altered by d(G-G). Photoaffinity labeling experiments with 32P-labeled 5'-phosphoryl deoxyguanylyl deoxyguanosine (pd(G-G], which also stimulates cleavage, show that pd(G-G) bound to the repressor under the conditions in which the repressor is cleaved by RecA protein. The binding increases the affinity of the repressor for RecA protein and thus greatly stimulates repressor cleavage. The cleavage reactions of LexA and lambda cI repressors by RecA protein are not affected by d(G-G).     

GW5‐Like,a homolog of GW5, negatively regulates grain width,weight and salt resistance in rice

Grain size is an important determinant of yield potential in crops. We previously demonstrated that natural mutations in the regulatory sequences of qSW5/GW5 confer grain width diversity in rice. However, the biological function of a GW5 homolog, named GW5-Like(GW5 L), remains unknown. In this study, we report on GW5 L knockout mutants in Kitaake, a japonica cultivar(cv.)considered to have a weak gw5 variant allele that confers shorter and wider grains. GW5 L is evenly expressed in various tissues, and its protein product is localized to the plasma membrane. Biochemical assays verified that GW5 L functions in a similar fashion to GW5. It positively regulates brassinosteroid(BR) signaling through repression of the phosphorylation activity of GSK2. Genetic data show that GW5 L overexpression in either Kitaake or a GW5 knockout line, Kasaorf3(indica cv. Kasalath background), causes more slender, longer grains relative to the wild-type. We also show that GW5 L could confer salt stress resistance through an association with calmodulin protein OsCa M1-1. These findings identify GW5 L as a negative regulator of both grain size and salt stress tolerance, and provide a potential target for breeders to improve grain yield and salt stress resistance in rice.     

Validation of gene based marker-QTL association for grain dimension traits in rice

Validation of marker-QTL association for genes grain size 3 (GS3), grain weight 2 (GW2), seed width 5 (qSW5) and a QTL qgrl7.1 for grain length was undertaken in a set of 242 diverse rice germplasm. Further, the study was extended to an F₂ mapping population derived from cross of Sonasal, a short grain aromatic rice landrace with Pusa Basmati 1121, a variety with extra long slender grains. Seven gene specific markers, namely, SF28, SR17, RGS1and RGS2 based on GS3, W004 for GW2, MS40671 for qSW5 and RM505 for qgrl7.1, were used for validation. Single marker analysis revealed significant association of these markers to grain size and shape. The marker SF28 explained highest phenotypic variance (37 %) while the marker W004 explained lowest variance (2.6 %) for grain length in the germplasm set at the significance level P?<?0.05. Three markers namely, SF28, MS40671 and RM505 were polymorphic between the parents Sonasal and Pusa Basmati 1121. In the F₂ population, the marker SF28 linked to gene GS3 explained highest phenotypic variance (32.5 %), while RM505 linked to qgrl7.1 explained 5.4 % of phenotypic variance for grain length. The marker SF28 was found to be most robust in the validation studies both in germplasm and F₂ population. The validated gene specific markers can be utilised in marker assisted selection for improving grain size and shape as these traits have significant contribution towards grain quality and grain yield. This is the first study on validation of gene based markers for grain dimension traits in Indian rice germplasm.     

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.     

The binding of guanine-guanine mismatched DNA to naphthyridine dimer immobilized sensor surfaces: kinetic aspects

Naphthyridine dimer composed of two naphthyridine chromophores and a linker connecting them strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. The kinetics for the binding of the G-G mismatch to the naphthyridine dimer was investigated by surface plasmon resonance assay. The sensor surface was prepared by immobilizing naphthyridine dimer through a long poly(ethylene oxide) linker with the ligand density of 9.1 x 10(-12) fmolnm(-2). The kinetic analyses revealed that the binding of the G-G mismatch was sequence dependent on the flanking base pairs, and the G-G mismatches flanking at least one G-C base pair bound to the surface via a two-step process with a 1:1 DNA-ligand stoichiometry. The first association rate constant for the binding of the G-G mismatch in the 5'-CGG-3'/3'-GGC-5' sequence to the naphthyridine dimer-immobilized sensor surface was 3.2 x 10(3)M(-1)s(-1) and the first dissociation rate constant was 1.4 x 10(-2)s(-1). The association and dissociation rate constants for the second step were insensitive to the flanking sequences, and were almost of the same order of magnitude as the first dissociation rate constant. This indicates that the second step had only a small energetic contribution to the binding. The association constant calculated from kinetic parameters was 2.7 x 10(5)M(-1), which is significantly smaller than the apparent association constants obtained from experiments in solution. Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on the complex produced from the G-G mismatch and naphthyridine dimer showed the formation of the 1:1 complex and a 1:2 DNA-ligand complex in solution. The latter complex became the dominant complex when a six-fold excess of naphthyridine dimer was added to DNA.     

Dectin-1 Polymorphism: A Genetic Disease Specifier in Autism Spectrum Disorders?

Introduction

In autism spectrum disorders (ASD), complex gene-environment interactions contribute to disease onset and progress. Given that gastro-intestinal dysfunctions are common in ASD, we postulated involvement of microbial dysbiosis in ASD and investigated, under a case-control design, the influence of DNA polymorphisms in the CLEC7A gene that encodes a pivotal fungal sensor, Dectin-1.

Material and methods

DNAs from 478 ASD patients and 351 healthy controls (HC) were analyzed for the CLEC7A rs16910631G/A and rs2078178 A/G single nucleotide polymorphisms (SNPs). Differences in the distribution of allele, genotype and haplotype by Chi-square testing and nonparametric analysis by Kruskal-Wallis/Mann–Whitney tests, where appropriate, were performed. The free statistical package R.2.13 software was used for the statistical analysis.

Results

We found that the CLEC7A rs2078178 G allele and GG genotype were more prevalent in HC as compared to ASD but failed to reach statistical significance for the latter (pc = 0.01, 0.06 respectively). However, after phenotype-based stratification, the CLEC7A rs2078178 G allele and GG genotype were found to be significantly more frequent in the Asperger group as compared to other ASD subsets (pc = 0.02, 0.01), a finding reinforced by haplotype analysis (rs2078178/rs16910631 G-G/G-G) (pc = 0.002). Further, intellectual quotient (IQ)-based stratification of ASD patients revealed that IQ values increase linearly along the CLEC7A rs2078178 AA, AG and GG genotypes (p = 0.05) and in a recessive manner (GG vs. AA+AG p = 0.02), further confirmed by haplotype distribution (CLEC7A rs2078178-16910631; A-G/A-G, A-G/G-G and G-G/G-G, p = 0.02, G-G/G-G vs. others, p = 0.01).

Conclusion

Our data suggest that the genetic diversity of CLEC7A gene influences the ASD phenotype by behaving as a disease specifier and imply that the genetic control of innate immune response could determine the ASD phenotype.     

籼稻稻米碾磨与外观品质性状的QTL定位

文章利用籼籼交组合特青/IRBB衍生的重组自交系群体, 在2个环境下对稻米碾磨品质和外观品质进行QTL定位。共计检测到控制稻米碾磨品质的QTL 12个和控制外观品质的QTL 18个, 包括糙米率8个、精米率2个、整精米率2个、粒长7个、粒宽5个和长宽比6个, 这些QTL分布于除第4和12染色体外的其他10条染色体上。其中, 第3染色体涵盖粒形基因GS3的区域对粒长、长宽比、糙米率和整精米率具有较大效应, 其献率分别为56.71%、42.23%、10.05%和4.91%; 第5染色体涵盖粒宽基因GW5的区域对粒宽、长宽比、糙米率和精米率具有较大效应, 表型变异贡献率分别为59.51%、36.68%、19.51%和4.56%。此外, 第6染色体涵盖直链淀粉含量基因Wx的区域对糙米率和精米率具有较小效应。GS3和GW5对糙米率和粒形具有重要作用。     

Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production

The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.     

水稻基因组加倍对籽粒大小调控基因表达的影响

水稻是最重要的粮食作物之一,提高水稻产量一直是育种的主要目标。水稻四倍体相对于二倍体具有籽粒变大、粒重增加的特点,研究基因组加倍后籽粒大小基因的调控模式,在育种应用方面具有十分重要的意义。本文以二倍体 -四倍体水稻为材料,分析6个控制籽粒大小基因在幼穗发育中的表达差异,同时结合转基因实验,探讨基因剂量增加对基因表达水平和籽粒大小的影响。结果发现：基因组加倍后,水稻的发育进程不变,但株高增加,叶片变宽,籽粒变大,增大后的籽粒在籼稻表现为长、宽均增加显著,而在粳稻中长度比宽度增加更为明显。进一步分析控制籽粒大小基因的表达差异情况,发现这些基因的表达不仅受发育时期的影响,在籼粳亚种间也明显不同,即受遗传背景的影响。在基因组加倍的情况下,正调控基因GS5、HGW的表达普遍高于对应的二倍体;负调控基因GS3在籼稻D9311中趋于下调或沉默,而在粳稻DBl中趋于上调,GW2在D9311中上调,而在DBl中趋于沉默。通过转基因实验分析负调控基因GW2在二倍体Bl中的表达趋势,发现其在基因剂量线性增加的情况下,表达水平高于二倍体和四倍体,导致其籽粒变小。本研究结果有助于了解水稻中控制籽粒大小的基因在二倍体和四倍体中的表达模式,为高产育种提供理论依据。     

BEAK-SHAPED GRAIN 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice

Grain shape and size both determine grain weight and therefore crop yield. However, the molecular mechanisms controlling grain shape and size are still largely unknown. Here, we isolated a rice mutant, beak-shaped grain1 (bsg1), which produced beak-shaped grains of decreased width, thickness and weight with a loosely interlocked lemma and palea that were unable to close tightly. Starch granules were also irregularly packaged in the bsg1 grains. Consistent with the lemma and palea shapes, the outer parenchyma cell layers of these bsg1 tissues developed fewer cells with decreased size. Map-based cloning revealed that BSG1 encoded a DUF640 domain protein, TRIANGULAR HULL 1, of unknown function. Quantitative PCR and GUS fusion reporter assays showed that BSG1 was expressed mainly in the young panicle and elongating stem. The BSG1 mutation affected the expression of genes potentially involved in the cell cycle and GW2, an important regulator of grain size in rice. Our results suggest that BSG1 determines grain shape and size probably by modifying cell division and expansion in the grain hull.     

Pathways of Androgenesis and Observations on Cultured Pollen Grains in Rice (Oryza saliva Subsp. Keng)

(1) Pathways A-V, A-G, A-GV, and B existed in androgenesis of rice. Usually B pathway was predominant. Degeneration could happen on androgenic grains via each pathway during the early stage of androgenesis. (2) Statistics suggested that the multinucleate grains might be converted to multicellular grains by a later formation of cell walls and were consequently one of the sources of callus. (3) Androgenic grains was usually of medium size (35–40 μm) with dense protoplasm. In isolated pollen culture, the diameter of androgenic grains increased 2.5–5μm per day. Before the breakdown of exine, the cell walls seemed to be thickened and the grains would contract abruptly. (4) Small, slowly-moving starch granules were usually contained within multicellular grains cultured isolately. They were speculated to participate actively in the metabolism of androgenic grains.     

小麦籽粒发育及淀粉晶体特性对花后高温胁迫的响应

选用2个品质类型和成熟期不同的新疆主栽小麦品种‘新春11号’和‘新春39号’,分别进行花后灌浆早期高温(花后5~8d,32℃,T_1)和中期高温(花后15~18d,38℃,T_2)处理,分析花后高温对小麦籽粒发育及淀粉晶体的影响。结果显示:(1)T_1处理明显降低了两品种籽粒长度和粒重,而T_2处理显著影响籽粒宽度和厚度;高温处理虽然降低了籽粒灌浆速率,但两品种灌浆最大峰值出现时间均在花后18d。(2)T_1处理对小麦籽粒A型淀粉粒形态的影响较大,中熟品种‘新春11号’的A型淀粉粒表面在花后10d时可观察到微孔,在花后15~20d时其粒径明显小于同期对照,在花后20~25d时淀粉粒表面压痕增多且A、B型淀粉粒表面出现明显缢缩;而早熟品种‘新春39号’淀粉粒形态和粒径大小受花后高温的影响相对较小。(3)两品种在不同高温处理下,其淀粉粒晶体特性衍射峰出现的位置相同,但淀粉粒的尖峰强度不同,表明高温胁迫不影响淀粉粒的晶体类型,但可能改变了淀粉粒内部的层状结构。研究表明,花后早期高温不仅对小麦籽粒外部形态有较大的影响,同时也影响到籽粒内部淀粉粒的形态和晶体的特性。     

Gains in QTL detection using an ultra-high density SNP map based on population sequencing relative to traditional RFLP/SSR markers

Huge efforts have been invested in the last two decades to dissect the genetic bases of complex traits including yields of many crop plants, through quantitative trait locus (QTL) analyses. However, almost all the studies were based on linkage maps constructed using low-throughput molecular markers, e.g. restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs), thus are mostly of low density and not able to provide precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, we constructed an ultra-high density genetic map based on high quality single nucleotide polymorphisms (SNPs) from low-coverage sequences of a recombinant inbred line (RIL) population of rice, generated using new sequencing technology. The quality of the map was assessed by validating the positions of several cloned genes including GS3 and GW5/qSW5, two major QTLs for grain length and grain width respectively, and OsC1, a qualitative trait locus for pigmentation. In all the cases the loci could be precisely resolved to the bins where the genes are located, indicating high quality and accuracy of the map. The SNP map was used to perform QTL analysis for yield and three yield-component traits, number of tillers per plant, number of grains per panicle and grain weight, using data from field trials conducted over years, in comparison to QTL mapping based on RFLPs/SSRs. The SNP map detected more QTLs especially for grain weight, with precise map locations, demonstrating advantages in detecting power and resolution relative to the RFLP/SSR map. Thus this study provided an example for ultra-high density map construction using sequencing technology. Moreover, the results obtained are helpful for understanding the genetic bases of the yield traits and for fine mapping and cloning of QTLs.     

Translational genomics of grain size regulation in wheat

Key message

Identifying and mapping grain size candidate genes in the wheat genome greatly empowers reverse genetics approaches to improve grain yield potential of wheat.

Abstract

Grain size (GS) or grain weight is believed to be a major driving force for further improvement of wheat yield. Although the large, polyploid genome of wheat poses an obstacle to identifying GS determinants using map-based cloning, a translational genomics approach using GS regulators identified in the model plants rice and Arabidopsis as candidate genes appears to be effective and supports a hypothesis that a conserved genetic network regulates GS in rice and wheat. In this review, we summarize the progress in the studies on GS in the model plants and wheat and identify 45 GS candidate loci in the wheat genome. In silico mapping of these GS loci in the diploid wheat and barley genomes showed (1) several gene families amplified in the wheat lineage, (2) a significant number of the GS genes located in the proximal regions surrounding the centromeres, and (3) more than half of candidate genes to be negative regulators, or their expression negatively related by microRNAs. Identifying and mapping the wheat GS gene homologs will not only facilitate candidate gene analysis, but also open the door to improving wheat yield using reverse genetics approaches by mining desired alleles in landraces and wild ancestors and to developing novel germplasm by TILLING and genome editing technologies.

     

GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein

The GS3 locus located in the pericentromeric region of rice chromosome 3 has been frequently identified as a major QTL for both grain weight (a yield trait) and grain length (a quality trait) in the literature. Near isogenic lines of GS3 were developed by successive crossing and backcrossing Minghui 63 (large grain) with Chuan 7 (small grain), using Minghui 63 as the recurrent parent. Analysis of a random subpopulation of 201 individuals from the BC₃F₂ progeny confirmed that the GS3 locus explained 80–90% of the variation for grain weight and length in this population. In addition, this locus was resolved as a minor QTL for grain width and thickness. Using 1,384 individuals with recessive phenotype (large grain) from a total of 5,740 BC₃F₂ plants and 11 molecular markers based on sequence information, GS3 was mapped to a DNA fragment approximately 7.9 kb in length. A full-length cDNA corresponding to the target region was identified, which provided complete sequence information for the GS3 candidate. This gene consists of five exons and encodes 232 amino acids with a putative PEBP-like domain, a transmembrane region, a putative TNFR/NGFR family cysteine-rich domain and a VWFC module. Comparative sequencing analysis identified a nonsense mutation, shared among all the large-grain varieties tested in comparison with the small grain varieties, in the second exon of the putative GS3 gene. This mutation causes a 178-aa truncation in the C-terminus of the predicted protein, suggesting that GS3 may function as a negative regulator for grain size. Cloning of such a gene provided the opportunity for fully characterizing the regulatory mechanism and related processes during grain development.     

灌浆期不同水分处理对玉米籽粒蛋白质及其组分和相关酶活性的影响

蛋白质作为氮素代谢的终极产物,与玉米(Zea mays)籽粒品质呈正相关关系,其生物合成主要在硝酸还原酶(NRase)、谷氨酰胺合成酶(GS)、谷氨酸脱氢酶(GDH)等一系列酶催化下完成,受制于品种自身遗传特性及环境因素,栽培管理措施和生态环境条件对玉米品质具有十分重要的影响。关于土壤水分供应状况对玉米籽粒主要品质成分的分布、积累动态和相关酶活性的影响的研究尚少见报道。以两种不同类型玉米:普通玉米‘掖单22’和高油玉米‘高油115’为研究对象,采用防雨棚池栽试验。水分处理以开花期为界线,设置3种水分处理,花后不浇水(W0)、花后浇1水(灌浆期,W1)、花后浇3水(灌浆期、乳熟期、蜡熟期,W2)。结果表明:两种类型玉米籽粒蛋白质及其组分含量的积累动态基本一致,且不受土壤水分供应状况的影响。玉米籽粒蛋白质及清蛋白、谷蛋白含量,均为‘高油115’较高,球蛋白含量为‘掖单22’较高,醇溶蛋白两类型玉米含量相近。在不同水分供应条件下,两种类型玉米叶片中NRase、GS酶活性和籽粒中GS、GDH酶活性的变化动态一致,NRase酶活性自灌浆初期至成熟期一直下降,GS、GDH酶活性呈单峰曲线,在授粉后20~40 d达到高峰,充足的水分供应有利于酶的活性维持较高水平;玉米叶片中NRase酶活性,‘掖单22’高于‘高油115’,叶片GS和籽粒GDH酶活性显著低于‘高油115’。研究表明:用玉米叶片中NRase和GS活性的高低表征籽粒蛋白质含量的高低不确切,土壤水分条件与不同类型玉米穗位叶和籽粒中GS 和GDH活性关系密切。     

The effect of temperature shock and grain morphology on alpha-amylase in developing wheat grain

Background and Aims

The premature production of alpha-amylase without visible germination has been observed in developing grain of many cereals. The phenomenon is associated with cool temperatures in the late stages of grain growth but the mechanisms behind it are largely unknown. The aim of this study was to replicate the phenomenon under controlled conditions and investigate the possibility of a mechanistic link with grain size or endosperm cavity size.

Methods

Five wheat (Triticum aestivum) genotypes differing in their susceptibility to premature alpha-amylase were subjected to a range of temperature shocks in controlled environments. A comparison was then made with plants grown under ambient conditions but with grain size altered by using degraining to increase the assimilate supply. At maturity, alpha-amylase, grain area and endosperm cavity area were measured in individual grains.

Key Results

Both cold and heat shocks were successful in inducing premature alpha-amylase in susceptible genotypes, with cold shocks the most effective. Cold shocks also increased grain area. Degraining resulted in increased grain area overall, but the larger grain did not have higher alpha-amylase. Analysis of individual grain found that instances of high alpha-amylase were not associated with differences in grain area or endosperm cavity area.

Conclusions

Pre-maturity alpha-amylase is associated with temperature shocks during grain filling. In some cases this coincides with an increase in grain area, but there is no evidence of a mechanistic link between high alpha-amylase and grain or endosperm cavity area.Key words: Alpha-amylase, pre-maturity alpha-amylase, late maturity alpha amylase, temperature, grain size, endosperm cavity, wheat, Triticum aestivum