Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight

Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci （QTLs）. We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.

OsDA1 positively regulates grain width in rice

The size and shape of rice grains influence their yield and commercial value.We investigated the role of OsDA1,a rice homolog of the Arabidopsis DA1 gene,in regulating grain size and shape.OsDA1 was highly expressed in young spikelets and glumes.Its overexpression led to enlarged seeds with increased width and decreased length/width ratio(LWR)and knocking out OsDA1 reduced grain width and increased grain length and LWR.A R310K point mutation in the DA1-like domain is a potential target for breeding for increased grain width and length.OsDA1 interacted with TCP gene-family proteins to regulate grain size and shape.Our findings deepen our understanding of the molecular mechanisms underlying grain size regulation and provide useful information for improving grain yield.

Fine-Mapping of qTGW1.2a, a Quantitative Trait Locus for 1000-Grain Weight in Rice

Thousand-grain weight (TGW) is a key component of grain yield in rice. This study was conducted to validate and fine-map qTGW1.2a, a quantitative trait locus for grain weight and grain size previously located in a 933.6-kb region on the long arm of rice chromosome 1. Firstly, three residual heterozygotes (RHs) were selected from a BC2F11 population of the indica rice cross Zhenshan 97 (ZS97)///ZS97//ZS97/Milyang 46. The heterozygous segments in these RHs were arranged successively in physical positions, forming one set of sequential residual heterozygotes (SeqRHs). In each of the populations derived, non-recombinant homozygotes were identified to produce near isogenic lines (NILs) comprising the two homozygous genotypes. The NILs were tested for grain weight, grain length and grain width. QTL analyses for the three traits were performed. Then, the updated QTL location was followed for a new run of SeqRHs identification-NIL development-QTL mapping. Altogether, 11 NIL populations derived from four sets of SeqRHs were developed and used. qTGW1.2a was finally delimitated into a 77.5-kb region containing 13 annotated genes. In the six populations segregating this QTL, which were in four generations and were tested across four years, the allelic direction of qTGW1.2a remained consistent and the genetic effects were stable. For TGW, the additive effects ranged from 0.23 to 0.38 g and the proportions of phenotypic variance explained ranged from 26.15% to 41.65%. These results provide a good foundation for the cloning and functional analysis of qTGW1.2a.

Functional Marker Development and Effect Analysis of Grain Size Gene GW2 in Extreme Grain Size Germplasm in Rice

GW2 is an important gene that regulates grain width and weight. We used cDNA clone to obtain the sequences of GW2 from large- and small-grained rice varieties, TD70 and Kasalath, respectively. Then, we developed a dCAPS （derived cleaved amplified polymorphic sequence） marker on the basis of the sequence difference between functional and nonfunctional GW2 genes to analyze the genotypes and phenotypes of recombinant inbred lines. Results showed that the sequence of GW2To7~ had a single nucleotide deletion at site 316 that generates a termination codon. This codon terminated the GW2 protein in advance. By contrast, the sequence of GW2Kasalath encoded an intact protein. A novel dCAPS marker was designed in accordance with a base A deletion at site 316 of the sequence. After the PCR product was digested by Apol, TD70 showed 21 and 30 bp fragments, and Kasalath showed a 51 bp fragment. Up to 82 lines contained GW2TDTO, and 158 lines contained GW2Kasalath. The lines that contained TD70 alleles displayed substantial increases in width and 1000-grain weight. This result suggested that GW2 played a critical role in rice breeding.

Natural variation of an autophagy-family gene among rice subspecies affects grain size and weight

Elucidating the genetic basis of natural variation in grain size and weight among rice varieties can help breeders develop high-yielding varieties.We identified a novel gene,GW3a(Grain Weight 3a)(LOC_Os03g27350),that affects rice grain size and weight.gw3a mutants showed higher total starch content and dry matter accumulation than the wild type(WT),Nipponbare,suggesting that GW3a negatively regulates grain size and weight.Moreover,our study found that GW3a interacted with OsATG8 by cleaving it,suggesting that GW3a may be involved in the assembly of autophagosomes and starch degradation in plants.The haplotype analysis of GW3a showed functional differences between indica and japonica rice.Taken together,we conclude that GW3a is expressed in the autophagosome pathway regulating starch metabolism in rice,affecting yield-related traits,such as grain size,grain weight and thousand grain weight(TGW).Our findings also shed new light on autophagy-mediated yield trait regulation,proposing a possible strategy for the genetic improvement of high-yield germplasm in rice.

基于染色体片段置换系的野生稻粒宽QTL-qGW8.1的精细定位

利用以栽培稻9311为受体、普通野生稻为供体的染色体单片段置换系CSSL182,检测到一个与粒宽相关的QTL。CSSL182与受体亲本9311粒型性状差异显著,且只在8号染色体有一个野生稻导入片段。构建CSSL182/9311的F_2次级分离群体,将粒宽QTL初定位在8号染色体的标记RM447和RM264之间,贡献率达22.49%,将该QTL命名为qGW8.1。随后进一步设计区间内多态性分子标记引物,检测F_2群体的2000株分离个体以及F_(2∶3)群体交换单株,结合后代表型验证,最终将qGW8.1精细定位到8号染色体10 kb区间内。该区间内含有3个候选基因,基因测序发现这3个基因在双亲之间均含有丰富的变异。对双亲子粒颖壳细胞电镜扫描观察发现,CSSL182的颖壳细胞宽度比9311减少16.7%。这一结果表明qGW8.1中来自野生稻的等位基因通过改变颖壳细胞形状影响粒型。

Identification and validation of major QTL for grain size and weight in bread wheat(Triticum aestivum L.)

Grain size and weight are key components of wheat yield.Exploitation of major underlying quantitative trait loci(QTL)can improve yield potential in wheat breeding.A recombinant inbred line(RIL)population was constructed to detect QTL for thousand-grain weight(TGW),grain length(GL)and grain width(GW)across eight environments.Genomic regions associated with grain size and grain weight were identified on chromosomes 4A and 6A using bulked segregant exome sequencing(BSE-Seq)analysis.After constructing genetic maps,six major QTL detected in at least four individual environments and in best linear unbiased estimator(BLUE)datasets,explained 7.50%-23.45%of the phenotypic variation.Except for QGl.cib-4A,the other five QTL were co-located in two regions,namely QTgw/Gw.cib-4A and QTgw/Gw/Gl.cib-6A.Interactions of these QTL were analyzed.Unlike QTgw/Gw/Gl.cib-6A,QTgw/Gw.cib-4A and QGl.cib-4A had no effect on grain number per spike(GNS).The QTL were validated in a second cross using Kompetitive Allele Specific PCR(KASP)markers.Since QTgw/Gw.cib-4A was probably a novel locus,it and the KASP markers reported here can be used in wheat breeding.TraesCS4A03G0191200 was predicted to be potential candidate gene for QTgw/Gw.cib-4A based on the sequence differences,spatiotemporal expression patterns,gene annotation and haplotype analysis.Our findings will be useful for fine mapping and for marker-assisted selection in wheat grain yield improvement.

Identification of QTLs for grain size and characterization of the beneficial alleles of grain size genes in large grain rice variety BL129

Grain size is one of the most important agronomic components of grain yield. Grain length, width and thickness are controlled by multiple quantitative trait loci （QTLs）. To understand genetic basis of large grain shape and explore the beneficial alleles for grain size improvement, we perform QTL analysis using an F2 population derived from a cross between the japonica variety Beilu 129 （BL129, wide and thick grain） and the elite indica variety Huazhan （HZ, narrow and long grain）. A total number of eight major QTLs are detected on three different chromosomes. QTLs for grain width （qGW）, grain thickness （qGT）, brown grain width （qBGW）, and brown grain thickness （qBGT） explained 77.67, 36.24, 89.63, and 39.41% of total phenotypic variation, respectively. The large grain rice variety BL129 possesses the beneficial alleles of GW2 and qSW5/ GW5, which have been known to control grain width and weight, indicating that the accumulation of the beneficial alleles causes large grain shape in BL129. Further results reveal that the rare gw2 allele from BL129 increases grain width, thickness and weight of the elite indica variety Huazhan, which is used as a parental line in hybrid rice breeding. Thus, our findings will help breeders to carry out molecular design breeding on rice grain size and shape.

水稻粒宽突变体gw4的鉴定与基因定位

【目的】水稻粒形是影响水稻产量和决定稻米外观品质的主要性状之一。筛选和鉴定新的粒形突变材料,可为研究水稻籽粒发育的调控机制奠定基础。【方法】粳稻品种中花11经1%的EMS处理,在诱变群体中获得一份窄粒突变体gw4(grain width on chromosome 4);分析粒形和其他主要农艺性状,在扫描电镜下观察颖壳细胞变化;利用突变体与籼稻品种台中本地1号配组的F2分离群体,选择隐性个体完成基因的精细定位;开展生物信息和测序分析,确定定位区间的候选基因;采用RT-PCR分析该基因在根、茎、叶、鞘、穗等组织中的表达模式及其他粒形相关基因的表达水平。【结果】与野生型相比,除了表现窄粒外,gw4的粒长、千粒重、每穗粒数、一次枝梗数和二次枝梗数等显著下降;扫描电镜发现gw4的颖壳内外表皮细胞均小于野生型;遗传分析表明该窄粒表型受一对单隐性核基因控制;通过开发的新标记最终将该基因定位在第4染色体BS6与EX49两个标记之间约31.74 kb的范围内;测序结果发现在LOC_Os04g01590基因编码区发生了一个由G至A的单碱基突变,导致原来编码的甘氨酸变成了天冬氨酸;qRT-PCR结果表明,LOC_Os04g01590主要在幼穗中表达,且在突变体中表达显著下降。【结论】GW4主要调控水稻粒宽的发育,预测LOC_Os04g01590为其候选基因。这为进一步丰富粒形的遗传调控网络打下了基础。

基于55K芯片小麦籽粒相关性状的QTL定位分析

为了进一步挖掘小麦籽粒相关性状的主效QTL位点,探索籽粒性状之间的遗传关系,利用籽粒性状差异较大的小麦品种安农859和武农988构建的124份DH群体为研究材料,分别测定2 a 7个环境下的粒长、粒宽及千粒质量表型值,开展籽粒性状多元回归分析,并基于DH群体的55K芯片数据进行籽粒相关性状QTL检测。结果表明,多元回归分析中,粒宽对千粒质量的贡献最大。通过完备区间作图对籽粒性状进行QTL定位,除6D和7B染色体外,其他19条染色体上共检测到69个有关籽粒性状的QTL,包括24个千粒质量QTL、28个粒长QTL、17个粒宽QTL,单个QTL的表型解释率为6.87%~27.74%。其中,7A染色体上粒长相关的Qgl.ahau-7A.1在7个环境及BLUP下均被检测到,表型解释率为9.48%~22.26%,加性效应为0.11~0.21 mm,物理区间4.91 Mb(AX-110430243~AX-110442528),可能为新的主效QTL。因此,Qgl.ahau-7A.1位点可作为后续精细定位和分子标记辅助育种重点关注的区域。

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.

深冷处理对纯银组织与性能的影响

采用深冷处理工艺,系统研究直径1.772 mm纯银显微组织、力学性能及电导率的变化。实验表明深冷处理后纯银晶粒尺寸与形貌并未发生显著变化,但其显微硬度由初始65.8 Hv增加至68.9 Hv,电导率由初始99.45%IACS增至101.28%IACS。X射线衍射仪分析得出,纯银经深冷处理后晶向峰值(220)、(311)均增强,组织均匀性得以提升,各晶向的半高宽产生一定程度降低。通过分析晶粒尺寸变化,得出半高宽值的降低主要由于深冷处理诱导微观应变的减小,而这有利于电导率的提升。

利用BC2F2高代回交群体定位水稻籽粒大小和形状QTL

以我国优良籼稻恢复系蜀恢527为轮回亲本,以来自菲律宾的Milagrosa为供体亲本,培育了样本容量为199株的BC2F2高代回交群体。选取85个均匀分布在12条染色体上的多态性SSR标记进行基因型分析,同时对粒长、粒宽、长宽比和千粒重4种性状进行了表型鉴定。采用性状-标记间的单向和双向方差分析对上述性状进行了QTL定位。单向方差分析(P<0.01)共检测到了10个控制粒长、粒宽、长宽比和千粒重的QTL,其中有3个具有多效性。由于粒长和长宽比的高度相关性,控制长宽比的2个QTL均能在粒长QTL中检测到。位于第3染色体着丝粒区域的qgl3b是一个控制粒长、长宽比和千粒重的主效QTL,它可以分别解释粒长、长宽比和千粒重表型变异的29.37%、26.15%和17.15%。该QTL对于粒长、长宽比和千粒重均表现较大的加性效应(来自蜀恢527的等位基因为增效)和负向超显性。位于第8染色体的qgw8位点是一个控制粒宽的主效QTL,同时也是控制千粒重的微效QTL,能解释粒宽表型变异的21.47%和千粒重表型变异的5.16%。该QTL对粒宽和千粒重均具有较大的加性效应(来自蜀恢527的等位基因为增效)和正向部分显性。双向方差分析(P<0.005)共检测到61对显著的上位性互作,涉及54个QTL,其中23个是能同时影响2～4个性状的多效位点,且有8个位点与单向方差分析检测到的相同。控制长宽比的13对上位性互作位点中,与控制粒长的上位性互作位点完全相同的有8对。以上结果为进一步开展水稻籽粒大小和形状有利基因的精细定位、克隆和分子设计育种奠定了基础。

利用籼粳回交群体分析水稻粒形性状相关QTLs

水稻谷粒的外观性状对稻米外观品质存在重要的影响。该研究利用SSR标记 ,以回交群体Balilla/NTH∥Balilla为作图群体 ,构建了水稻 12条染色体的连锁图 ,该遗传图谱包括 10 8个分子标记 ,平均图距为 11 9cM。以构建的遗传图谱为基础 ,采用区间作图法对谷粒外观性状 ,包括粒长、粒宽和粒形进行了数量性状基因 (QTL)定位。结果表明 ,粒长、粒宽和粒形在回交群体中均呈近似的正态分布 ,表现出典型的数量性状特征。QTL定位结果表明 ,第 12染色体上RM10 1 RM2 70区间内存在一个与粒长性状相关的QTL ,(qGL 12 ) ,加性效应约为 0 2 6mm ,贡献率为 16 7%。在第 2和第 3染色体上RM15 4 RM2 11和RM2 5 7 RM175区间内 ,分别检测到qGW 2和qGW 3两个位点与粒宽性状有关 ,加性效应为分别为 - 0 10mm和 - 0 12mm ,贡献率分别为 11 5 %和 16 6 %。对于粒形性状 ,共检测到 3个QTLs,qLW 2 ,qLW 6和qLW 7,分别位于第 2、6和 7染色体上。其中qLW 2和qLW 7的加性效应分别约为0 0 9和 0 10 ,两个QTLs分别可解释表型变异的 12 7%和 18 3% ;而qLW 6的加性效应约为 - 0 13,可解释粒形变异的 11 5 %。文中还讨论了粒形和稻米外观品质同时改良的可能性。

不同生态环境下冬小麦籽粒大小相关性状的QTL分析

【目的】鉴定影响籽粒大小相关性状的QTL,并估计QTL的表型效应;分析不同环境下QTL的稳定性。【方法】以冬小麦小粒地方品种和尚麦为母本,大粒育成品种豫8679为父本及其F7:8重组自交系的142个株系为试验材料,分析籽粒长度、宽度、厚度、体积及千粒重在北京(2006、2007)、合肥(2007)和成都(2007)4个不同环境下的性状表现,并利用已构建的含有170个SSR标记和2个EST标记的遗传图谱,对这5个性状进行QTL定位分析。【结果】4个环境下共检测到93个影响籽粒长度、宽度、厚度、体积及千粒重的QTL,这些QTL分布在除1D和6A之外的所有小麦染色体上。在检测到的QTL中,与籽粒长度、宽度、厚度、体积和千粒重相关的QTL分别为17、16、18、21和21个。另外,本研究还在1A、1B、2A、2D、3A、3B、5A、5B、5D、6A、6D、7B和7D染色体上共发现了18个QTL富集区。【结论】获得93个影响小麦籽粒大小相关性状的QTL,这些QTL可作为利用分子标记辅助育种途径进行小麦遗传改良的依据。

利用水稻重组自交系群体定位谷粒外观性状的数量性状基因

用区间作图和混合线性模型的复合区间作图两种方法 ,对水稻 (OryzasativaL .)珍汕 97和明恢 6 3组合的重组自交系群体的谷粒外观性状———粒长、粒宽和粒形进行了数量性状基因 (QTL)定位。用区间作图法在LOD≥ 2 .4水平上 (近似于α =0 .0 0 5) ,1 998年对粒长、粒宽和粒形分别检测到 6、2和 2个QTLs ;1 999年对以上 3个性状分别检测到 3、2和 2个QTLs。其中 7个QTLs在两年均检测到。位于第 3染色体RG393-C1 0 87区间的QTL效应大 ,同时影响粒长和粒形 ,两年贡献率分别为 57.5%、6 1 .4%和 2 6 .7%、2 9.9%。位于第 5染色体RG36 0 -C734B区间的QTL效应大 ,同时影响粒宽和粒形 ,两年贡献率分别为 44.2 %、53.2 %和 32 .1 %、36 .0 %。用混合线性模型的复合区间作图法在P =0 .0 0 5水平上 ,对粒长、粒宽和粒形分别检测到 8、5和 5个QTLs ,共解释各自性状变异的 58.81 %、44.75%和 57.47%。只检测到 1个QTL与环境之间存在显著互作。

煤层气储层煤粉运移规律试验研究

在研究煤粉形成及运移规律的基础上,通过室内人造裂缝的方法,开展了不同粒径的煤粉在不同裂缝宽度、不同流动速度下的运移规律试验,研究了裂缝宽度、流动速度、煤粉粒径与出粉量、渗透率的关系,对制定生产中煤粉控制措施具有重要的指导意义。研究结果表明,随着流量的增大,煤心出口端煤粉含量增多,煤岩的渗透率下降,渗透率伤害率升高;在排水的初始阶段,在相同裂缝宽度、相同颗粒直径下,随着排水体积的增加,渗透率伤害率非常大,但是排出一定体积之后再继续排水,渗透率下降速度减慢。

稻米粒形与主要品质性状的关系

以65个水稻品系为试材,研究了稻米粒形(长宽比)与主要品质性状的关系。结果表明,糙米率和整精米率与长宽比呈显著负相关,精米率与长宽比呈正相关,但是没有达到显著水平,减小长宽比有利于改善加工品质。垩白粒率和垩白度与长宽比呈负相关,没有达到显著水平,稻米粒形不是决定外观品质的主要因素。直链淀粉和蛋白质含量与长宽比虽然呈显著正相关,但是点的分布比较分散,并无必然联系。

二棱大麦与六棱大麦籽粒性状的差异性及其相关性

为了解不同棱型大麦籽粒性状的差异性及其千粒重与粒型性状的相关性,对98份二棱大麦和89份六棱大麦品种(系)在不同试点的千粒重、粒长、粒宽进行测定和分析。结果表明,二棱大麦千粒重、粒长和粒宽普遍高于六棱大麦,不同棱型大麦千粒重、粒长和粒宽在基因型间及环境间差异均达到显著或极显著水平。经过聚类分析,依据千粒重将二棱大麦和六棱大麦聚为高、中、低粒重3类。随着千粒重的减小,二棱大麦千粒重与粒长之间由显著正相关变为不显著负相关,六棱大麦则由不显著正相关变为显著正相关。二棱大麦和六棱大麦各类品种(系)的平均千粒重与粒宽均呈极显著正相关,而且相关系数均高于千粒重与粒长的相关系数。

利用DH群体定位水稻谷粒外观性状的QTL

采用混合线性模型的复合区间作图方法,对水稻“圭630”和“02428”组合的DH群体的谷粒外观性状——粒长、粒宽和粒形进行了数量性状基因定位,同时对定位的主效应和上位性进行了环境效应分析。2002年对粒长、粒宽和粒形分别检测到5、4和2个QTLs;2003年对以上3个性状分别检测到3、4和4个QTLs。其中4个QTLs在2年均检测到,且其贡献率较大。位于第4染色体C22-RG449d区间的QTL效应大,同时影响粒长和粒宽,2年内均被检测到。联合2年数据分析分别检测到6个粒长QTLs、6个粒宽QTLs和3个粒形QTLs,共解释各自性状变异的67.71%、50.08%和29.17%,且影响粒形的3个QTLs同时影响粒长或粒宽。对粒形和粒宽分别检测到4个QTLs与环境之间存在显著互作。本实验中检测到主效应和上位性对谷粒外观性状均具有重要作用,但上位性贡献率相对主效应较小,环境互作效应更小。