Fine mapping and characterization of a major QTL for grain length,QGl.cau-2D.1,that has pleiotropic effects in synthetic allohexaploid wheat

Grain size is one of the determinants of grain yield,and identifying the genetic loci that control grain size will be helpful for increasing grain yield.In our previous study,a quantitative trait locus(QTL)for grain length(GL),QGl.cau-2D.1,was identified from an F2 population developed from the cross between the natural(TAA10)and synthetic(XX329)allohexaploid wheat.In the present study,we mainly fine mapped and validated its genetic effects.To this end,multiple near-isogenic lines(NILs)were obtained through marker-assisted selection with TAA10 as the recurrent parent.The secondary populations derived from 25 heterozygous recombinants were used for fine mapping of QGl.cau-2D.1,and the allele from XX329 significantly increased GL,thousand-grain weight(TGW),total spikelet number per spike(TSN)and spike compactness(SC).Using NILs for XX329(2D+)and TAA10(2D−),we determined the genetic and pleiotropic effects of QGl.cau-2D.1.The target sequences were aligned with the wheat reference genome RefSeq v2.1 and spanned an~0.9 Mb genomic region.TraesCS2D03G0114900(ortholog of Os03g0594700)was predicted as the candidate gene based on whole-genome re-sequencing and expression analyses.In summary,the map-based cloning of QGl.cau-2D.1 will be useful for improving grain weight with enhanced GL and TSN.

Poaceae Orthologs of Rice OsSGL,DUF1645 Domain-Containing Genes,Positively Regulate Drought Tolerance,Grain Length and Weight in Rice

Grain yield is a polygenic trait that can be influenced by environmental factors and genetic compositions at all plant growth stages.Currently,the molecular mechanisms behind the coordination of the interaction between grain yield-related traits remain unknown.In this study,we characterized the function of four STRESS_tolerance and GRAIN_LENGTH(Os SGL)Poaceae ortholog genes that are transcribed into DUF1645 domain-containing proteins in relation to the grain length,grain weight,and drought stress-tolerance of rice.The transgenic plants with overexpressing or heterologous high levels of Poaceae OsSGL ortholog genes exhibited longer grain size than the wild type plants.Larger cells were seen in panicles of the four transgenic lines with paraffin sectioning and scanning electron microscopy analyses.In addition,four Poaceae OsSGL ortholog genes positively affected the drought tolerance of rice.Four transgenic plants displayed higher resistance to drought stress at the seedling and vegetative stages.RNA-sequencing and qRT-PCR results indicated that over-or heterologous-expression of four Poaceae OsSGL ortholog genes also affected the transcriptome of rice plants.These genes may play a role in auxin and cytokinin biosynthesis and their transduction pathways.Taken together,these results suggested that the four OsSGL orthologs have a conserved function in the regulation of stress-tolerance and cell growth by modulating hormonal biosynthesis and signaling.

Fine mapping and candidate gene analysis of qGL10 affecting rice grain length

Grain size influences the yield and quality of rice(Oryza sativa L.),and grain length is one of the component traits of grain size.In this study,a near-isogenic line LB3 with long grain size was constructed using japonica rice cultivar 02428,with short grain size,as the recipient parent and indica rice cultivar ZYX,with long grain size,as the donor parent,by multi-generation backcrossing and selfing.BSA-seq was used for preliminary QTL mapping and InDel markers were developed to fine map the locus.The major QTL,tentatively named qGL10,for grain length was located in a 128.45 kb region of chromosome 10.Combined with haplotype analysis of rice varieties,expression pattern analysis of candidate genes suggested LOC_Os10g39130(OsMADS56)as a candidate gene.Sequence alignment of OsMADS56 in 02428 and LB3 revealed that there were 15 SNPs in the promoter region and four in the coding region.Further haplotype analysis suggested that SNP9(G/A)located in the TGTCACA motif might account for the different expression levels of OsMADS56 in 02428 and LB3.These results lay a foundation for the application of qGL10 in molecular breeding of new rice varieties.

QTL-Seq Identified a Major QTL for Grain Length and Weight in Rice Using Near Isogenic F_2 Population

Mapping and isolation of quantitative trait loci(QTLs)or genes controlling grain size or weight is very important to uncover the molecular mechanisms of seed development and crop breeding.To identify the QTLs controlling grain size and weight,we developed a near isogenic line F_2(NIL-F_2)population,which was derived from a residual heterozygous plant in an F_7 generation of recombinant inbred line(RIL).With the completion of more than 30×whole genome re-sequencing of the parents,two DNA bulks for large and small grains,a total of 58.94 Gb clean nucleotide data were generated.A total of455 262 single nucleotide polymorphisms(SNPs)between the parents were identified to perform bulked QTL-seq.A candidate genomic region containing SNPs strongly associated with grain length and weight was identified from 15 to 20 Mb on chromosome 5.We designated the major QTL in the candidate region as q TGW5.3.Then,q TGW5.3 was further validated with PCR-based conventional QTL mapping method through developing simple sequence repeat and Insertion/Deletion markers in the F_2 population.Furthermore,recombinants and the progeny tests delimited the candidate region of q TGW5.3 to 1.13 Mb,flanked by HX5009(15.15 Mb)and HX5003(16.28 Mb).A set of NILs,selected from the F_2 population,was developed to evaluate the genetic effect of q TGW5.3.Significant QTL effects were detected on grain length,grain width and 1000-grain weight of H12-29 allele with 1.14 mm,-0.11 mm and 3.11 g,which explained 99.64%,95.51%and 97.32%of the phenotypic variations,respectively.

Effects of GS3 and GL3.1 for Grain Size Editing by CRISPR/Cas9 in Rice

Grain size is one of key agronomic traits associated with grain yield and grain quality. Both major quantitative trait loci GS3 and GL3.1 play a predominant role in negative regulation of grain size. In this study, a CRISPR/Cas9-mediated multiplex genome editing system was used to simultaneously edit GS3 and GL3.1 in a typical japonica rice Nipponbare. In T1 generation, we found that gs3 formed slender grain with lower chalkiness percentage, while gs3gl3.1 produced larger grain with higher chalkiness percentage. In terms of other agronomic traits, flag leaf size, grain number and grain yield of both gs3 and gs3gl3.1 mutants were affected. It is noteworthy that gs3 and gs3gl3.1 mutants both led to dramatical reduction of grain number, thereby decreased grain yield. In conclusion, these results indicated that knockout of GS3 and GL3.1 could rapidly improve grain size, but probably have some negative influences on grain quality and grain yield.

GL9 from Oryza glumaepatula controls grain size and chalkiness in rice

Grain size is a key factor influencing grain yield and appearance quality in rice.We identified twelve quantitative trait loci(QTL)for grain length(GL),nine for grain width(GW),and nine for 1000-kernel weight(TKW)using GLU-SSSLs,which are single-segment substitution lines with Oryza glumaepatula as donor parent and Huajingxian 74(HJX74)as recipient parent.Among the QTL,qGL1-2,qGL1-4,qGL9-2,qGW2-2,qGW9-1 and qTKW9-2 contributed to high grain yield.GL9 was identified as a candidate gene for qGL9-2 by map-based cloning and sequencing,and is a novel allele of GS9.The kernel of NIL-gl9was slenderer and longer than that of HJX74,and the TKW and grain yield per plant of NIL-gl9 were higher than those of HJX74.The proportion of grain chalkiness of NIL-gl9 was much lower than that of HJX74.Thus,gl9 increased grain yield and appearance quality simultaneously.Three pyramid lines,NIL-gs3/gl9,NIL-GW7/gl9 and NIL-gw8/gl9,were developed and the kernel of each was longer than that of the corresponding recipient parent lines.The gl9 allele may be beneficial for breeding rice varieties with high grain yield and good appearance quality.

Effect of Silicon Content and Shake-Out Time on Hardness and Grain Size Properties of GL 250 Cast Iron

The properties of cast iron grade GL 250 are dependent on the microstructures developed during casting. These microstructures are in turn dependent on the composition of the alloy, type of mould and other numerous casting practice variables such as shake-out time, pouring temperature, mould ambient conditions and inoculating technique. In this work, the effect of silicon content and shake-out time on the grain size (GS) and hardness properties of GL 250 cast iron was studied using a pouring temperature of 1400℃ and sand mould casting. Using charge materials consisting of pig iron and other additives, GL 250 castings containing silicon contents of 1.7, 2.1 and 2.7% were casted using a constant pouring temperature of 1400℃, molding sand of specified properties and ambient mould temperature of 32℃. Results showed that type A flake type was obtained at 30mins shakeout time for all samples for the C.I composition under study. Increasing shake-out time decreased hardness and increased carbide grain size. Increasing silicon content was observed to increase grain size and reduce free graphite but with resultant decrease in hardness. Two mathematical relationships were derived. One related grain-size to silicon content and shakeout time while the second related Brinnel Hardness to Silicon content and shake-out time. They are: Grain Size=0.40 Si+0.17Shake-out Time-0.15 and BHN=-60.53Si-7.15Shake-out Time+329.35 at 1400℃ pouring temperature in a molding sand of specified properties and sand mould ambient temperature of 32℃.

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.

水稻粒质量和粒形QTL定位及粒长位点qGL3.2的鉴定

[目的]通过水稻粒质量和粒形相关基因的QTL分析,挖掘普通野生稻(OryzarufipogonGriff.)中优异基因,为高产水稻资源的鉴定与品种改良提供研究基础和创新材料。[方法]以籼稻品种‘9311’为受体亲本,普通野生稻为供体亲本构建的染色体片段置换系群体为研究材料,考察2015、2016和2017年水稻种子千粒质量、粒长、粒宽和长宽比等性状,利用QTLIciMapping4.1软件对控制籽粒大小的QTL进行定位分析。并利用‘9311’与小粒家系Q8衍生的次级F2群体验证第3染色体1个效应明显的QTL(qGL3.2)。[结果]3年共定位到16个QTL,分布于第2、3、6、8和11染色体上,解释遗传变异的4.52%~13.08%。‘9311’与Q8家系衍生的次级F2群体验证了标记Indel3-17和RM3646之间qGL3.2位点的真实性,其对千粒质量、粒宽和长宽比的贡献率分别为33.18%、8.76%和59.92%;对粒长的效应尤为明显,LOD值高达39.29,可解释表型变异的贡献率达61.43%。精细定位和测序分析表明qGL3.2在基因Os03g0407400编码区发生1个C-A的替换,导致蛋白翻译提前终止。对130份种质资源等位变异类型和籽粒长度的分析表明C-A等位变异与粒长高度相关。[结论]qGL3.2位点Os03g0407400基因C-A突变对水稻粒质量和粒形有重要影响,这为该基因的进一步育种利用和分子标记辅助选择提供依据。

水稻粒长新基因GL12-1定位与利用分析

水稻的粒长是水稻粒型构成的因素之一,直接影响水稻的单产,而粒长又是稻米品质的重要指标之一。该研究利用选育含有稻瘟病抗性基因Pigm-1的长粒恢复系R20-4为研究材料,通过图位克隆的方法,对粒长基因进行鉴定,并对该长粒性状在育种中的应用进行了分析。结果显示:(1)该长粒为显性基因控制的性状。(2)将粒长基因GL12-1初步定位在水稻第12染色体上Indel-12-3和Indel-12-7之间,物理距离约4.5 Mb。(3)通过进一步开发标记,最终将该粒长基因GL12-1定位在标记Indel-10和Indel-16之间,物理距离约900 kb。(4)长粒恢复系R20-4与‘庆源A’、‘定源A’和‘启源A’测交组合的粒长均表现R20-4表型,而与‘靓香A’测交组合的粒长比R20-4更长。研究表明,粒长基因GL12-1为显性基因控制的性状,可能为一个新的粒长控制基因,该研究为后期GL12-1基因的克隆、功能研究以及粒型分子育种奠定了基础。

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.

Fine mapping of qTGW10-20.8, a QTL having important contribution to grain weight variation in rice

Grain weight is one of themost important determinants of grain yield in rice.In this study,QTL analysis for grain weight,grain length,and grainwidthwas performed using populations derived from crosses between major parental lines of three-line indica hybrid rice.A total of 27 QTL for grain weight were detected using three recombinant inbred line populations derived from the crosses Teqing/IRBB lines,Zhenshan 97/Milyang 46,and Xieqingzao/Milyang 46.Of these,10 were found in only a single population and the other 17 in two or all three populations.Nine of the 17 common QTL were located in regions where no QTL associated with grain weight have been cloned and onewas selected for fine-mapping.Eight populations segregating in an isogenic background were derived from one F7 residual heterozygote of Teqing/IRBB52.The target QTL,qTGW10-20.8 controlling grain weight,grain length,and grain width,was localized to a 70.7-kb region flanked by InDel markers Te20811 and Te20882 on the long arm of chromosome 10.The QTL region contains seven annotated genes,ofwhich six encode proteins with known functional domains and one encodes a hypothetical protein.One of the genes,Os10g0536100 encoding the MIKC-type MADS-box protein OsMADS56,is the most likely candidate for qTGW10-20.8.These results provide a basis for cloning qTGW10-20.8,which has an important contribution to grain weight variation in rice.

Effects of Interface and Grain Boundary on the Electrical Resistivity of Cu/Ta Multilayers

The electrical resistivity of Cu/Ta multilayers deposited by radio-frequency magnetron sputtering on a polyimide substrate was investigated as a function of monolayer thickness. It is found that the resistivity of the multilayer increases with decreasing monolayer thickness from 500 nm to 10 nm. Two significant effects of layer interface scattering and grain boundary scattering were identified to dominate electronic transportation behavior in the Cu/Ta multilayers at different length scales. The electrical resistivity of the multilayer with monolayer thickness ranging from nanometer to submicron scales can be well described by a newly-proposed Fuchs-Sandheimair （F-S） and Mayadas-Shatzkes （M-S） combined model.

Identification of long-grain chromosome segment substitution line Z744 and QTL analysis for agronomic traits in rice

Length of grain affects the appearance, quality, and yield of rice. A rice long-grain chromosome segment substitution line Z744, with Nipponbare as the recipient parent and Xihui 18 as the donor parent, was identified. Z744 contains a total of six substitution segments distributed on chromosomes(Chrs.) 1, 2, 6, 7, and 12, with an average substitution length of 2.72 Mb. The grain length, ratio of length to width, and 1 000-grain weight of Z744 were significantly higher than those in Nipponbare. The plant height, panicle number, and seed-set ratio in Z744 were significantly lower than those in Nipponbare, but they were still 78.7 cm, 13.5 per plant, and 86.49%, respectively. Furthermore, eight QTLs of different traits were identified in the secondary F2 population, constructed by Nipponbare and Z744 hybridization. The grain weight of Z744 was controlled by two synergistic QTLs(qGWT1 and q GWT7) and two subtractive QTLs(qGWT2 and qGWT6), respectively. The increase in the grain weight of Z744 was caused mainly by the increase in grain length. Two QTLs were detected, qGL1 and qGL7-3, which accounted for 25.54 and 15.58% of phenotypic variation, respectively. A Chi-square test showed that the long-grain number and the short-grain number were in accordance with the 3:1 separation ratio, which indicates that the long grain is dominant over the short-grain and Z744 was controlled mainly by the principal effect qGL1. These results offered a good basis for further fine mapping of qGL1 and further dissection of other QTLs into single-segment substitution lines.

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.

A natural allele of TAW1 contributes to high grain number and grain yield in rice

Grain number per panicle (GNP) is a complex trait controlled by quantitative trait loci (QTL),directly determining grain yield in rice.Identifying GNP-associated QTL is desirable for increasing rice yield.A rice chromosome segment substitution line (CSSL),F771,which showed increased panicle length and GNP,was identified in a set of CSSLs derived from a cross between two indica cultivars,R498 (recipient) and WY11327 (donor).Genetic analysis showed that the panicle traits in F771 were semidominant and controlled by multiple QTL.Six QTL were consistently identified by QTL-seq analysis.Among them,the major QTL q PLN10 for panicle length and GNP was localized to a 121-kb interval between markers N802 and N909 on chromosome 10.Based on quantitative real-time PCR and sequence analysis,TAWAWA1(TAW1),a known regulator of rice inflorescence architecture,was identified as the candidate gene for q PLN10.A near-isogenic line,NIL-TAW1,was developed to evaluate its effects.In comparison with the recurrent parent R498,NIL-TAW1 showed increased panicle length (14.0%),number of secondary branches (20.9%) and GNP (22.0%),and the final grain yield per plant of NIL-TAW1 was increased by18.6%.Transgenic experiments showed that an appropriate expression level of TAW1 was necessary for panicle development.Haplotype analysis suggested that the favorable F771-type (Hap 13) of TAW1was introduced from aus accessions and had great potential value in high-yield breeding both in indica and japonica varieties.Our results provide a promising genetic resource for rice grain yield improvement.

Root Length Density in Maize/Cowpea Intercropping under a Basin Tillage System in a Semi-Arid Area of Zimbabwe

A study to assess the effect of intercropping maize (Zea mays L.) and cowpea (Vigna unguiculata L.) within the same basin or outside the basin on root length density (RLD) was conducted at the International Crop Research Institute for Semi-Arid Tropics (ICRISAT) Matopos Research Station from December 2009 to April 2010. The experiment was laid out in a Randomised Complete Block Design (RCBD) with four treatments replicated four times namely;sole maize, sole cowpea, maize-cowpea intercrop with cowpea and maize planted within the same basin and maize-cowpea intercrop with cowpea planted 20 cm outside the maize basin. There was significant difference (P < 0.001) in RLD, grain yield and stover yield. Maize-cowpea intercropped within the same basin achieved higher RLD, grain yield and stover yield than cowpea that was intercropped outside the basin and the sole crops. The land equivalent ratio (LER) in both intercrop designs showed that intercropping had better grain yield performance when compared to sole cropping. It can be concluded that intercropping maize and cowpeas within the same basin can result in an environment around the crop achieving higher RLD which translates to better grain yield compared to the sole cropping and intercropping cowpeas outside the basin.

A gain-of-function mutation of OsMAPK6 leads to long grain in rice

Grain size is one of the most important agronomic traits controlling grain yield. Development of novel germplasm with large grains would be beneficial for crop improvement. We report the genetic identification and functional analysis of the LONG GRAIN 6(LOG6) gene, which is identical to MITOGENACTIVATED PROTEIN KINASE 6(OsMAPK6), affecting grain length of rice. Map-based cloning revealed that the long-grain phenotype of log6-D results from a glutamine(E) to lysine(K) mutation in the conserved TEY motif of OsMAPK6. In near-isogenic lines(NILs), the log6-D allele increased grain length and grain yield of Guichao 2(GC2), Teqing(TQ), and 93–11. Sequence analysis revealed 10 OsMAPK6 haplotypes,with xian(indica) and geng(japonica) harboring different haplotypes. Our findings shed light on the function of MAPKs and offer a novel dominant allele for improving the grain yield of rice.

利用剩余杂合体衍生的近等基因系精细定位水稻粒长微效QTL qGL1.1

【目的】本研究旨在对前期在水稻第1染色体长臂521.8 kb的区间内定位到的q TGW1.1b进行精细定位。【方法】从qTGW1.1和qTGW1.2所在区间分别呈杂合的2个BC2F9单株配组衍生的F4群体中,筛选到Wn28826-RM1231区间内杂合片段呈梯系排列的3个单株,构建了3套F5:6近等基因系。2017年种植于浙江杭州,考查千粒重、粒长和粒宽。利用SAS软件的GLM程序进行双因素方差分析,对qTGW1.1b的效应进行了验证。在此基础上,筛选出杂合片段更小且呈交迭排列的6个剩余杂合体,发展了6套F8:9近等基因系,2018年种植于海南陵水。对每套近等基因系中双亲基因型株系的表型差异进行双因素方差分析。【结果】qTGW1.1b在2个试验中对粒长和千粒重均呈极显著差异,效应方向一致且大小稳定。密阳46等位基因能分别增加粒长0.027 mm和提高千粒重0.17 g,贡献率分别达到27.12%和19.09%。【结论】鉴于qTGW1.1b在前后试验中对粒长影响最为显著,而对粒宽作用不显著,故将qTGW1.1b重新命名为qGL1.1。通过比较各套近等基因系的分离区间的基因组位置,最终将qGL1.1定位于Wn29077和Wn29154之间约76.8 kb的区间内。

Relationship between the eating quality and the physico-chemical properties of high grain quality rice in China

Relationship between eating quality(EQ)andphysico-chemical properties of 78 high grainquality rice varieties,45 indica and 33 japoni-ca,were studied in 1995.The samples werecollected from different rice growing regions in20 provinces.The physico-chemical proper-ties,including grain length(GL),length towidth ratio(L/W),chalky grain rate(CG),chalkiness(CH),translucency(TR),gela-tinization temperature(GT,measured by alka-li spreading value),gel consistency(CA2),amylase content(AC),and protein content(PC)were measured according to the standard