Effect of delayed sowing on grain number, grain weight, and protein concentration of wheat grains at specific positions within spikes

Delays in sowing have significant effects on the grain yield,yield components,and grain protein concentrations of winter wheat.However,little is known about how delayed sowing affects these characteristics at different positions in the wheat spikes.In this study,the effects of sowing date were investigated in a winter wheat cultivar,Shannong 30,which was sown in 2019 and 2020 on October 8(normal sowing)and October 22(late sowing)under field conditions.Delayed sowing increased the partitioning of ^(13)C-assimilates to spikes,particularly to florets at the apical section of a spike and those occupying distal positions on the same spikelet.Consequently,the increase in grain number was the greatest for the apical sections,followed by the basal and central sections.No significant differences were observed between sowing dates in the superior grain number in the basal and central sections,while the number in apical sections was significantly different.The number of inferior grains in each section also increased substantially in response to delayed sowing.The average grain weights in all sections remained unchanged under delayed sowing because there were parallel increases in grain number and ^(13)C-assimilate partitioning to grains at specific positions in the spikes.Increases in grain number m^(–2) resulted in reduced grain protein concentrations as the limited nitrogen supply was diluted into more grains.Delayed sowing caused the greatest reduction in grain protein concentration in the basal sections,followed by the central and apical sections.No significant differences in the reduction of the grain protein concentration were observed between the inferior and superior grains under delayed sowing.In conclusion,a 2-week delay in sowing improved grain yield through increased grain number per spike,which originated principally from an increased grain number in the apical sections of spikes and in distal positions on the same spikelet.However,grain protein concentrations declined in each section because of the increased grain number and reduced N uptake.

The 218th amino acid change of Ser to Ala in TaAGPS-7A increases enzyme activity and grain weight in bread wheat

ADP-glucose pyrophosphorylase(AGPase)influences cereal productivity.There are few reports on the function of cytosolic AGPase small subunit in bread wheat(TaAGPS).In the present study,TaAGPS was preferentially expressed in developing endosperm during grain-filling stages in bread wheat.TaAGPS allelic variations were characterized in 143 wheat accessions by PacBio RS II sequencing.Two haplotypes(TaAGPS-7A-TG and TaAGPS-7A-CT)of TaAGPS-7A were identified and corresponding functional markers were developed,whereas no variants of TaAGPS-7B and TaAGPS-7D were detected.TaAGPS-7A was associated with thousand-kernel weight(TKW)by haplotype–trait association analysis in two populations.Near-isogenic lines(NILs)with TaAGPS-7A-TG showed higher TKW and total kernel starch content than those with TaAGPS-7A-CT,owing to the higher AGPase activity of TaAGPS-7A-TG than TaAGPS-7A-CT both in vitro and in vivo.Overexpression of TaAGPS-7A-TG in bread wheat doubled the transcription levels of TaAGPS and increased AGPase activity by 55.7%,resulting in a 3.0-g higher TKW than in the wild type(WT).Knockdown of TaAGPS led to reduced expression of TaAGPS,AGPase activity,and TKW than in the WT.Thus,owing to the 218th amino acid change of Ser to Ala in TaAGPS-7A,the favorable haplotype TaAGPS-7A-TG showed higher AGPase activity,resulting in higher kernel starch content and grain weight.This finding could be applied to increasing starch content and grain weight in bread wheat.

Fine Mapping of qTGW3-1,a QTL for 1000-Grain Weight on Chromosome 3 in Rice

The QTL qTGW3-1 was located on chromosome 3 of rice （Oryza sativa L.） and associated with the 1 000-grain weight （TGW） according to the result of our earlier study. With the objective of fine mapping of this locus, we developed a F2 population consisting of 3 428 plants derived from the cross between TGW-related near isogenic line DL017 （BC3F4 generation of GSL 156×Nipponbare） and the recurrent parent Nipponbare. Using six microsatellites, this QTL was delimited between RM5477 and RM6417. Markers MM 1455 and MM 1456 within this region were used for further mapping of this QTL. Finally, qTGW3-1 was fine-mapped into a 89-kb interval between RM5477 and MM1456, which locates in the BAC clone AC107226 harboring five putative candidate genes.

QTL mapping of grain appearance quality traits and grain weight using a recombinant inbred population in rice(Oryza sativa L.)

supported by a grant from the National High-Tech R＆D Program of China （2014AA10A603, 2014AA10A604）;a grant from the Youth Foundation in Sichuan, China （2011JTD0022）;the special fund for China Agricultural Research System （CARS-01-08）;the Provincial Specialized Funds for Innovation Ability Promotion in Sichuan, China （2013GXJS005）

Mapping of qTGW1.1,a Quantitative Trait Locus for 1000-Grain Weight in Rice (Oryza sativa L.)

1000-grain weight （ TGW） is one ot the three component traits ot the grain yiela in rice （Oryza sativa L）. This study was conducted to validate and fine-map qTGWl. 1, a minor QTL for TGW which was previously located in a 3.7-Mb region on the long arm of rice chromosome 1. Five sets of near isogenic lines （NILs） were developed from two BC2F4 populations of the indica rice cross Zhenshan 973/Milyang 46 The NIL sets consisted of two homozygous genotypic groups differing in the regions RM11448-RM11522, RM11448-RM11549, RM1232-RM11615, RM11543-RM11554 and RM11569-RM11621, respectively. Four traits, including TGW, grain length, grain width and heading date, were measured. Phenotypic difference between the two genotypic groups in each NIL population was analyzed using SAS procedure GLM. Significant QTL effects were detected on TGW with the Zhenshan 97 allele increasing grain weight by 0.12 g to 0.14 g and explaining 8.30% to 15.19% of the phenotypic variance. Significant effects were also observed for grain length and width, whereas no significant effect was found for heading date. Based on comparison among the five NILs on the segregating regions and the results of QTL analysis, qTGWl. 1 was delimited to a 376.9-kb region flanked by DNA markers Wn28382 and RMl1554. Our results indicate that the effects of minor QTLs could be steadily detected in a highly isogenic background and suggest that such QTLs could be utilized in the breeding of high-yielding rice varieties.

QTL Mapping of Grain Weight Trait in Rice

To provide new experimental materials for QTL analysis of rice yield trait, we constructed a mapping population of 150 1ines （recombination inbred lines, R1L） derived from a cross between rice varieties V20B and CPSLO17, and localized QTLs and evaluated the genetic effects in the two parents and 150 RILs for thousand-grain weight trait by using internal mapping method of software MapQTL5 combining thousand-grain weight phenotypic data of the RILs. The results showed that a new QTL （qTGW-3） related to thousand-grain weight trait was detected. Individual QTL （LOD=4.14） explained 11.9% of the observed phenotypic variance. And the QTL alleles came from the parent V20B.

Analysis of digenic epistatic effects and QE interaction effects QTL controlling grain weight in rice

Immortalized F2 population of rice (Oryza sativa L.) was developed by randomly mating F1 among recombinant inbred (RI) lines derived from (Zhenshan 97B×Minghui 63),which allowed replications within and across environments.QTL (quantitative trait loci) mapping analysis on kilo-grain weight of immortalized F2 population was performed by using newly developed software for QTL mapping,QTL Mapper 2.0. Eleven distinctly digenic epistatic loci included a total of 15 QTL were located on eight chromosomes.QTL main effects of additive,dominance,and additive×additive,additive×dominance,and dominance×dominance interactions were estimated.Interaction effects between QTL main effects and environments (QE) were predicted.Less than 40% of single effects,most of which were additive effects,for identified QTL were significant at 5% level.The directional difference for QTL main effects suggested that these QTL were distributed in parents in the repulsion phase.This should make it feasible to improve kilo-grain weight of both parents by selecting appropriate new recombinants. Only few of the QE interaction effects were significant.Application prospect for QTL mapping achievements in genetic breeding was discussed.

Dissection of two quantitative trait loci for grain weight linked in repulsion on the long arm of chromosome 1 of rice(Oryza sativa L.)

Grain weight is a key determinant of grain yield in rice. Three sets of rice populations with overlapping segregating regions in isogenic backgrounds were established in the generations of BC2 F5, BC2 F6 and BC2 F7, derived from Zhenshan 97 and Milyang 46, and used for dissection of quantitative trait loci(QTL) for grain weight. Two QTL linked in repulsion phase on the long arm of chromosome 1 were separated. One was located between simple sequence repeat(SSR) markers RM11437 and RM11615, having a smaller additive effect with the enhancing allele from the maintainer line Zhenshan 97 and a partially dominant effect for increasing grain weight. The other was located between SSR markers RM11615 and RM11800, having a larger additive effect with the enhancing allele from the restorer line Milyang 46 and a partially dominant effect for increasing grain weight. When the two QTL segregated simultaneously, a residual additive effect with the enhancing allele from Milyang 46 and an over-dominance effect for increasing grain weight were detected. This suggests that dominant QTL linked in repulsion phase might play an important role in heterosis in rice. Our study also indicates that the use of populations with overlapping segregating regions in isogenic backgrounds is helpful for the dissection of minor linked QTL.

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.

Characterization of a new hexaploid triticale 6D(6A) substitution line with increased grain weight and decreased spikelet number

Hexaploid triticale(×Triticosecale,AABBRR)is an important forage crop and a promising energy plant.Transferring D-genome chromosomes or segments from common wheat(Triticum aestivum)into hexaploid triticale is attractive in improving its economically important traits.Here,a hexaploid triticale 6D(6A)substitution line Lin 456 derived from the cross between the octoploid triticale line H400 and the hexaploid wheat Lin 56 was identified and analyzed by genomic in situ hybridization(GISH),fluorescence in situ hybridization(FISH),and molecular markers.The GISH analysis showed that Lin 456 is a hexaploid triticalewith 14 rye(Secale cereale)chromosomes and 28 wheat chromosomes,whereas non-denaturing fluorescence in situ hybridization(ND-FISH)and molecular marker analysis revealed that it is a 6D(6A)substitution line.In contrast to previous studies,the signal of Oligo-pSc119.2 was observed at the distal end of 6DL in Lin 456.The wheat chromosome 6D was associatedwith increased grain weight and decreased spikelet number using the genotypic data combined with the phenotypes of the F2 population in the three environments.The thousand-grain weight and grain width in the substitution individuals were significantly higher than those in the non-substitution individuals in the F2 population across the three environments.We propose that the hexaploid triticale 6D(6A)substitution line Lin 456 can be a valuable and promising donor stock for genetic improvement during triticale breeding.

Post-transcriptional regulation of grain weight and shape by the RBP-A-J-K complex in rice

RNA-binding proteins(RBPs)are components of the post-transcriptional regulatory system,but their regulatory effects on complex traits remain unknown.Using an integrated strategy involving map-based cloning,functional characterizations,and transcriptomic and population genomic analyses,we revealed that RBP-K(LOC_Os08g23120),RBP-A(LOC_Os11g41890),and RBP-J(LOC_Os10g33230)encode proteins that form an RBP-A-J-K complex that negatively regulates rice yield-related traits.Examinations of the RBP-A-J-K complex indicated RBP-K functions as a relatively non-specific RBP chaperone that enables RBP-A and RBP-J to function normally.Additionally,RBP-J most likely affects GA pathways,resulting in considerable increases in grain and panicle lengths,but decreases in grain width and thickness.In contrast,RBP-A negatively regulates the expression of genes most likely involved in auxin-regulated pathways controlling cell wall elongation and carbohydrate transport,with substantial effects on the rice grain filling process as well as grain length and weight.Evolutionarily,RBP-K is relatively ancient and highly conserved,whereas RBP-J and RBP-A are more diverse.Thus,the RBP-A-J-K complex may represent a typical functional model for many RBPs and protein complexes that function at transcriptional and post-transcriptional levels in plants and animals for increased functional consistency,efficiency,and versatility,as well as increased evolutionary potential.Our results clearly demonstrate the importance of RBP-mediated post-transcriptional regulation for the diversity of complex traits.Furthermore,rice grain yield and quality may be enhanced by introducing various complete or partial loss-of-function mutations to specific RBP genes using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9 technology and by exploiting desirable natural tri-genic allelic combinations at the loci encoding the components of the RBP-A-J-K complex through marker-assisted selection.

GSW3.1,a novel gene controlling grain size and weight in rice

Grain size and weight are closely related traits determining yield in rice(Oryza sativa L.).Since indica and japonica rice varieties differ significantly in multiple traits,a high-generation recombinant inbred line(RIL)population derived from the crossing LH9(indica)and RPY(japonica)was used to map grainrelated traits in six environments.Pyramiding of the quantitative trait loci(QTL)for thousand-grain weight showed that combinations of multiple QTL significantly increased the phenotypic effect.A novel gene named GSW3.1 controlling grain size and weight was discovered using the major QTL for the colocalization of grain width and thousand-grain weight on chromosome 3.Gene editing revealed that GSW3.1(LOC_Os03g16850)was pleiotropic,positively regulating grain size and weight while affecting several other agronomic traits.Haplotype analysis indicated that some traits,including grain width and weight,were highly correlated with indica-japonica differentiation.

Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice

Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.

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.

TaABI19 positively regulates grain development in wheat

Starch is the most important component in the endosperm, and its synthesis is regulated by multiple transcription factors(TFs) in cereals. However, whether the functions of these TFs are conserved among cereals remains unclear. In this study,we cloned a B3 family TF in wheat, named TaABI19, based on its orthologous sequence in maize(Zea mays L.). Alignment of the DNA and protein sequences showed that ABI19 is conserved in maize and wheat(Triticum aestivum L.). We found that TaABI19 is highly expressed in young spikes and developing grains, and encodes a nucleus-localized transcriptional activator in wheat. The taabi19-b1 null mutants obtained by EMS exhibited a down-regulation of starch synthesis, shorter grain length and lower thousand-grain weight(TGW). Furthermore, we proved that TaABI19 could bind to the promoters of TaPBF homologous genes and enhance their expression. Haplotype association showed that TaABI19-B1 is significantly associated with TGW. We found that Hap2 and Hap3 were favored and had undergone positive selection in China’s wheat breeding programs. Less than 50% of the modern cultivars convey the favored haplotypes, indicating that TaABI19 still can be considered as a target locus for marker-assisted selection breeding to increase TGW in China.

OsSPL18 controls grain weight and grain number in rice

Grain weight and grain number are two important traits directly determining grain yield in rice. To date,a lot of genes related to grain weight and grain number have been identified; however, the regulatory mechanism underlying these genes remains largely unknown. In this study, we studied the biological function of OsSPL18 during grain and panicle development in rice. Knockout (KO) mutants of OsSPL18exhibited reduced grain width and thickness, panicle length and grain number, but increased tiller number. Cytological analysis showed that OsSPL18 regulates the development of spikelet hulls by affecting cell proliferation. qRT-PCR and GUS staining analyses showed that OsSPL18 was highly expressed in developing young panicles and young spikelet hulls, in agreement with its function in regulating grain and panicle development. Transcriptional activation experiments indicated that OsSPL18is a functional transcription factor with activation domains in both the N-terminus and C-terminus, and both activation domains are indispensable for its biological functions. Quantitative expression analysis showed that DEP1, a major grain number regulator, was significantly down-regulated in OsSPL18 KO lines.Both yeast one-hybrid and dual-luciferase (LUC) assays showed that OsSPL18 could bind to the DEP1promoter, suggesting that OsSPL18 regulates panicle development by positively regulating the expression of DEP1. Sequence analysis showed that OsSPL18 contains the OsmiR156k complementary sequence in the third exon; 5?RLM-RACE experiments indicated that OsSPL18 could be cleaved by OsmiR156k. Taken together, our results uncovered a new OsmiR156k-OsSPL18-DEP1 pathway regulating grain number in rice.

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

谷物重量是庄稼谷物产量的一个主要决定因素并且被自然地发生的量的特点 loci (QTL ) 控制。我们更早识别了控制米饭谷物宽度和重量的主要 QTL， GW5，它在米饭染色体 5 上被印射到一个再结合热点。获得 GW5 怎么控制米饭谷物宽度的更好的理解，我们进行了这个地点的好印射并且揭开在米饭栽培变种 Asominori 与增加的谷物宽度联系的 1 212-bp 删除，与苗条谷物米饭 IR24 比较。另外， 46 米饭栽培变种的 genotyping 分析表明这删除高度与谷物宽度显型被相关，建议 GW5 删除可能在米饭驯服期间被选择了。GW5 编码对原子核局部性的 144 氨基酸的新奇原子蛋白质。而且，我们证明 GW5 身体上在酵母与 polyubiquitin 交往二混血儿的试金。一起，我们的结果建议 GW5 代表主要 QTL 内在的米饭宽度和重量，并且它多半在 ubiquitin-proteasome 小径行动在种子开发期间调整房间分割。这研究提供新奇卓见进控制米饭谷物开发的分子的机制并且建议 GW5 能为庄稼的产量很高的繁殖用作一个潜在的工具。

Genetic dissection of a thousand-grain weight quantitative trait locus on rice chromosome 1

Thousand-grain weight (TGWT) is an important factor affecting grain yield as well as grain quality in rice. A quantitative trait locus (QTL) qTGWT1-1 for TGWT was detected previously near DNA marker RG532 on the short arm of chromosome 1 in a recombinant inbred line (RIL) population derived from the indica-indica rice cross Zhengshan97B (ZS97B)/Milyang46 (MY46). In this study, two residual het-erozygous lines (RHLs), Ch1 and Ch2, derived from the ZS97B/MY46 RIL F7 population, were used to develop two F6 populations, RIL-1 and RIL-2. The genome of Ch1 and Ch2 contains a heterozygous region flanked by RM1―RM3746 and RM151―RM243 on the short arm of chromosome 1, respectively, but is homozygous in other regions. Two tightly linked QTLs, Gw1-1 and Gw1-2, with the same additive direction and similar effect on TGWT, were detected in the region of QTL qTGWT1-1 in population RIL-2. No QTL was detected in the population RIL-1. Four individual RHLs from the population RIL-2 carrying heterozygous segments flanked by RM151―RM10404, RM10381―RM243, RM10435―RM259 and RM10398―RM5359, respectively, were chosen to develop four F2 populations. Ten maternal homozy-gotes and 10 paternal homozygotes were selected from each of the four F2 populations derived from the four RHLs. The four sets of near isogenic lines (NILs) were grown for phenotyping of TGWT and delimitation of Gw1-1 and Gw1-2. Results showed that Gw1-1 and Gw1-2 were located in the intervals RM10376―RM10398 and RM10404―RM1344 which cover 392.9 and 308.5 kb regions, respectively. The enhancing alleles were from ZS97B at both loci, and no significant interactions were detected. Genetic dissection of Gw1-1 and Gw1-2 has laid a foundation for their cloning and molecular breeding of grain yield and quality in rice.

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.