Dissecting the genetic basis of grain color and pre-harvest sprouting resistance in common wheat by association analysis

Pre-harvest sprouting(PHS) adversely affects wheat quality and yield, and grain color(GC) is associated with PHS resistance. However, the genetic relationship between GC and PHS resistance remains unclear. In this study, 168 wheat varieties(lines) with significant differences in GC and PHS resistance were genotyped using an Illumina 90K iSelect SNP array. Genome-wide association study(GWAS) based on a mixed linear model showed that 67 marker-trait associations(MTAs) assigned to 29 loci, including 17 potentially novel loci, were significantly associated with GC, which explained 1.1–17.0% of the phenotypic variation. In addition, 100 MTAs belonging to 54 loci, including 31 novel loci, were significantly associated with PHS resistance, which accounted for 1.1–14.7% of the phenotypic variation. Subsequently, two cleaved amplified polymorphic sequences(CAPS) markers, 2B-448 on chromosome 2B and 5B-301 on chromosome 5B,were developed from the representative SNPs of the major common loci Qgc.ahau-2B.3/Qphs.ahau-2B.4controlling GC/PHS resistance and PHS resistance locus Qphs.ahau-5B.4, respectively. Further validation in 171 Chinese mini-core collections confirmed significant correlations of the two CAPS markers with GC and PHS resistance phenotypes under different environments(P<0.05). Furthermore, the wheat public expression database, transcriptomic sequencing, and gene allelic variation analysis identified TraesCS5B02G545100, which encodes glutaredoxin, as a potential candidate gene linked to Qphs.ahau-5B.4. The new CAPS marker CAPS-356 was then developed based on the SNP(T/C) in the coding sequences(CDS) region of TraesCS5B02G545100. The high-density linkage map of the Jing 411/Hongmangchun 21 recombinant inbred lines(RILs) constructed based on specific locus amplified fragment sequencing markers showed that CAPS-356 collocated with a novel QTL for PHS resistance, supporting the role of TraesCS5B02G545100 as the potential candidate gene linked to Qphs.ahau-5B.4. These results provide valuable information for the map-based cloning of Qphs.ahau-5B.4 and breeding of PHS resistant white-grained varieties.

Identification of the candidate gene controlling tiller angle in common wheat through genome-wide association study and linkage analysis

Wheat tiller angle(TA)is an important agronomic trait that contributes to grain production by affecting plant architecture.It also plays a crucial role in high-yield wheat breeding.An association panel and a recombinant inbred line(RIL)population were used to map quantitative trait loci(QTL)for TA.Results showed that 470 significant SNPs with 10.4%–28.8%phenotypic variance explained(PVE)were detected in four replicates by a genome-wide association study(GWAS).Haplotype analysis showed that the TA_Hap_4B1 locus on chromosome 4B was a major QTL to regulate wheat TA.Ten QTL were totally detected by linkage mapping with the RIL population,and QTA.hau-4B.1 identified in six environments with the PVE of 7.88%–18.82%was a major and stable QTL.A combined analysis demonstrated that both TA_Hap_4B1 and QTA.hau-4B.1 were co-located on the same region.Moreover,QTA.hau-4B.1 was confirmed by bulked segregant RNA-Seq(BSR-Seq)analysis.Phenotypic analysis showed that QTA.hau-4B.1was also closely related to yield traits.Furthermore,Traes CS4B02G049700 was considered as a candidate gene through analysis of gene sequence and expression.This study can be potentially used in cloning key genes modulating wheat tillering and provides valuable genetic resources for improvement of wheat plant architecture.

Study on Genomic Changes in Partial Amphiploids of Common Wheat_Wheatgrass

According to conventional theory, little genomic changes should occur in homozygous and stable amphiploids of the grass family, particularly those involving polyploid wheat as a parent. In the present study, however, extensive genomic changes were detected in two octoploid partial amphiploids of common wheat (Triticum aestivum L.)_wheatgrass (Agropyron intermedium (Host) P.B.=Elytrigia intermedia (Host) Nevski=Thinopyrum intermedium (Host) Barkworth and Dewey), namely Zhong 3 and Zhong 5, by RFLP analysis using 10 low_copy, wheat chromosome_specific sequences and 33 representative homoeologous group_specific sequences as probes. Genomic changes involved loss of wheat hybridization fragment(s) and/or acquisition of new fragment(s). Uniformity of the RFLP patterns among 5 individual plants taken respectively from Zhong 3 and Zhong 5 in two successive generations, suggested that genomic changes probably had occurred in the early few generations after octoploid amphiploid formation, and remained essentially static thereafter. The highly similar RFLP patterns between Zhong 3 and Zhong 5, which had identical genomic constitution but differed from each other due to involvement of different wheat varieties as parents imply that genomic changes were probably not at random. Possible causes for the extensive and rapid genomic changes in the newly formed plant amphiploids, as well as their implications for polyploid genome evolution and breeding application are discussed.

Isolation and Characterization of a BBC1 cDNA from Common Wheat

A breast basic conserved 1 (BBC1) cDNA has been isolated from common wheat (Triticum aestivum L.). Analysis of amino acid sequence derived from the cDNA showed that the wheat BBC1 was highly hydrophilic and rich in alanine, lysine, glutamic acid and arginine residues. The transcription of wheat BBC1 mRNA was regulated by low temperature. Southern blotting analysis showed that BBC1 existed as a small family in common wheat genome.

Cloning and Sequence Analysis of PBF Encoding Genes from Hexaploid Common Wheat Chinese Spring (Triticum aestivum L.)

[Objective] This study aimed to investigate the polymorphism of PBF en- coding genes from common wheat Chinese Spring （Triticum aestivum L.）. [Method] Using common wheat Chinese Spring as the experimental material, gene-specific primers were designed and applied to amplify the genomic DNA of Chinese Spring. PCR products were isolated, purified and ligated into the cloning vector. Positive clones were randomly selected for sequencing. A series of softwares including DNAMAN, Signalp, PSIPRED, Nuc_PLoc and MEGA were employed for sequence assembly and alignment, signal peptide prediction, primary and secondary structure prediction, as well as analyses of subcellular location and phylogenetic relationships between the PBF family members in Poaceae. [Result] Twenty-five target sequences were obtained from the genome of hexaploid common wheat Chinese Spring, which were classified into three clusters based on the sequence similarity. SNPs exist at two loci of the subunit, resulting in the change of encoded amino acid residues and affecting the secondary structure of final product encoded. [Conclusion] PBF encoding sequences are extremely conservative in Chinese Spring with certain variations. This study provides theoretical reference to evaluate the expression efficiency of wheat storage proteins.

Identification and genetic analysis of multiple P chromosomes of Agropyron cristatum in the background of common wheat

Agropyron cristatum, a wild relative of common wheat(Triticum aestivum L.), provides many desirable genetic resources for wheat improvement, such as tolerance to cold, drought, and disease. To transfer and utilize these desirable genes, in this study, two wheat-A. cristatum derivatives II-13 and II-23 were identified and analyzed. We found that the number of root tip cell chromosomes was 44 in both II-13 and II-23, but there were four and six P genome chromosomes in II-13 and II-23, respectively, based on genomic in situ hybridization(GISH). The chromosome configurations of II-13 and II-23 were both 2 n=22 II by the meiotic analysis of pollen mother cells(PMCs) at metaphase I, indicating that there were two and three pairs of P chromosomes in II-13 and II-23, respectively. Notably, wheat chromosome 7 D was absent in derivative line II-13 while II-23 lacked chromosomes 4 B and 7 A based on SSR analysis combining fluorescence in situ hybridization(FISH) analysis with p As1 and p Sc119.2 as probes. Chromosomes 2 P and 7 P were detected in both II-13 and II-23. Another pair of P genome chromosomes in II-23 was determined to be 4 P based on expressed-sequences tags-sequence tagged sites(EST-STS) markers specific to A. cristatum and FISH with probes p Ac TRT1 and p Acp CR2. Overall, these results suggest that II-13 was a 7 P(7 D) substitution line with one pair of additional 2 P chromosomes and II-23 was a multiple 4 P(4 B), 7 P(7 A) substitution line with one pair of additional 2 P chromosomes. Moreover, we obtained six alien disomic addition lines and five alien disomic substitution lines by backcrossing. These new materials will allow desirable genes from A. cristatum to be used in common wheat.

Application of Synthetic Hexaploid Wheat Derived from T.Durum, Ae.taushii in Common Wheat Breeding for FHB Resistance

The F 1 and F 4 plants of 'synthetic hexaploid wheat/common wheat'crosses and part of their parents were inoculated with Fusarium graminearum to evaluate FHB resistance.The results showed tht the scab resistance in the F 1 varied with the synthetic wheat accessions used as crossing parents.In the F 4,some resistant head lines were generated from the crosses,although their parents had different scab resistance levels.It indicated that synthetic hexaploid wheat are useful in wheat breeding for scab resistance.

A MITE insertion into the 3′-UTR regulates the transcription of TaHSP16.9 in common wheat

Miniature inverted-repeat transposable elements(MITEs) are a type of DNA transposon frequently inserted into promoters, untranslated regions(UTR), introns, or coding sequences of genes. We found a 276-bp tourist-like MITE insertion in the 3′-UTR of a 16.9 k Da small heat shock protein gene(TaH SP16.9-3A) on chromosome 3A of common wheat. Haplotype analysis revealed two haplotypes, s HSP-W(wild type without MITE insertion) and s HSP-M(mutant with MITE insertion), present in wheat germplasm. Both semiquantitative PCR and quantitative real-time PCR analyses showed increased transcription levels of TaH SP16.9-3A in s HSP-M compared with those of s HSP-W after heat treatment at 42 °C. It appeared that the MITE insertion into the 3′-UTR enhances the transcription of TaH SP16.9-3A.

Molecular differences in genotroph forms of common wheat (Triticum aestivum L.) and their initial cultivars

By the modified ISSR-amplification method the comparison of the structural organization of enzyme loci in the initial common wheat cultivars and their genotrophs induced with epimutagens: plant niacin acid and niacin acid and its derivatives produced from β-picoline fraction of coal-tar pitch (niacin acid nitrile, isocinchomeronic and benzoic acids) was carried out. It is shown that niacin acid influence causes in genotrophs specific band appearance in PCR-profiles obtained on the DNA of enzyme loci. All these before mentioned epimutagens induced in limits of each genotroph differently directed changes in number and intensity of bands of PCR-profiles.

Changes in the organization of isozyme loci Me1 and Adh1 in-duced with triton X-100 in common wheat lines

The study of PCR-profiles of isozyme loci Me1 and Adh1 in common wheat lines obtained by means of treatment of initial cultivar Alem with detergent Tri-ton X-100 was carried out by the modified ISSR-amplification method. It was demonstrated that exposure to Triton X-100 causes changes of PCR-profiles of enzyme loci. The obtained data are suggestive of the role of chromatin and nuclear mem-brane interaction in structural-functional genome organization.

A cell wall invertase modulates resistance to fusarium crown rot and sharp eyespot in common wheat

Fusarium crown rot(FCR) and sharp eyespot(SE)are serious soil-borne diseases in wheat and its relatives that have been reported to cause wheat yield losses in many areas. In this study, the expression of a cell wall invertase gene, TaCWI-B1,was identified to be associated with FCR resistance through a combination of bulk segregant RNA sequencing and genome resequencing in a recombinant inbred line population. Two biparental populations were developed to further verify TaCWI-B1 association with FCR resistance.Overexpression lines and ethyl methanesulfonate(EMS) mutants revealed TaCWI-B1 positively regulating FCR resistance. Determination of cell wall thickness and components showed that the TaCWI-B1-overexpression lines exhibited considerably increased thickness and pectin and cellulose contents. Furthermore, we found that TaCWI-B1 directly interacted with an alphagalactosidase(TaGAL). EMS mutants showed that TaGAL negatively modulated FCR resistance. The expression of TaGAL is negatively correlated with TaCWI-B1 levels, thus may reduce mannan degradation in the cell wall, consequently leading to thickening of the cell wall. Additionally, TaCWI-B1-overexpression lines and TaGAL mutants showed higher resistance to SE;however, TaCWI-B1 mutants were more susceptible to SE than controls.This study provides insights into a FCR and SE resistance gene to combat soil-borne diseases in common wheat.

A Genetic Map Constructed Using a Doubled Haploid Population Derived from Two Elite Chinese Common Wheat Varieties

Genetic mapping provides a powerful tool for the analysis of quantitative trait loci （QTLs） at the genomic level. Herein, we report a new genetic linkage map developed from an F1-derived doubled haploid （DH） population of 168 lines, which was generated from the cross between two elite Chinese common wheat （Triticum aestivum L.） varieties, Huapei 3 and Yumai 57. The map contained 305 loci, represented by 283 simple sequence repeat （SSR） and 22 expressed sequence tag （EST）-SSR markers, which covered a total length of 2141.7 cM with an average distance of 7.02 cM between adjacent markers on the map. The chromosomal locations and map positions of 22 new SSR markers were determined, and were found to distribute on 14 linkage groups. Twenty SSR loci showed different chromosomal locations from those reported in other maps. Therefore, this map offers new information on the SSR markers of wheat. This genetic map provides new opportunities to detect and map QTLs controlling agronomically important traits. The unique features of this map are discussed.

Comparative study of symmetric and asymmetric somatic hybridization between common wheat and Haynaldia villosa

Symmetric and asymmetric protoplast fusion between long term cell suspension-derived protoplasts of Triticum aestivum (cv. Jinan 177) and protoplasts of Haynaldia villosa prepared from one-year-old embryogeneric calli was performed by PEG method. In asymmetric fusion, donor calli were treated with gamma ray at a dose of 40, 60, 80 Gy (1.3 Gy/min) respectively and then used to isolate protoplasts. Results of morphological, cytological, biochemical (isozyme) and 5S rDNA spacer sequence analysis revealed that we obtained somatic hybrid lines at high frequency from both symmetric and asymmetric fusion. Hybrid plants were recovered from symmetric and low dose γ-fusion combinations. GISH (genomic in situ hybridization) analysis proved exactly the existence of both parental chromosomes and the common occurrence of several kinds of translocation between them in the hybrid clones regenerated from symmetric and asymmetric fusion. And the elimination of donor DNA in hybrid clones regenerated from asymmetric fusion combinations was found to increase with the increasing gamma doses. It is concluded that transference and recombination of nuclear DNA can be achieved effectively by symmetric and asymmetric fusion, hybrids with small fragment translocation which are valuable in plant breeding can be obtained directly by asymmetric fusion.

Genetic Analysis of Chromosomal Loci Affecting the Content of Insoluble Glutenin in Common Wheat

In common wheat, insoluble glutenin （IG） is an important fraction of flour glutenin macropolymers, and insoluble glutenin content （IGC） is positively associated with key end-use quality parameters. Here, we present a genetic analysis of the chromosomal loci affecting IGC with the data collected from 90 common wheat varieties cultivated in four environments. Statistical analysis showed that IGC was controlled mainly genetically and influenced by the environment. Among the major genetic components known to affect end-use quality, 1BL/1RS translocation had a significantly negative effect on IGC across all four environments. As to the different alleles of Glu-A1, -B1 and -D1 loci, Glu-Ala, Glu-Blb and Glu-Dld exhibited relatively strong positive effects on IGC in all environments. To identify new loci affecting IGC, association mapping with 1355 DArT markers was conducted. A total of 133 markers were found associated with IGC in two or more environments （P 〈 0.05）, ten of which consistently affected IGC in all four environments. The phenotypic variance explained by the ten markers varied from 4.66% to 8.03%, and their elite alleles performed significantly better than the inferior counterparts in enhancing IGC. Among the ten markers, wPt-3743 and wPt-733835 reflected the action of Glu-D1, and wPt-664972 probably indicated the effect of GIu-A1. The other seven markers, forming three clusters on 2AL. 3BL or 7BL chromosome arms, represented newly identified genetic determinants of IGC. Our work provided novel insights into the genetic control of IGC, which may facilitate wheat end- use quality improvement through molecular breeding in the future.

Effects on Genome Constitution and Novel Cell Wall Formation Caused by the Addition of 5RS Rye Chromosome to Common Wheat

The cytological instability of common wheat-rye addition lines was investigated in the present study. The chromosome numbers of almost all addition lines were considerably stable, but those of CS ＋ 5R were very variable. The rye chromosome added in this line was found to be much shorter than expected. Fluorescent in situ hybridization with 5S rDNA and the centromere-speciflc probes clearly revealed that the short rye chromosome contains only a short arm of chromosome 5R （5RS）. In this line, chromosome numbers of both 5RS and common wheat were changeable. The chromosome numbers ranged from 2n = 36 to 2n = 44 in the cells carrying two 5RS, and ranged from 2n = 31 to 2n = 44 in one 5RS cells. In addition to the chromosome instability, the muIticells wrapped in a sac-like structure were frequently observed in the root meristematic tissues of CS ＋5RS after the enzyme treatment for chromosome preparation. Genomic in situ hybridization with rye DNA as a probe showed that all cells in sacs investigated were at the interphase stage and contained one or two 5RS chromosomes. An electron microscopic analysis revealed that the cells of CS＋5RS, particularly in sacs, have abnormal （irregular and curved） cell walls. These results indicate that 5RS has （a） specific factor（s） influencing the cell wall development as well as the genome stability.

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.

Global Profiling of 2-hydroxyisobutyrylome in Common Wheat

As a novel post-translational modification(PTM),lysine 2-hydroxyisobutyrylation(Khib)is considered to regulate gene transcriptional activities in eukaryotic cells;however,the functions of Khib-modified proteins in plants remain unknown.Here,we report that Khib is an evolutionarilyconserved PTM in wheat and its progenitors.A total of 3348 Khib sites on 1074 proteins are identified in common wheat(Triticum aestivum L.)by using affinity purification and mass spectroscopy of 2-hydroxyisobutyrylome.Bioinformatic data indicate that Khib-modified proteins participate in a wide variety of biological and metabolic pathways.Immunoprecipitation confirms that Khibmodified proteins are present endogenously.A comparison of Khib and other main PTMs shows that Khib-modified proteins are simultaneously modified by multiple PTMs.Using mutagenesis experiments and co-immunoprecipitation assays,we demonstrate that Khib on K206 of phosphoglycerate kinase(PGK)is a key regulatory modification for its enzymatic activity,and mutation on K206 affects the interactions of PGK with its substrates.Furthermore,Khib modification of low-molecular-weight proteins is a response to the deacetylase inhibitors nicotinamide and trichostatin.This study provides evidence to promote our current understanding of Khib in wheat plants,including the cooperation between Khib and its metabolic regulation.

Rapid identification of Psathyrostachys huashanica Keng chromosomes in wheat background based on ND-FISH and SNP array methods

Psathyrostachys huashanica Keng(2n=2x=14,NsNs)is regarded as a valuable wild relative species for common wheat cultivar improvement because of its abundant beneficial agronomic traits.However,although the development of many wheat–P.huashanica-derived lines provides a germplasm base for the transfer of excellent traits,the lag in the identification of P.huashanica chromosomes in the wheat background has limited the study of these lines.In this study,three novel nondenaturing fluorescence in situ hybridization(ND-FISH)-positive oligo probes were developed.Among them,HS-TZ3 and HS-TZ4 could specifically hybridize with P.huashanica chromosomes,mainly in the telomere area,and HS-CHTZ5 could hybridize with the chromosomal centromere area.We sequentially constructed a P.huashanica FISH karyotype and idiogram that helped identify the homologous groups of introduced P.huashanica chromosomes.In detail,1Ns and 2Ns had opposite signals on the short and long arms,3Ns,4Ns,and 7Ns had superposed two-color signals,5Ns and 6Ns had fluorescent signals only on their short arms,and 7Ns had signals on the intercalary of the long arm.In addition,we evaluated different ways to identify alien introgression lines by using low-density single nucleotide polymorphism(SNP)arrays and recommended the SNP homozygosity rate in each chromosome as a statistical pattern.The 15K SNP array is widely applicable for addition,substitution,and translocation lines,and the 40K SNP array is the most accurate for recognizing transposed intervals between wheat and alien chromosomes.Our research provided convenient methods to distinguish the homologous group of P.huashanica chromosomes in a common wheat background based on ND-FISH and SNP arrays,which is of great significance for efficiently identifying wheat–P.huashanica-derived lines and the further application of Ns chromosomes.

Characterization and distribution of novel alleles of the vernalization gene Vrn-A1 in Chinese wheat(Triticum aestivum L.) cultivars

The ability of wheat to adapt to a wide range of environmental conditions is determined mostly by allelic diversity among genes regulating vernalization requirement.Vrn-1 is a major regulator of this requirement.In this study,two novel alleles of Vrn-A1 were discovered in Chinese cultivars:vrn-A1n was identified in two landraces,Jiunong 2 and Ganchun 16,and Vrn-A1o was detected in Duanhongmangmai.Both novel alleles showed a linked duplication in the promoter region.The common copy of these two alleles was identical to the recessive allele vrn-A1.Compared with the recessive allele vrn-A1,the other copy of vrn-A1n contained a 54-bp deletion in the promoter region and the distinct copy of Vrn-A1o contained an11-bp deletion in the promoter region.In segregating populations in the greenhouse under nonvernalizing(20–25°C)and long-day(16 h light)conditions,plants with the novel vrn-A1n allele did not head earlier than those with the recessive vrn-A1 allele.However,plants that were either homozygous or heterozygous for the novel Vrn-A1o allele headed earlier than those with the recessive vrn-A1 allele.To identify the novel allele with the small-sized product and facilitate screening,a DNA marker for the novel dominant allele Vrn-A1o was designed.Analysis of the novel-allele distribution showed that two cultivars carrying the vrn-A1n allele were dispersed in the northwestern spring wheat zone,and 12 cultivars carrying the dominant Vrn-A1o allele were widely distributed in the northwestern spring wheat zone,Xinjiang winter and spring wheat zone,Yellow and Huai River valley winter wheat zone,and QinghaiTibetan Plateau spring and winter wheat zone.Our study identifies useful germplasm and a DNA marker for wheat breeding.

Development and characterization of wheat–Aegilops kotschyi 1U^(k)(1A)substitution line with positive dough quality parameters

Exploring novel high molecular weight glutenin subunits(HMW-GSs)from wild related species is a strategy to improve wheat processing quality.The objective of the present investigation was to identify the chromosomes of the wheatalien introgression line N124,derived from the hybridization between Triticum aestivum with Aegilops kotschyi,and characterize the effects on quality-related traits.Fluorescence in situ hybridization karyotypes showed that N124 is a disomic 1U^(k)(1A)substitution line.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and reversedphase high-performance liquid chromatography verified N124 expressed two HMW-GSs of the Ae.kotschyi parent.PacBio RNA sequencing and phylogenetic analysis confirmed that the two HMW-GSs were U^(k)x and U^(k)y.Compared to the wheat parent,the substitution line had no obvious agronomic defects except fewer grains per spike but improved several major quality parameters.It can be served as a donor or bridge material for wheat quality improvement.