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A co-located KL and TKW-related QTL with no negative effect on PH and AD was rapidly identified using BSA and wheat 660 K SNP array. Its effect was validated in a panel of 218 wheat accessions. Kernel length (KL) and thousand-kernel weight (TKW) of wheat (Triticum aestivum L.) contribute significantly to kernel yield. In the present study, a recombinant inbred line (RIL) population derived from the cross between the wheat line S849-8 with larger kernels and more spikelets per spike and the line SY95-71 was developed. Further, of both the bulked segregant analysis (BSA) and the wheat 660 K single nucleotide polymorphism (SNP) array were used to rapidly identify genomic regions for kernel-related traits from this RIL population. Kompetitive Allele Specific PCR markers were further developed in the SNP-enriched region on the 2D chromosome to construct a genetic map. Both QKL.sicau-SSY-2D for KL and QTKW.sicau-SSY-2D for TKW were identified at multiple environments on chromosome arm 2DL. These two QTLs explained 9.68-23.02% and 6.73-18.32% of the phenotypic variation, respectively. The effects of this co-located QTL were successfully verified in a natural population consisting of 218 Sichuan wheat accessions. Interestingly, the major QTL was significantly and positively correlated with spike length, but did not negatively affect spikelet number per spike (SNS), plant height, or anthesis date. These results indicated that it is possible to synchronously improve kernel weight and SNS by using this QTL. Additionally, several genes associated with kernel development and filling rate were predicted and sequenced in the QTL-containing physical intervals of reference genomes of 'Chinese spring' and Aegilops tauschii. Collectively, these results provide a QTL with great breeding potential and its linked markers which should be helpful for fine mapping and molecular breeding.

Summary Pre‐harvest sprouting (PHS), the germination of grain before harvest, is a serious problem resulting in wheat yield and quality losses.Here, we mapped the PHS resistance gene PHS‐3D from synthetic hexaploid wheat to a 2.4 Mb presence–absence variation (PAV) region and found that its resistance effect was attributed to the pleiotropic Myb10‐D by integrated omics and functional analyses.Three haplotypes were detected in this PAV region among 262 worldwide wheat lines and 16 Aegilops tauschii, and the germination percentages of wheat lines containing Myb10‐D was approximately 40% lower than that of the other lines. Transcriptome and metabolome profiling indicated that Myb10‐D affected the transcription of genes in both the flavonoid and abscisic acid (ABA) biosynthesis pathways, which resulted in increases in flavonoids and ABA in transgenic wheat lines. Myb10‐D activates 9‐cis‐epoxycarotenoid dioxygenase (NCED) by biding the secondary wall MYB‐responsive element (SMRE) to promote ABA biosynthesis in early wheat seed development stages.We revealed that the newly discovered function of Myb10‐D confers PHS resistance by enhancing ABA biosynthesis to delay germination in wheat. The PAV harboring Myb10‐D associated with grain color and PHS will be useful for understanding and selecting white grained PHS resistant wheat cultivars.

A novel, stably expressed, and plant height-independent QTL for spike extension length on 5AS was identified and validated in different populations using a newly developed and tightly linked KASP marker. As an important component of plant height (PH), spike extension length (SEL) plays a significant role in formation of an ideotype in wheat. Despite the fact that numerous loci for SEL in wheat have been reported, our knowledge on PH-independent loci remains to be limited. In this study, two recombinant inbred line (RIL) populations genotyped using the Wheat55K SNP were used to detect quantitative trait loci (QTL) controlling SEL across six environments. A total of 30 QTL for SEL were detected in these two RIL populations, and four of them, i.e., QSEL.sicau-2CN-4D, QSEL.sicau-2SY-4B.2, QSEL.sicau-2SY-4D.1, and QSEL.sicau-2CN-5A, were stably expressed. Genetic and conditional QTL analysis showed that the first three were significantly associated with PH, while the last one, QSEL.sicau-2CN-5A, is independent of PH. Comparison of genetic and physical maps suggested that only QSEL.sicau-2CN-5A located on chromosome arm 5AS is likely a novel QTL. A Kompetitive Allele-Specific PCR (KASP) marker, KASP-AX-110413733, tightly linked to this novel QTL was developed to successfully confirm its effect in three different genetic populations. Further, in the interval where QSEL.sicau-2CN-5A was located on 'Chinese Spring' wheat reference genome, three promising genes mainly expressed in wheat stem were predicated and they all encode the cytochrome P450 that was demonstrated to be closely associated with SEL elongation in rice. In addition, significant correlations between SEL and PH, spikelet number per spike, and thousand-grain weight were also detected. Altogether, our results broaden our understanding on genetic basis of SEL and will be useful for marker-based selection of lines with different SELs and fine mapping the novel and PH-independent QTL QSEL.sicau-2CN-5A.

Black point (BP) disease of wheat has become a noticeable problem in China. The symptoms are spots that are brown to black in color around the wheat kernel embryo or in the endosperm, resulting in a significant reduction of wheat grain quality. Here, we evaluated 272 Chinese wheat landraces for BP reaction and performed a genome-wide association study to identify BP resistance quantitative trait loci (QTLs) in five field environments without artificial inoculation. The BP incidence data showed continuous distributions and had low to moderate correlations between environments (r = 0.094 to 0.314). Among the 272 landraces, 11 had 0.1 to 4.9%, 144 had 5 to 14.9%, 100 had 15 to 29.9%, and 17 had >30% incidence. We found three resistant accessions: WH094 (3.33%), AS661463 (2.67%), and AS661231 (2.67%), which can be used in breeding programs to enhance BP resistance. We identified 11 QTLs, which explained 8.22 to 10.99% phenotypic BP variation, and mapped them to eight wheat chromosomes. Three of the QTLs were novel. The molecular markers for the BP resistance could facilitate molecular breeding for developing BP-resistant cultivars.

High yield and quality are essential goals of wheat (Triticum aestivum L.) breeding. Kernel length (KL), as a main component of kernel size, can indirectly change kernel weight and then affects yield. Identification and utilization of excellent loci in wheat genetic resources is of great significance for cultivating high yield and quality wheat. Genetic identification of loci for KL has been performed mainly through genome-wide association study in natural populations or QTL mapping based on genetic linkage map in high generation populations.In this study, an FA major, novel and stable QTL (Qkl.sicau-BLE18-4A) for KL was identified and verified in two F

Plant transcription factors (TFs), such as basic helix-loop-helix (bHLH) and AT-rich zinc-binding proteins (PLATZ), play critical roles in regulating the expression of developmental genes in cereals. We identified the bHLH protein TaPGS1 (T. aestivum Positive Regulator of Grain Size 1) specifically expressed in the seeds at 5-20 days post-anthesis in wheat. TaPGS1 was ectopically overexpressed (OE) in wheat and rice, leading to increased grain weight (up to 13.81% in wheat and 18.55% in rice lines) and grain size. Carbohydrate and total protein levels also increased. Scanning electron microscopy results indicated that the starch granules in the endosperm of TaPGS1 OE wheat and rice lines were smaller and tightly embedded in a proteinaceous matrix. Furthermore, TaPGS1 was bound directly to the E-box motif at the promoter of the PLATZ TF genes TaFl3 and OsFl3 and positively regulated their expression in wheat and rice. In rice, the OsFl3 CRISPR/Cas9 knockout lines showed reduced average thousand-grain weight, grain width, and grain length in rice. Our results reveal that TaPGS1 functions as a valuable trait-associated gene for improving cereal grain yield.

Chinese wheat landrace Anyuehong (AYH) has displayed high levels of stable adult plant resistance (APR) to stripe rust for15 years. To identify quantitative trait loci (QTLs) for stripe rust resistance in AYH, a set of 110 recombinant inbred lines (RILs) was developed from a cross between AYH and susceptible cultivar Taichung 29. The parents and RILs were evaluated for final disease severity (FDS) in six field tests with a mixture of predominant

Stripe rust (yellow rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive diseases of wheat worldwide. Chinese wheat landrace Guangtoumai (GTM) exhibited a high level of resistance against predominant P. striiformis f. sp. tritici races in China at the adult plant stage. The objective of this research was to identify and map the major locus/loci for stripe rust resistance in GTM. A set of 212 recombinant inbred lines (RILs) was developed from a cross between GTM and Avocet S. The parents and RILs were evaluated in three field tests (2018, 2019, and 2020 at Chongzhou, Sichuan) with the currently predominant P. striiformis f. sp. tritici races for final disease severity and genotyped with the Wheat 55K single nucleotide polymorphism (SNP) array to construct a genetic map with 1,031 SNP markers. A major locus, named QYr.GTM-5DL, was detected on chromosome 5DL in GTM. The locus was mapped in a 2.75-cM interval flanked by SNP markers AX-109855976 and AX-109453419, explaining up to 44.4% of the total phenotypic variation. Since no known Yr genes have been reported on chromosome 5DL, QYr.GTM-5DL is very likely a novel adult plant resistance locus. Haplotype analysis revealed that the resistance allele displayed enhanced levels of stripe rust resistance and is likely present in 5.3% of the 247 surveyed Chinese wheat landraces. The derived cleaved amplified polymorphic sequence (dCAPS) marker dCAPS-5722, converted from a SNP marker tightly linked to QYr.GTM-5DL with 0.3 cM, was validated on a subset of RILs and 48 commercial wheat cultivars developed in Sichuan. The results indicated that QYr.GTM-5DL with its linked dCAPS marker could be used in marker-assisted selection to improve stripe rust resistance in breeding programs, and this quantitative trait locus will provide new and possibly durable resistance to stripe rust.

Background Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a serious foliar disease of wheat. Identification of novel stripe rust resistance genes and cultivation of resistant cultivars are considered to be the most effective approaches to control this disease. In this study, we evaluated the infection type (IT), disease severity (DS) and area under the disease progress curve (AUDPC) of 143 Chinese wheat landrace accessions for stripe rust resistance. Assessments were undertaken in five environments at the adult-plant stage with Pst mixture races under field conditions. In addition, IT was assessed at the seedling stage with two prevalent Pst races (CYR32 and CYR34) under a controlled greenhouse environment. Results Seventeen accessions showed stable high-level resistance to stripe rust across all environments in the field tests. Four accessions showed resistance to the Pst races CYR32 and CYR34 at the seedling stage. Combining phenotypic data from the field and greenhouse trials with 6404 markers that covered the entire genome, we detected 17 quantitative trait loci (QTL) on 11 chromosomes for IT associated with seedling resistance and 15 QTL on seven chromosomes for IT, final disease severity (FDS) or AUDPC associated with adult-plant resistance. Four stable QTL detected on four chromosomes, which explained 9.99–23.30% of the phenotypic variation, were simultaneously associated with seedling and adult-plant resistance. Integrating a linkage map of stripe rust resistance in wheat, 27 QTL overlapped with previously reported genes or QTL, whereas four and one QTL conferring seedling and adult-plant resistance, respectively, were mapped distantly from previously reported stripe rust resistance genes or QTL and thus may be novel resistance loci. Conclusions Our results provided an integrated overview of stripe rust resistance resources in a wheat landrace diversity panel from the southern autumn-sown spring wheat zone of China. The identified resistant accessions and resistance loci will be useful in the ongoing effort to develop new wheat cultivars with strong resistance to stripe rust.

Anthocyanins and selenium have vital biological functions for human and plants, they were investigated thoroughly and separately in plants. Previous studies indicated pigmented fruits and vegetables had higher selenium concentration, but whether there is a relationship between anthocyanins and selenium is unclear. In this study, a combined phenotypic and genotypic methodological approach was undertaken to explore the potential relationship between anthocyanins and selenium accumulation by using phenotypic investigation and RNA-seq analysis. The results showed that pigmented cultivars enrichment in Se is a general phenomenon observed for these tested species, this due to pigmented cultivars have higher Se efficiency absorption. Se flow direction mainly improve concentration of S-rich proteins of LMW-GS. This may be a result of the MYB and bHLH co-regulate anthocyanins biosynthesis and Se metabolism at the transcriptional level. This thesis addresses a neglected aspect of the relevant relationship between anthocyanins and selenium.

Eight major and stably expressed QTL for flag leaf morphology across eleven environments were identified and validated using newly developed KASP markers in seven biparental populations with different genetic backgrounds. Flag leaf morphology is a determinant trait influencing plant architecture and yield potential in wheat (Triticum aestivum L.). A recombinant inbred line (RIL) population with a 55 K SNP-based constructed genetic map was used to map quantitative trait loci (QTL) for flag leaf length (FLL), width (FLW), area (FLA), angle (FLANG), opening angle (FLOA), and bend angle (FLBA) in eleven environments. Eight major QTL were detected in 11 environments with 5.73–54.38% of explained phenotypic variation. These QTL were successfully verified using the newly developed Kompetitive Allele Specific PCR (KASP) markers in six biparental populations with different genetic backgrounds. Among these 8 major QTL, two co-located intervals were identified. Significant interactions for both FLL- and FLW-related QTL were detected. Comparison analysis showed that QFll.sau-SY-2B and QFla.sau-SY-2B are likely new loci. Significant relationships between flag leaf- and yield-related traits were observed and discussed. Several genes associated with leaf development including the ortholog of maize ZmRAVL1, a B3-domain transcription factor involved in regulation of leaf angle, were predicted in physical intervals harboring these major QTL on reference genomes of bread wheat ‘Chinese spring’, T. turgidum, and Aegilops tauschii. Taken together, these results broaden our understanding on genetic basis of flag leaf morphology and provide clues for fine mapping and marker-assisted breeding wheat with optimized plant architecture for promising loci.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the most devastating foliar diseases in wheat. Host resistance is the most effective strategy for the management of the disease. To screen for accessions with stable resistance and identify effective stripe rust resistance loci, a genome-wide association study (GWAS) was conducted using a panel of 140 Chinese wheat landraces. The panel was evaluated for stripe rust response at the adult-plant stage at six field-year environments with mixed races and at the seedling stage with two separate predominant races of the pathogen, and genotyped with the genome-wide Diversity Arrays Technology markers. The panel displayed abundant phenotypic variation in stripe rust responses, with 9 landraces showing stable resistance to the mixture of Pst races at the adult-plant stage in the field and 10 landraces showing resistance to individual races at the seedling stage in the greenhouse. GWAS identified 12 quantitative trait loci (QTL) significantly (P ≤ 0.001) associated to stripe rust resistance using the field data of at least two environments and 18 QTL using the seedling data with two races. Among these QTL, 10 were presumably novel, including 4 for adult-plant resistance mapped to chromosomes 1B (QYrcl.sicau-1B.3), 4A (QYrcl.sicau-4A.3), 6A (QYrcl.sicau-6A.2) and 7B (QYrcl.sicau-7B.2) and 6 for all-stage resistance mapped to chromosomes 2D (QYrcl.sicau-2D.1), 3B (QYrcl.sicau-3B.3), 3D (QYrcl.sicau-3D), 4B (QYrcl.sicau-4B), 6A (QYrcl.sicau-6A.1) and 6D (QYrcl.sicau-6D). The landraces with stable resistance can be used for developing wheat cultivars with effective resistance to stripe rust.

In this study, we successfully expressed the active 1Ay subunit of Triticum urartu in barley by Agrobacterium-mediated transformation with a transformation efficiency of 19.9%. The results of SDS-PAGE revealed that the expressed proteins of 1Ay subunit were present at some grains of each of 46 original T0 plants, showing identical mobility to those of positive standards of T. urartu grain protein and bacteria expressional proteins. In the T2 generation, three homozygous lines, 2-28, 3-11, and 5-6, were identified. The results of scanning electron microscopy showed an increased amount of protein bodies in these transgenic lines. The main effects in the expression of the 1Ay subunits was a considerable increase in the glutenin content, but a decrease in the contents of gliadins while there were no effects in the contents of albumin, globulin and the total protein. We found that the gluten could not be washed out from the flour obtained from transgenic barley lines when using a Gluten index analyzer and a Farinograph indicating that the transgenic barley lines could not form dough. The lack of x-type HMW-GS and the reduction in number of subunit were inferred as the possible reasons for the inability to form gluten polymer.

The jasmonate-regulated protein Ta-JA1 belongs to the monocot chimeric jacalin (MCJ) family and plays a vital role in stress resistance in wheat. However, the impact of wheat polyploidization on Ta-JA1 remains unclear. In this study, 149 members of the MCJ family were identified among members of Triticeae using a genome-wide approach. The genes were resolved into three clades; MCJ genes in each clade were derived from different donor genes during evolution. Segmental duplication may have been the primary driver, compared with tandem duplication, of expansion in the MCJ family of wheat. Gene loss and acquisition occurred during tetraploidization, and the core expansion of the family occurred after tetraploidization. Sequencing data for 2104 accessions of T. aestivum and 99 accessions of T. dicoccoides showed that Ta-JA1-2A and Ta-JA1 were highly conserved in common wheat, and four alleles (TdJA1-Ax2, TdJA1-Ay2, TdJA1-Ax3, and TdJA1-Ay3) were detected in T. dicoccoides. Using gene-specific markers, one AsJA1-B allele was detected in 11 Ae. speltoides accessions and one TuJA1-Ax1 allele was detected in 70 T. urartu accessions. Six alleles were detected on chromosome 2A: TdJA1-Ax1 (13 accessions), TdJA1-Ay1 (57 accessions), TdJA1-Ax2 (23 accessions), TdJA1-Ay2 (42 accessions), TdJA1-Ax3 (29 accessions), and TdJA1-Ay3 (251 accessions). Only one allele (TdJA1-B) on chromosome 2B was detected in 415 T. dicoccoides accessions. A geographical distribution analysis revealed that Israel hosted higher allelic variation than other regions. Quantitative reverse transcription PCR analysis indicated that divergence in expression has occurred among Ta-JA1 alleles and, notably, TdJA1-Ax1 and TdJA1-Ay1 showed significantly higher expression levels than the other four allelic types in T. dicoccoides. The present results contribute to an improved understanding of the effects of polyploidization on the MCJ gene family and the functions of Ta-JA1, and may be useful to enrich common wheat germplasm resources.

A novel Wx-B1 allele was characterized; a transposon insertion resulted in the loss of its function, which is different from the previously reported gene silencing mechanisms at the Wx-B1 locus. The waxy protein composition of 53 Chinese wheat landraces was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis; of these, 10 did not show the expression of Wx-A1 (four accession) or Wx-B1 (six accessions) protein. The results of molecular marker detection revealed that the Wx-B1 allele (Wx-B1n) showed normal expression, inconsistent with the findings of SDS-PAGE for the Xiaobaipi accession. Further cloning of the 9160-bp region covering the Wx-B1 coding region and 3′-downstream region revealed that a 2178-bp transposon fragment had been inserted at 2462 bp within the tenth exon of Wx-B1n ORF, leading to the absence of Wx-B1 protein. Sequence analysis indicated that the insertion possessed the structural features of invert repeat and target repeat elements, we deduced that it was a transposon. Further PCR analysis revealed that this fragment had moved, but not copied itself, from 3B chromosome to the current location in Wx-B1n. Therefore, the reason for the inactivation of Wx-B1n was considerably different from those for the inactivation of Wx-B1b, Wx-B1k, and Wx-B1m; to our knowledge, this kind of structural mutation has never been reported in Wx-B1 alleles. This novel allele is interesting, because it was not associated with the deletion of other quality-related genes included in the 67 kb region lost with the common null allele Wx-B1b. The null Wx-B1n might be useful for investigating gene inactivation and expression as well as for enriching the genetic resource pool for the modification of the amylose/amylopectin ratio, thereby improving wheat quality.

In our previous work, a novel high-molecular-weight glutenin subunit (HMW-GS) with an extremely large molecular weight from

Background Kernel length (KL), kernel width (KW) and thousand-kernel weight (TKW) are key agronomic traits in wheat breeding. Chuannong16 (‘CN16’) is a commercial cultivar with significantly longer kernels than the line ‘20828’. To identify and characterize potential alleles from CN16 controlling KL, the previously developed recombinant inbred line (RIL) population derived from the cross ‘20828’ × ‘CN16’ and the genetic map constructed by the Wheat55K SNP array and SSR markers were used to perform quantitative trait locus/loci (QTL) analyses for kernel traits. Results A total of 11 putative QTL associated with kernel traits were identified and they were located on chromosomes 1A (2 QTL), 2B (2 QTL), 2D (3 QTL), 3D, 4A, 6A, and 7A, respectively. Among them, three major QTL, QKL.sicau-2D, QKW.sicau-2D and QTKW.sicau-2D, controlling KL, KW and TKW, respectively, were detected in three different environments. Respectively, they explained 10.88–18.85%, 17.21–21.49% and 10.01–23.20% of the phenotypic variance. Further, they were genetically mapped in the same interval on chromosome 2DS. A previously developed kompetitive allele-specific PCR (KASP) marker KASP-AX-94721936 was integrated in the genetic map and QTL re-mapping finally located the three major QTL in a 1- cM region flanked by AX-111096297 and KASP-AX-94721936. Another two co-located QTL intervals for KL and TKW were also identified. A few predicted genes involved in regulation of kernel growth and development were identified in the intervals of these identified QTL. Significant relationships between kernel traits and spikelet number per spike and anthesis date were detected and discussed. Conclusions Three major and stably expressed QTL associated with KL, KW, and TKW were identified. A KASP marker tightly linked to these three major QTL was integrated. These findings provide information for subsequent fine mapping and cloning the three co-localized major QTL for kernel traits.

Terpenoids play important roles in plant defense. Although some terpene synthases have been characterized, terpenoids and their biosynthesis in wheat (Triticum aestivum L.) still remain largely unknown. Here, we describe the identification of a terpene synthase gene in wheat. It encodes a sesquiterpene synthase that catalyzes &beta, patchoulene formation with E,E-farnesyl diphosphate (FPP) as the substrate, thus named as TaPS. TaPS exhibits inducible expression in wheat in response to various elicitations. Particularly, alamethicin treatment strongly induces TaPS gene expression and &beta, patchoulene accumulation in wheat. Overexpression of TaPS in Arabidopsis successfully produces &beta, patchoulene, verifying the biochemical function of TaPS in planta. Furthermore, these transgenic Arabidopsis plants exhibit resistance against herbivory by repelling beet armyworm larvae feeding, thereby indicating anti-herbivory activity of &beta, patchoulene. The catalytic mechanism of TaPS is also explored by homology modeling and site-directed mutagenesis. Two key amino acids are identified to act in protonation and stability of intermediates and product formation. Taken together, one wheat sesquiterpene synthase is identified as &beta, patchoulene synthase. TaPS exhibits inducible gene expression and the sesquiterpene &beta, patchoulene is involved in repelling insect infestation.

A major and stably expressed QTL for spikelet number per spike identified in a 2-cM interval on chromosome arm 2DS was validated using two populations with different genetic backgrounds. Spikelet number per spike (SNS) plays a key role in wheat yield improvement. Numerous genetic and environmental factors influencing SNS have been documented, but the number of major, stably expressed and validated loci underlying SNS is still limited. In this study, a recombinant inbred line (RIL) population derived from a normal spikelet cultivar and a multiple-spikelet wheat line (with a longer spike with more canonically oriented apical spikelets) was genotyped using a Wheat55K single-nucleotide polymorphism (SNP) array and simple sequence repeat (SSR) markers. SNS was measured for this RIL population in eight environments. Five QTL were each identified in two or more environments. One of them, QSns.sau-2D (LOD = 3.47–38.24, PVE = 10.16–45.68%), was detected in all the eight environments. The QTL was located in a 2-cM interval on chromosome arm 2DS flanked by the markers AX-109836946 and AX-111956072. This QTL, QSns.sau-2D, significantly increased SNS by up to 14.72%. Several genes associated with plant growth and development were identified in the physical interval of QSns.sau-2D. This QTL was further validated by the tightly linked Kompetitive Allele Specific PCR (KASP) marker, KASP-AX-94721936, in two other populations with different genetic backgrounds. The significant correlation between SNS and anthesis date, plant height, spike length, grain number per spike and thousand-grain weight were detected and discussed. These results lay the foundation for fine mapping and cloning gene(s) underlying QSns.sau-2D.

An EMS-induced single-base mutation at a splice site caused abnormal RNA splicing and resulted in the gene inactivation and the lack of Wx-A1 protein in a wheat EMS mutant line. An EMS-mutagenized population was generated using common wheat cv. SM126 consisting of 10,600 M2 plants. One Wx-A1 null mutant was identified through analyses of 390 grains produced from 130 M2 plants using electrophoresis analyses. The Wx-A1 sequences of parental line SM126 and M2-31 mutant were determined as 2781 bp, and there was only one SNP mutation between them. The SNP was a mutation from G to A at nucleotide sequence position 2168 bp (G2168A) downstream of the start codon which was located at the splicing site within the eighth intron. All 52 cDNA transcripts were found to be incorrectly spliced and can be summarized as five types of variations. The deletion of the exon and the exclusion of intron were structural features in abnormal splicing RNA. Together with the prediction of potential splice regulatory motifs, the mutation G2168A happened within the 5′ splice site of the eighth intron and destroyed the splice donor site from GU to AU, which may have brought about a barrier against correct RNA splice, and generated abnormal mRNA, which was the mechanism of the inactivation of Wx-A1 in M2-31. The lack of Wx-A1 has resulted in changes in starch properties in the M2-31 mutant, with the reduction in amylose and starch contents. The increased grains hardness was observed in M2-31, which may be related to the lower expression level of Pinb-D1 gene. As the waxy wheat foods have a lot of advantages, the null waxy genes will be widely applied in breeding waxy wheat for varied amylose contents.

Chromosome translocation is an important driving force in shaping genomes during evolution. Detailed knowledge of chromosome translocations in a given species and its close relatives should increase the efficiency and precision of chromosome engineering in crop improvement. To identify genes flanking the breakpoints of translocations and inversions as a step toward identifying breakpoints in bread wheat, we systematically analysed genes in the Brachypodium genome against wheat survey sequences and bin-mapped ESTs (expressed sequence tags) derived from the hexaploid wheat genotype ‘Chinese Spring’. In addition to those well-known translocations between group 4, 5, and 7 chromosomes, this analysis identified genes flanking the three pericentric inversions on chromosomes 2B, 4B, and 5A. However, numerous chromosomal rearrangements reported in early studies could not be confirmed. The genes flanking the breakpoints reported in this study are valuable for isolating these breakpoints.

In this study, the ARF genes were cloned, sequenced and located on the chromosomes. The gene expression of various stress conditions were analyzed through RT-PCR. Two important features of ARF in wheat were found: (1) High sequences homology among species in mammalian and plant and (2) Four exons and three introns were conserved in Poaceae. In this study the coding genes of ADP-ribosylation Factors (ARF) were characterized and they were located on chromosomes 3AL and 2DL in common wheat and its diploid progenitors. Forty-seven candidate SNPs in ARF were detected which were located in exons (17 SNPs) and introns (30 SNPs), respectively. As expected, most of the SNPs (66.34%) in ARF were transitions and the rest (33.66%) were transversions. The expression difference of ARF under various environmental stresses (low-temperature, Abscisic Acid (ABA), Polyethylene Glycol (PEG), NaCl, stripe rust), in two stages (seedling and maturity) and in different tissues (root, stem, flag leaf and immature embryo) of 15 days post-flowering were investigated. The results revealed that the expression levels of ARF were affected by environmental stresses. PEG stress induced the highest level of ARF expression, followed by the stripe rust and ABA stresses.

Triticum turgidum ssp. dicoccon PI94668 and PI349045 were identified as containing null alleles at Glu-A1 and Glu-B1 loci in previous investigation. Sequencing of the respective HMW-GS genes Ax, Bx, Ay and By in both accessions indicated equal DNA lengths with gene silencing caused by 1 to 4 in-frame stop codon(s) in the open reading frames. Six synthetic hexaploid wheat lines were produced by crossing PI94668 or PI349045 with six Aegilops tauschii by spontaneous chromosome doubling of unreduced gametes. As expected, these amphiploids had three different HMW-GS: Dx 3.1(t) + Dy11(*t), Dx2.1(t) +10(t) and Dx2(t) +Dy12(t) in Glu-D1 but double nulls in Glu-A1 and Glu-B1. Quality tests showed that most quality parameters in two T. turgidum ssp. dicoccon parents were very low due to the lack of HMW-GSs. However, incorporation of HMW-GS from Ae. tauschii in six synthetic hexaploid wheat lines significantly increased most quality related parameters. The potential values of these wheat lines in improving the quality of wheat are discussed.

Gene loss during the formation of hexaploid bread wheat has been repeatedly reported. However, our knowledge on genome-wide analysis of the genes present on a single subgenome (SSG) in bread wheat is still limited. In this study, by analysing the ‘Chinese Spring’ chromosome arm shotgun sequences together with high-confidence gene models, we detected 433 genes on a SSG. Greater gene loss was observed in A and D subgenomes compared with B subgenome. More than 79% of the orthologs for these SSG genes were detected in diploid and tetraploid relatives of hexaploid wheat. Unexpectedly, no bias in expression breadth or in the distribution patterns of GO (gene ontology) terms for these genes was detected among the high-confidence genes. Further, network and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses indicated that most of these genes were not functionally related to each other. Interestingly, 30.7% of these SSG genes were most highly expressed in root, showing biased distribution given the distribution of the whole high-confidence genes. Collectively, these results facilitate our understanding of the loss of the genes that were retained in a SSG during the formation of hexaploid wheat.

Yellow or stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating foliar disease that affects common wheat (Triticum aestivum L.) around the world. In China, common wheat landraces are potential sources of disease and abiotic stress resistance genes for wheat improvement. Yilongtuomai (YL), a wheat landrace from Yilong County, Sichuan Province, shows high levels of resistance against most Chinese Pst races. In this study, the resistance of YL to stripe rust disease was examined in detail. Parent strains, YL and Taichung 29, a variety susceptible to Pst race CYR32, and their F1, F2, and F2:3 offspring, were inoculated with CYR32 during the seedling stage in the field or adult-plant stage in the greenhouse. Results indicated that resistance to CYR32 in YL is conferred by a single dominant gene, designated YrYL The segregating F2 population (352 plants), was analyzed in terms of its resistance locus using simple sequence repeats (SSRs), resistance gene analog polymorphisms (RGAPs), and sequence-related amplified polymorphism (SRAP). A linkage group of 6 SSRs, 2 RGAPs, and 1 SRAP was constructed for the YrYL gene. Using the identified SSRs associated with physical mapping of RGAP using Chinese Spring nullisomic-tetrasomic stocks, the YrYL gene was localized to the short arm of chromosome 7D. The gene was flanked by 1 SSR marker, Xbarc92, and 1 RGAP marker, CLRRfor/Ptokin4, at genetic distances of 5.35 and 9.86 cM, respectively. The YrYL gene was compared to other stripe rust resistance genes reported on chromosome 7D by evaluating its reaction patterns to CYR32 and its pedigree relationship. Our results suggest that the YrYL gene is a new stripe rust resistance gene.

Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) of wheat and barley and is considered to be one of the most devastating plant diseases worldwide. Chitin is a critical component of the fungal cell wall and is polymerized from UDP-N-acetyl-alpha-D-glucosamine by chitin synthase. We characterized FgCHS8, a new class of the chitin synthase gene in F. graminearum. Disruption of FgCHS8 resulted in reduced accumulation of chitin, decreased chitin synthase activity, and had no effect on conidia growth when compared with the wild-type isolate. ΔFgCHS8 had a growth rate comparable to that of the wild-type isolate in vitro. However, ΔFgCHS8 had reduced growth when grown on agar supplemented with either 0.025% SDS or 0.9 mM salicylic acid. ΔFgCHS8 produced significantly less deoxynivalenol and exhibited reduced pathogenicity in wheat spikes. Re-introduction of a functional FgCHS8 gene into the ΔFgCHS8 mutant strain restored the wild-type phenotypes. Fluorescence microscopy revealed that FgCHS8 protein was initially expressed in the septa zone, and then gradually distributed over the entire cellular membrane, indicating that FgCHS8 was required for cell wall development. Our results demonstrated that FgCHS8 is important for cell wall sensitivity to environmental stress factors and deoxynivalenol production in F. graminearum.

The identification and characterization of resistance genes effective against stripe rust of wheat is beneficial for modern wheat breeding programs. Molecular markers to such genes facilitate their deployment. The variety Milan has resistance that is effective against the predominant stripe rust races in the Sichuan region. Two resistant and two susceptible F8 lines from a cross between Milan and the susceptible variety Chuannong 16 were used to investigate inheritance of the Milan resistance. Three F2 populations were developed from crosses between the resistant lines and their susceptible sibling lines (LM168a × LM168c, LM168c × LM168a, LM168b × LM168d) and used for genetic analysis and molecular mapping of the genes for resistance. The stripe rust resistance in LM168a and LM168b was conferred by a single dominant gene, temporarily designated as YrLM168a. Forty-five extreme susceptible plants from the F2 families of LM168d × LM168b were genotyped with 836 simple sequence repeat (SSR) markers to map YrLM168a. YrLM168a was mapped in chromosome 6BL. The nearest flanking markers Xwmc756 and Xbarc146 were 4.6 and 4.6 cM away from the gene at both sides, respectively. The amplification results of twenty extreme resistant (IT 0) and susceptible (IT 4) F2 plants of LM168c × LM168a and LM168a × LM168c with marker Xwmc756 further validated the mapping results. The study suggested that extreme individuals and recessive phenotype class can be successfully used for mapping genes, which should be efficient and reliable. In addition, the flanking markers near YrLM168a should be helpful in marker-assisted breeding.

The function of starch phosphorylase has long been debated on the regulation of starch metabolism during the growth and development of plants. In this study, we isolated starch phosphorylase genes (Pho1 and Pho2) from barley, characterized their gene and protein structures, predicated their promoter’s cis-elements and analyzed expression patterns. Multiple alignments of these genes showed that (1) both Pho1 and Pho2 genes possess 15 exons and 14 introns in all but three of the species analyzed, Aegilops tauschii (for Pho1 which contains 16 exons and 15 introns), potato (for Pho1b which contains 14 exons and 13 introns), and Triticum uraru (for Pho2 which contains 15 exons and 14 introns); (2) the exon–intron junctions of Pho1 and Pho2 flanking the ligand-binding sites are more conservative than the other regions. Analysis of protein sequences revealed that Pho1 and Pho2 were highly homologous except for two regions, the N terminal domain and the L78 insertion region. The results of real-time quantitative PCR (RT-qPCR) indicated that Pho2 is mainly expressed in germinating seeds, and the expression of Pho1 is similar to that of starch synthesis genes during seed development in barley. Microarray-based analysis indicated that the accumulation of Pho1 or Pho2 transcripts exhibited uniform pattern both in various tissues and various stages of seed development among species of barley, rice, and Arabidopsis. Pho1 of barley was significantly down-regulated under cold and drought treatments, and up-regulated under stem rust infection. Pho2 exhibited similar expression to Pho1 in barley. However, significant difference in expression was not detected for either Pho1 or Pho2 under any of the investigated abiotic stresses. In Arabidopsis, significant down-regulation was detected for Pho1 (PHS1) under abscisic acid (ABA) and for Pho2 (PHS2) under cold, salt, and ABA. Our results provide valuable information to genetically manipulate phosphorylase genes and to further elucidate their regulatory mechanism in the starch biosynthetic pathway.

To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.