Introgression of QTL from Aegilops tauschii enhances yield-related traits in common wheat

To break the narrow diversity bottleneck of the wheat D genome, a set of Aegilops tauschii-wheat introgression(A-WI) lines was developed by crossing Ae. tauschii accession T015 with common wheat elite cultivar Zhoumai 18(Zhou18). A high-density genetic map was constructed based on Single Nucleotide Polymorphism(SNP) markers and 15 yield-related traits were evaluated in 11 environments for detecting quantitative trait loci(QTL). A total of 27 environmentally stable QTL were identified in at least five environments, 20 of which were derived from Ae. tauschii T015, explaining up to 24.27% of the phenotypic variations. The major QTL for kernel length(KL), QKl-2D.5, was delimited to a physical interval of approximately 2.6 Mb harboring 52 candidate genes. Three Kompetitive Allele Specific PCR(KASP)markers were successfully developed based on nonsynonymous nucleotide mutations of candidate gene AetT093_2Dv1G100900.1 and showed that A-WI lines with the T015 haplotype had significantly longer KL than the Zhou18 haplotype across all 11 environments. Four primary valuable A-WIs with good trait performance and carrying yield-related QTL were selected for breeding improvement. The results will facilitate the efficient transfer of beneficial genes from Ae. tauschii into wheat cultivars to improve wheat yield and other traits.

A Unique Aegilops tauschii Genotype Needless to Immature Embryo Culture in Cross with Wheat

Common or bread wheat ( Triticum aestivum L., AABBDD, 2n=42) originated ca. 8 000 years ago from hybridization of tetraploid wheat ( Triticum turgidum L., AABB, 2n=28) and diploid Aegilops tauschii Coss. (DD, 2n=14). An essential prerequisite for this evolutionary step is that the natural hybrids between tetraploid wheat and diploid Aegilops tauschii can produce relatively many filled seeds which germinated well. In this study, without special techniques, e.g. immature embryo culture, out of 22 Ae. tauschii accessions, the genotype AS60 produced relatively many filled seeds which germinated well. The seed germination percentages in the crosses of Ae. tauschii ×tetraploid wheat, tetraploid wheat× Ae. tauschii and Ae. tauschii ×common wheat were, respectively, 50.0%, 57.1% and 45.5%. It seems that Ae. tauschii accession AS60 has a unique genotype which facilitate hybrid seed development and viability, and which meets with the prerequisite for wheat evolutionary. Furthermore, the significance of this finding for common wheat improvement and evolution was discussed.

Production of aneuhaploid and euhaploid sporocytes by meiotic restitution in fertile hybrids between durum wheat Langdon chromosome substitution lines and Aegilops tauschii

Fertile F1 hybrids were obtained between durum wheat （Triticum durum Desf.） Langdon （LDN） and its 10 disomic substitution （LDN DS） lines with Aegilops tauschii accession AS60 without embryo rescue. Selfed seedset rates for hybrids of LDN with AS60 were 36.87% and 49.45% in 2005 and 2006, respectively. Similar or higher selfed seedset rates were observed in the hybrids of 1D （1A）, 1D （1B）, 3D （3A）, 4D （4B）, 7D （7A）, and 2D （2B） with AS60, while lower in hybrids of 3D （3B） ＋ 3BL, 5D （5A） ＋ 5AL, 5D （5B） ＋ 5B and 6D （6B） ＋ 6BS with AS60 compared with the hybrids of LDN with AS60. Observation of male gametogenesis showed that meiotic restitution, both first-division restitution （FDR） and single-division meiosis （SDM） resulted in the formation of functional unreduced gametes, which in turn produced seeds. Both euhaploid and aneuhaploid gametes were produced in F1 hybrids. This suggested a strategy to simultaneously transfer and locate major genes from the ancestral species T. turgidum or Ae. tauschii. Moreover, there was no significant difference in the aneuhaploid rates between the F1 hybrids of LDN and LDN DS lines with AS60, suggesting that meiotic pairing between the two D chromosomes in the hybrids of LDN DS lines with AS60 did not promote the formation of aneuhaploid gametes.

Tolerant Mechanism and Chromosome Location of Gene Controlling Sprouting Tolerance in Aegilops Tauschii Cosson

An artificial amphiploid RSP (2n = 42, AABBDD) between tetraploid landrace Ailanmai (Triticum turgidum L., 2n= 28, AABB) and Aegllops tauschii (DD, 2n = 14) expressed high tolerance to preharvest sprouting which derived from Ae. tauschii. Tolerance to preharvest sprouting of RSP was examined by four ways in six varying periods after anthesis. The germination percentages of preharvest intact spikes were only 6.06% in its high peak period of germination. Its tolerance was mainly decided by the seed dormancy. It was showed that the tolerance to sprouting in ' RSP' derived from Ae. tauschii was inherited as a recessive trait which was controlled by one gene, located on chromosome 2D.

Association analysis of grain traits with SSR markers between Aegilops tauschii and hexaploid wheat(Triticum aestivum L.)

Seven important grain traits, including grain length（GL）, grain width（GW）, grain perimeter（GP）, grain area（GA）, grain length/width ratio（GLW）, roundness（GR）, and thousand-grain weight（TGW）, were analyzed using a set of 139 simple sequence repeat（SSR） markers in 130 hexaploid wheat varieties and 193 Aegilops tauschii accessions worldwide. In total, 1 612 alleles in Ae. tauschii and 1 360 alleles in hexaploid wheat（Triticum aestivum L.） were detected throughout the D genome. 197 marker-trait associations in Ae. tauschii were identified with 58 different SSR loci in 3 environments, and the average phenotypic variation value（R2） ranged from 0.68 to 15.12%. In contrast, 208 marker-trait associations were identified in wheat with 66 different SSR markers in 4 environments and the average phenotypic R2 ranged from 0.90 to 19.92%. Further analysis indicated that there are 6 common SSR loci present in both Ae. tauschii and hexaploid wheat, which are significantly associated with the 5 investigated grain traits（i.e., GA, GP, GR, GL, and TGW） and in total, 16 alleles derived from the 6 aforementioned SSR loci were shared by Ae. tauschii and hexaploid wheat. These preliminary data suggest the existence of common alleles may explain the evolutionary process and the selection between Ae. tauschii and hexaploid wheat. Furthermore, the genetic differentiation of grain shape and thousand-grain weight were observed in the evolutionary developmental process from Ae. tauschii to hexaploid wheat.

Genome-wide characterization of early response genes to abscisic acid coordinating multiple pathways in Aegilops tauschii

The diploid wild goat grass Aegilops tauschii(Ae. tauschii, 2 n = 14;DD), as the D-sub genome of common wheat, provides rich germplasm resources for many aspects of wheat breeding. Abscisic acid(ABA) is an essential phytohormone that plays a pivotal role in plant adaptation to abiotic stresses. However,the gene regulation network of Ae. tauschii in response to ABA stress remains unclear. Here, we conducted a time-course strand-specific RNA-sequencing study to globally profile the transcriptome that responded to ABA treatment in Ae. tauschii. We identified 4818 differentially expressed transcription units/genes with time-point-specific induction/repression patterns. Using functional annotation, one-to-one ortholog and comparative transcriptome profiling analyses, we identified 319 ABA-responsive Ae. tauschii orthologs that were also induced/repressed under ABA treatment in hexaploid wheat. On the quantitative trait loci(QTL) used in wheat marker-assisted breeding, we found that the ABA-responsive expression patterns of eight Ae. tauschii orthologs were associated with drought stress tolerance, flowering process and/or grain quality. Of them, the ABA-responsive gene encoding sucrose:sucrose 1-fructosyltransferase in fructan and glucose metabolism pathways showed the most significant association with wheat drought tolerance. The characterization of ABA early-responsive genes in this study provides valuable information for exploring the molecular functions of the regulatory genes and will assist in wheat breeding.

Characterization of WAP2 gene in Aegilops tauschii and comparison with homoeologous loci in wheat

The Q/q gene, also known as WAP2, is an important gene for wheat domestication and is a member of the AP2 （APETALA2） class of transcription factors. In the present study, we first isolated the WRAP2 allele （where the superscript ＂t＂ refers to the speciese source, in this case ＂tauschii＂） on chromosome 5D from Aegilops tauschii Coss., the D-genome donor species of common wheat. We found that WRAP2 and the AP2 gene from Arabidopsis share a central core of the AP2 polypeptide, a highly basic 10-amino acid domain, and an AASSGF box, although there are many differences in the 37-amino acid serine-rich acidic domain and the remaining regions. In addition, WRAP2 was highly homologous to the homoeologous loci on 5A and 5B of wheat at both the nucleotide and amino acid level. However, there were some variations that are probably related to gene function. In the first AP2 domain, the amino acids VYL on the 5D and 5A loci were replaced with LLR on 5B. In the 37-amino acid serine-rich acidic domain, WRAP2 on 5D had an extra amino acid insertion. There was also a variation at the 329 amino acid position, which is thought to be related to the appearance of free-threshing wheat. At this position, the amino acid is isoleucine on 5A for the Q allele and valine for the q allele, whereas the amino acid is leucine on 5D and 5B. Furthermore, a Stowaway miniature terminal inverted repeat element （MITE） insertion was present in the ninth intron of WAP2 on 5B of all common wheats and partial tetraploid Triticum turgidum wheats. These results provide new clues for studies into the evolutionary biology of WAP2 and the origin of common wheat.

Mapping of the heading date gene HdAey2280 in Aegilops tauschii

An optimum heading date is essential for sustainable crop productivity and ensuring high yields. In the present study, F2：3 populations were generated by crossing an early-heading accession, Y2280, with a late-heading accession, Y2282. The heading dates of the F2 and F3 populations were investigated in a field study. Using publicly available simple sequence repeat （SSR） markers, the early heading date gene HdAey2280 was mapped onto Aegilops tauschfi chromosome 7DS between the flanking markers wmc438 and barc126 at distances of 15 and 9.1 cM, respectively. Further analysis indicated that HdAey2280 is a novel heading date gene. New SSR markers were developed based on the Ae. tauschfi draft genome sequence, resulting in four new markers that were linked to the heading date gene HdAey2280. The closest distance of these markers was 1.9 cM away from the gene. The results collected in this study will serve as a framework for map-based cloning and marker-assisted selection in wheat breeding programs in the future.

Identification and Mapping of Two New Genes Conferring Resistance to Powdery Mildew from Aegilops tauschii （Coss,） Schmal

Two powdery mildew resistance genes were Identified from Aegilops tauschll accessions Y201 and Y212 and mapped using two different F2 populations derived from the crosses between susceptible accession Y2272 and Y201, and susceptible accession Y2263 and Y212. Genetic analysis of resistance to powdery mildew Indicated that the resistance of Y201 was controlled by a single dominant gene, whereas the resistance of Y212 was controlled by a single recessive gene. We have temporarily designated these genes as PmY201 and PmY212, respectively. By bulk segregation analysis, six mlcrosatelllte markers Including Xgwm174, cfd26, cfd57, cfdl02, Xgwm583 and Xgwm639 were found to be linked to PraY201 with genetic distances of 5.2, 7.7, 9.6, 12.5, 20.2 and 22.1 cM, respectively. Five SSR markers, including cfd57, Xgwm182, cfd7, cfd102, and cfd12, were found to be linked to PmY212 with distances of 5.6, 7.2, 11.5, 14.7, and 18.5 cM, respectively. According to the locations of the linked markers, the two resistance genes were located In the 5DL region. Based on the chromosomal locations and the resistance patterns of the two genes, we propose that PmY201 and PmY212 are two novel powdery mildew resistance genes, and are suitable for marker-assisted selection.

Sequencing and comparative analyses of Aegilops tauschii chromosome arm 3DS reveal rapid evolution of Triticeae genomes

Bread wheat （Triticum aestivum, AABBDD） is an allohexaploid species derived from two rounds of interspecific hybridizations. A high-quality genome sequence assembly of diploid Aegilops tauschii, the donor of the wheat D genome, will provide a useful platform to study polyploid wheat evolution. A combined approach of BAC pooling and next-generation sequencing technology was employed to sequence the minimum tiling path （MTP） of 3176 BAC clones from the short arm ofAe. tauschii chromosome 3 （At3DS）. The final assembly of 135 super-scaffolds with an N50 of 4.2 Mb was used to build a 247-Mb pseudomolecule with a total of 2222 predicted protein-coding genes. Compared with the orthologous regions of rice, Brachypodium, and sorghum, At3DS contains 38.67% more genes. In comparison to At3DS, the short arm sequence of wheat chromosome 3B （Ta3BS） is 95-Mb large in size, which is primarily due to the expansion of the non-centromeric region, suggesting that transposable element （TE） bursts in Ta3B likely occurred there. Also, the size increase is accompanied by a proportional increase in gene number in Ta3BS. We found that in the sequence of short arm of wheat chromosome 3D （Ta3DS）, there was only less than 0.27% gene loss compared to At3DS. Our study reveals divergent evolution of grass genomes and provides new insights into sequence changes in the polyploid wheat genome.

New insights into the dispersion history and adaptive evolution of taxon Aegilops tauschii in China

Aegilops tauschii,the wild progenitor of wheat D-genome and a valuable germplasm for wheat improvement,has a wide natural distribution from eastern Turkey to China.However,the phylogenetic relationship and dispersion history of Ae.tauschii in China has not been scientifically clarified.In this study,we genotyped 208 accessions(with 104 in China)using dd RAD sequencing and 55K SNP array,and classified the population into six sublineages.Three possible spreading routes or events were identified,resulting in specific distribution patterns,with four sublineages found in Xinjiang,one in Qinghai,two in Shaanxi and one in Henan.We also established the correlation of SNP-based,karyotypebased and spike-morphology-based techniques to demonstrate the internal classification of Ae.tauschii,and developed consensus dataset with 1245 putative accessions by merging data previously published.Our analysis suggested that eight inter-lineage accessions could be assigned to the putative Lineage 3and these accessions would help to conserve the genetic diversity of the species.By developing the consensus phylogenetic relationships of Ae.tauschii,our work validated the hypothesis on the dispersal history of Ae.tauschii in China,and contributed to the efficient and comprehensive germplasm-mining of the species.

QTL Mapping for Important Agronomic Traits in Synthetic Hexaploid Wheat Derived from Aegiliops tauschii ssp. tauschii

Aegiliops tauschii is classified into two subspecies： Ae. tauschii ssp. tauschii and Ae. tauschii ssp. strangulata. Novel genetic variations exist in Ae. tauschii ssp. tauschii that can be utilized in wheat improvement. We synthesized a hexaploid wheat genotype（SHW-L1） by crossing an Ae. tauschii ssp. tauschii accession（AS60） with a tetraploid wheat genotype（AS2255）. A population consisting of 171 F8 recombinant inbred lines was developed from SHW-L1 and Chuanmai 32 to identify QTLs associated with agronomic traits. A new genetic map with high density was constructed and used to detect the QTLs for heading date, kernel width, spike length, spikelet number, and thousand kernel weight. A total of 30 putative QTLs were identified for five investigated traits. Thirteen QTLs were located on D genomes of SHW-L1, six of them showed positive effect on agronomic traits. Chromosome region flanked by wPt-6133–wPt-8134 on 2D carried five environment-independent QTLs. Each QTL accounted for more than 10% phenotypic variance. These QTLs were highly consistent across environments and should be used in wheat breeding.

Development of diagnostic markers for a wheat leaf rust resistance gene Lr42 using RNA-sequencing

Wheat leaf rust is a prevalent foliar disease in wheat worldwide. Growing resistant cultivars is an effective strategy to minimize the impact of leaf rust on yield and grain quality. Lr42 is a leaf rust resistance gene identified from Aegilops tauschii and is still effective against current predominant leaf rust races in the United States and many other countries. In this study, we developed diagnostic DNA markers for Lr42 using the sequence polymorphisms of a differentially expressed gene(TaRPM1) encoding a putative NBARC protein in the Lr42 candidate region identified by RNA-sequencing of two near-isogenic lines contrasting in Lr42 alleles. Markers were designed based on a deletion mutation and a single nucleotide polymorphism(SNP) in the gene. Haplotype analyses of the newly developed markers in the three diversity panels demonstrated that they are diagnostic for Lr42, and superior to previously used markers in selection accuracy. These markers have the advantages of low cost and easy assay, and they are suitable for marker-assisted selection in breeding programs with either high-or low-throughput marker screening facilities.

Synthetic hexaploid wheat and its utilization for wheat genetic improvement in China

Synthetic hexaploid wheat （Triticum turgidum x Aegilops tauschii） was created to explore for novel genes from T. turgidum and Ae. tauschii that can be used for common wheat improvement. In the present paper, research advances on the utilization of synthetic hexaploid wheat for wheat genetic improvement in China are reviewed. Over 200 synthetic hexaploid wheat （SHW） accessions from the International Maize and Wheat Improvement Centre （CIMMYT） were introduced into China since 1995. Four cultivars derived from these, Chuanmai 38, Chuanmai 42, Chuanmai 43 and Chuanmai 47, have been released in China. Of these, Chuanmai 42, with large kernels and resistance to stripe rust, had the highest average yield （〉 6 t/ha） among all cultivars over two years in Sichuan provincial yield trials, outyielding the commercial check cultivar Chuanmai 107 by 22,7%. Meanwhile, by either artificial chromosome doubling via colchicine treatment or spontaneous chromosome doubling via a union of unreduced gametes （2n） from T. turgidum-Ae, tauschii hybrids, new SHW lines were produced in China. Mitotic-like meiosis might be the cytological mechanism of spontaneous chromosome doubling. SHW lines with genes for spontaneous chromosome doubling may be useful for producing new SHW-alien amphidiploids and double haploid in wheat genetic improvement.

粗山羊草响应盐胁迫转录组分析

利用转录组测序(RNA-Seq)技术对粗山羊草‘AL8/78'在200 mmol/L NaCl人工模拟盐胁迫0h和96h进行了转录组分析。结果表明:盐胁迫处理下上调和下调表达的差异表达基因(DEGs)分别为546个和876个;GO富集分析发现,DEGs主要集中在代谢过程、细胞过程、细胞连接、催化活性等生物过程;KEGG富集分析发现,DEGs主要富集在苯丙酮生物合成、类黄酮生物合成、植物激素信号转导等信号通路。通过对DEGs进行转录因子注释,共注释到41个转录因子,主要包括WRKY、MYB、bHLH、NAC和HSP等类型。利用实时荧光定量PCR(qRT-PCR)对DEGs进行了表达模式验证,发现其与RNA-Seq测序结果一致,证明了RNA-Seq结果的准确性。本研究为挖掘粗山羊草耐盐基因提供了理论基础。