Genome-Wide Identification and Expression Profiling Analysis of WOX Family Protein-Encoded Genes in Triticeae Species

The WOX family is a group of plant-specific transcription factors which regulate plant growth and development, cell division and differentiation. From the available genome sequence databases of nine Triticeae species, 199 putative WOX genes were identified. Most of the identified WOX genes were distributed on the chromosomes of homeologous groups 1 to 5 and originated via the orthologous evolution approach. Parts of WOX genes in Triticum aestivum were confirmed by the specific PCR markers using a set of Triticum. durum-T. aestivum genome D substitution lines. All of these identified WOX proteins could be grouped into three clades, similar to those in rice and Arabidopsis. WOX family members were conserved among these Triticeae plants; all of them contained the HOX DNA-binding homeodomain, and WUS clade members contained the characteristic WUS-box motif, while only WUS and WOX9 contained the EAR motif. The RNA-seq and qPCR analysis revealed that the TaWOX genes had tissue-specific expression feature. From the expression patterns of TaWOX genes during immature embryo callus production, TaWOX9 is likely closely related with the regulation of regeneration process in T. aestivum. The findings in this study could provide a basis for evolution and functional investigation and practical application of the WOX family genes in Triticeae species.

Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in polyploid wheat and related species

Background Wheat (Triticum aestivum) originated from three different diploid ancestral grass species and experienced two rounds of polyploidization. Exploring how certain wheat gene subfamilies have expanded during the evolutionary process is of great importance. The Lateral Organ Boundaries Domain (LBD) gene family encodes plant-specific transcription factors that share a highly conserved LOB domain and are prime candidates for this, as they are involved in plant growth, development, secondary metabolism and stress in various species. Methods Using a genome-wide analysis of high-quality polyploid wheat and related species genome sequences, a total of 228 LBD members from five Triticeae species were identified, and phylogenetic relationship analysis of LBD members classified them into two main classes (classes I and II) and seven subgroups (classes I a–e, II a and II b). Results The gene structure and motif composition analyses revealed that genes that had a closer phylogenetic relationship in the same subgroup also had a similar gene structure. Macrocollinearity and microcollinearity analyses of Triticeae species suggested that some LBD genes from wheat produced gene pairs across subgenomes of chromosomes 4A and 5A and that the complex evolutionary history of TaLBD4B-9 homologs was a combined result of chromosome translocation, polyploidization, gene loss and duplication events. Public RNA-seq data were used to analyze the expression patterns of wheat LBD genes in various tissues, different developmental stages and following abiotic and biotic stresses. Furthermore, qRT-PCR results suggested that some TaLBDs in class II responded to powdery mildew, regulated reproductive growth and were involved in embryo sac development in common wheat.

Comparative Analysis of the Glutathione S-Transferase Gene Family of Four Triticeae Species and Transcriptome Analysis of GST Genes in Common Wheat Responding to Salt Stress

Glutathione S-transferases (GSTs) are ancient proteins encoded by a large gene family in plants, which play multiple roles in plant growth and development. However, there has been little study on the GST genes of common wheat (Triticum aestivum) and its relatives (Triticum durum, Triticum urartu, and Aegilops tauschii), which are four important species of Triticeae. Here, a genome-wide comprehensive analysis of this gene family was performed on the genomes of common wheat and its relatives. A total of 346 GST genes in T. aestivum, 226 in T. durum, 104 in T. urartu, and 105 in Ae. tauschii were identified, and all members were divided into ten classes. Transcriptome analysis was used to identify GST genes that respond to salt stress in common wheat, which revealed that the reaction of GST genes is not sensitive to low and moderate salt concentrations but is sensitive to severe concentrations of the stressor, and the GST genes related to salt stress mainly come from the Tau and Phi classes. Six GST genes which respond to different salt concentrations were selected and validated by a qRT-PCR assay. These findings will not only provide helpful information about the function of GST genes in Triticeae species but also offer insights for the future application of salt stress resistance breeding in common wheat.

Mediation of a GDSL Esterase/Lipase in Carotenoid Esterification in Tritordeum Suggests a Common Mechanism of Carotenoid Esterification in Triticeae Species

Carotenoids are essential in human diet, so that the development of programs toward carotenoid enhancement has been promoted in several crops. The cereal tritordeum, the amphiploid derived from the cross between Hordeum chilense Roem. et Schulz. and durum wheat has a remarkable carotenoid content in the endosperm. Besides, a high proportion of these carotenoids are esterified with fatty acids. The identification of the gene(s) responsible for xanthophyll esterification would be useful for breeding as esterified carotenoids show an increased ability to accumulate within plant cells and have a higher stability during post-harvest storage. In this work, we analyzed five genes identified as candidates for coding the xanthophyll acyltransferase (XAT) enzyme responsible for lutein esterification in H. chilense genome. All these genes were expressed during grain development in tritordeum, but only HORCH7HG021460 was highly upregulated. Sequence analysis of HORCH7HG021460 revealed a G-to-T transversion, causing a Glycine to Cysteine substitution in the protein of H290 (the only accession not producing quantifiable amounts of lutein esters, hereinafter referred as zero-ester) of H. chilense compared to the esterifying genotypes. An allele-specific marker was designed for the SNP detection in the H. chilense diversity panel. From the 93 accessions, only H290 showed the T allele and the zero-ester phenotype. Furthermore, HORCH7HG021460 is the orthologue of XAT-7D, which encodes a XAT enzyme responsible for carotenoid esterification in wheat. Thus, HORCH7HG021460 (XAT-7Hch) is a strong candidate for lutein esterification in H. chilense and tritordeum, suggesting a common mechanism of carotenoid esterification in Triticeae species. The transference of XAT-7Hch to wheat may be useful for the enhancement of lutein esters in biofortification programs.

GRAS-Di system facilitates high-density genetic map construction and QTL identification in recombinant inbred lines of the wheat progenitor Aegilops tauschii

Due to large and complex genomes of Triticeae species, skim sequencing approaches have cost and analytical advantages for detecting genetic markers and building linkage maps. Here, we develop a high-density linkage map and identify quantitative trait loci (QTLs) for recombinant inbred lines of Aegilops tauschii, a D-genome donor of bread wheat, using the recently developed genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) system, which facilitates skimming of the large and complicated genome and generates a large number of genetic markers. The deduced linkage groups based on the GRAS-Di genetic markers corresponded to the chromosome number of Ae. tauschii. We successfully identified stable QTLs for flowering time and spikelet shape-related traits. Genotype differences of RILs at the QTL-linked markers were significantly associated with the trait variations. In particular, one of the QTL-linked markers for flowering time was mapped close to VRN3 (also known as FLOWERING LOCUS T), which controls flowering. The GRAS-Di system is, therefore, an efficient and useful application for genotyping and linkage mapping in species with large and complex genomes, such as Triticeae species.

Characterization of the Heavy-Metal-Associated Isoprenylated Plant Protein (HIPP) Gene Family from Triticeae Species

Heavy-metal-associated (HMA) isoprenylated plant proteins (HIPPs) only exist in vascular plants. They play important roles in responses to biotic/abiotic stresses, heavy-metal homeostasis, and detoxification. However, research on the distribution, diversification, and function of HIPPs in Triticeae species is limited. In this study, a total of 278 HIPPs were identified from a database from five Triticeae species, and 13 were cloned from Haynaldia villosa. These genes were classified into five groups by phylogenetic analysis. Most HIPPs had one HMA domain, while 51 from Clade I had two, and all HIPPs had good collinear relationships between species or subgenomes. In silico expression profiling revealed that 44 of the 114 wheat HIPPs were dominantly expressed in roots, 43 were upregulated under biotic stresses, and 29 were upregulated upon drought or heat treatment. Subcellular localization analysis of the cloned HIPPs from H. villosa showed that they were expressed on the plasma membrane. HIPP1-V was upregulated in H. villosa after Cd treatment, and transgenic wheat plants overexpressing HIPP1-V showed enhanced Cd tolerance, as shown by the recovery of seed-germination and root-growth inhibition by supplementary Cd. This research provides a genome-wide overview of the Triticeae HIPP genes and proved that HIPP1-V positively regulates Cd tolerance in common wheat.

Genomics-Enabled Analysis of Puroindoline b2 Genes Identifies New Alleles in Wheat and Related Triticeae Species

Kernel hardness is a key trait of wheat seeds, largely controlled by two tightly linked genes Puroindoline a and b (Pina and Pinb). Genes homologous to Pinb, namely Pinb2, have been studied. Whether these genes contribute to kernel hardness and other important seed traits remains inconclusive. Using the high-quality bread wheat reference genome, we show that PINB2 are encoded by three homoeologous loci Pinb2 not syntenic to the Hardness locus, with Pinb2-7A locus containing three tandem copies. PINB2 proteins have several features conserved for the Pin/Pinb2 phylogenetic cluster but lack a structural basis of significant impact on kernel hardness. Pinb2 are seed-specifically expressed with varied expression levels between the homoeologous copies and among wheat varieties. Using the high-quality genome information, we developed new Pinb2 allele specific markers and demonstrated their usefulness by 1) identifying new Pinb2 alleles in Triticeae species; and 2) performing an association analysis of Pinb2 with kernel hardness. The association result suggests that Pinb2 genes may have no substantial contribution to kernel hardness. Our results provide new insights into Pinb2 evolution and expression and the new allele-specific markers are useful to further explore Pinb2’s contribution to seed traits in wheat.