Baolong Liu

Genetic map of Triticum turgidum based on a hexaploid wheat population without genetic recombination for D genome

BackgroundA synthetic doubled-haploid hexaploid wheat population, SynDH1, derived from the spontaneous chromosome doubling of triploid F1 hybrid plants obtained from the cross of hybrids Triticum turgidum ssp. durum line Langdon (LDN) and ssp. turgidum line AS313, with Aegilops tauschii ssp. tauschii accession AS60, was previously constructed. SynDH1 is a tetraploidization-hexaploid doubled haploid (DH) population because it contains recombinant A and B chromosomes from two different T. turgidum genotypes, while all the D chromosomes from Ae. tauschii are homogenous across the whole population. This paper reports the construction of a genetic map using this population.ResultsOf the 606 markers used to assemble the genetic map, 588 (97%) were assigned to linkage groups. These included 513 Diversity Arrays Technology (DArT) markers, 72 simple sequence repeat (SSR), one insertion site-based polymorphism (ISBP), and two high-molecular-weight glutenin subunit (HMW-GS) markers. These markers were assigned to the 14 chromosomes, covering 2048.79 cM, with a mean distance of 3.48 cM between adjacent markers. This map showed good coverage of the A and B genome chromosomes, apart from 3A, 5A, 6A, and 4B. Compared with previously reported maps, most shared markers showed highly consistent orders. This map was successfully used to identify five quantitative trait loci (QTL), including two for spikelet number on chromosomes 7A and 5B, two for spike length on 7A and 3B, and one for 1000-grain weight on 4B. However, differences in crossability QTL between the two T. turgidum parents may explain the segregation distortion regions on chromosomes 1A, 3B, and 6B.ConclusionsA genetic map of T. turgidum including 588 markers was constructed using a synthetic doubled haploid (SynDH) hexaploid wheat population. Five QTLs for three agronomic traits were identified from this population. However, more markers are needed to increase the density and resolution of this map in the future study.

Synthesizing double haploid hexaploid wheat populations based on a spontaneous alloploidization process

Doubled haploid (DH) populations are useful to scientists and breeders in both crop improvement and basic research. Current methods of producing DHs usually need in vitro culture for extracting haploids and chemical treatment for chromosome doubling. This report describes a simple method for synthesizing DHs (SynDH) especially for allopolyploid species by utilizing meiotic restitution genes. The method involves three steps: hybridization to induce recombination, interspecific hybridization to extract haploids, and spontaneous chromosome doubling by selfing the interspecific F(1)s. DHs produced in this way contain recombinant chromosomes in the genome(s) of interest in a homogeneous background. No special equipment or treatments are involved in the DH production and it can be easily applied in any breeding and/or genetic program. Triticum turgidum L. and Aegilops tauschii Coss, the two ancestral species of common wheat (Triticum aestivum L.) and molecular markers were used to demonstrate the SynDH method.

Transcriptome Analysis of Purple Pericarps in Common Wheat (Triticum aestivum L.).

Wheat (Triticum aestivum L.) cultivars possessing purple grain arethought to be more nutritious because of high anthocyanin contents in the pericarp. Comparative transcriptome analysis of purple (cv Gy115) and white pericarps was carried out using next-generation sequencing technology. There were 23,642 unigenes significantly differentially expressed in the purple and white pericarps, including 9945 up-regulated and 13,697 down-regulated. The differentially expressed unigenes were mainly involved in encoding components of metabolic pathways, The flavonoid biosynthesis pathway was the most represented in metabolic pathways. In the transcriptome of purple pericarp in Gy115, most structural and regulatory genes biosynthesizing anthocyanin were identified, and had higher expression levels than in white pericarp. The largestunigene of anthocyanin biosynthesis in Gy115 was longer than the reference genes, which implies that high-throughput sequencing could isolate the genes of anthocyanin biosynthesis in tissues or organs with high anthocyanin content. Based on present and previous results, three unigenes of MYB gene on chromosome 7BL and three unigenes of MYC on chromosome 2AL were predicted as candidate genes for the purple grain trait. This article was the first to provide a systematic overview comparing the transcriptomes of purple and white pericarps in common wheat, which should be very valuable for identifying the key genes for the purple pericarp trait.

Identification of a candidate gene for Rc-D1, a locus controlling red coleoptile colour in wheat

Red coleoptile is an easily observed agronomic trait of wheat and has been extensively studied. However, the molecular mechanism of this trait has not yet been revealed. In this study, the MYB gene TaMYB-D1 was isolated from the wheat cultivar ‘Gy115’, which possesses red coleoptiles. This gene resided at the short arm of the homoelogous group 7 chromosomes. TaMYB-D1 was the only gene expressed in the coleoptiles of ‘Gy115’ and was not expressed in ‘Opata’ and ‘CS’, which have uncoloured coleoptiles. Phylogenetic analysis placed TaMYB-D1 very close to ZmC1 and other MYB proteins regulating anthocyanin biosynthesis. The encoded protein of TaMYB-D1 had an integrated DNA binding domain of 102 amino acids and a transcription domain with 42 amino acids, similar to the structure of ZmC1. Transient expression analysis in onion epidermal cells showed that TaMYB-D1 was located at the plant nucleus, which suggested its role as a transcription factor. The expression of TaMYB-D1 was accompanied with the expression of TaDFR and anthocyanin biosynthesis in the development of the coleoptile of ‘Gy115’. Transient expression analysis showed that only TaMYB-D1 induced a few ‘Opata’ coleoptile cells to synthesize anthocyanin in light, and the gene also induced a colour change to red in many cells with the help of ZmR. All of these results suggested TaMYB-D1 as the candidate gene for the red coleoptile trait of ‘Gy115’.

Assessing genetic diversity and its changes of bread wheat in Qinghai Province, China, using agronomic traits and microsatellite markers

Little is known about the diversity of wheat in Qinghai Province, China. Agronomic traits and microsatellite markers were used to survey genetic diversity and its change with time in 66 wheat cultivars registered from 1957 to 2009 in Qinghai Province. The average values of plant height, ear length, spikelets per ear, effective spikelets per ear, effective tillers per plant, internode length under spike, kernels per spike, grain weight per ear, 1,000-grain weight, and distance of spike base to auricle of flag leaf were studied. The mean Shannon-Weaver diversity index (H’) was 1.67 and increased with time. One hundred and eighty nine microsatellite markers also were used to examine genetic diversity at a molecular level which showed that the average number of alleles (Na), genetic diversity index (He), and allelic richness (Rs) were 3.69, 0.5, and 3.45, respectively, and also increased with time but did not lead to a significant differentiation among the decades. These results suggest that the modern wheat breeding practice did not cause a genetic reduction in Qinghai Province.

Allelic Variation and Transcriptional Isoforms of Wheat TaMYC1 Gene Regulating Anthocyanin Synthesis in Pericarp

Recently the TaMYC1 gene encoding bHLH transcription factor has been isolated from the bread wheat (Triticum aestivum L.) genome and shown to co-locate with the Pp3 gene conferring purple pericarp color. As a functional evidence of TaMYC1 and Pp3 being the same, higher transcriptional activity of the TaMYC1 gene in colored pericarp compared to uncolored one has been demonstrated. In the current study, we present additional strong evidences of TaMYC1 to be a synonym of Pp3. Furthermore, we have found differences between dominant and recessive Pp3(TaMyc1) alleles. Light enhancement of TaMYC1 transcription was paralleled with increased AP accumulation only in purple-grain wheat. Coexpression of TaMYC1 and the maize MYB TF gene ZmC1 induced AP accumulation in the coleoptile of white-grain wheat. Suppression of TaMYC1 significantly reduced AP content in purple grains. Two distinct TaMYC1 alleles (TaMYC1p and TaMYC1w) were isolated from purple- and white-grained wheat, respectively. A unique, compound cis-acting regulatory element had six copies in the promoter of TaMYC1p, but was present only once in TaMYC1w. Analysis of recombinant inbred lines showed that TaMYC1p was necessary but not sufficient for AP accumulation in the pericarp tissues. Examination of larger sets of germplasm lines indicated that the evolution of purple pericarp in tetraploid wheat was accompanied by the presence of TaMYC1p. Our findings may promote more systematic basic and applied studies of anthocyanins in common wheat and related Triticeae crops.

Frame-shift mutation causes the function loss of TaMYB-A1 regulating anthocyanin biosynthesis in Triticum aestivum

Red coleoptile is an easily observed trait in Triticum aestivum and can provide some protection against stress. Here, TaMYB-A1 or TuMYB-A1, homologous to TaMYB-D1, which controls red coleoptile formation in the common wheat cultivar ‘Gy115’, was isolated from eight T. aestivum and 34 T. urartu cultivars. The genome sequence of TaMYB-A1 was 867 bp with an intron of 93 bp, which was similar to the MYBs regulating anthocyanin biosynthesis in T. aestivum but different from other MYB transcription factors regulating anthocyanin biosynthesis. TaMYB-A1 had an integrated DNA-binding domain of 102 amino acids and a transcriptional domain of 42 amino acids, which was responsible for regulating anthocyanin biosynthesis. TaMYB-A1 was assigned to the same branch as the MYBs regulating anthocyanin biosynthesis in a phylogenetic tree. A transient expression analysis showed that TaMYB-A1 induced ‘Opata’ coleoptile cells to synthesize anthocyanin with the help of ZmR. A non-functional allele of TaMYB-a1 existed in common wheat cultivars containing rc-a1. One single nucleotide was deleted 715 bp after the start codon in TaMYB-a1 compared with TaMYB-A1. The deletion caused a frame shift mutation, destroyed the DNA transcription activator domain, and resulted in TaMYB-a1 losing its ability to regulate anthocyanin biosynthesis in ‘Opata’ coleoptile cells. Those cultivars with functional TaMYB-A1 or TuMYB-A1 have red coleoptiles. The isolation of TaMYB-A1 should aid in understanding the molecular mechanisms of coleoptile traits in T. aestivum.

Diversity and distribution of Rc1 alleles in Triticum urartu from the Middle East

Rc1 (red coleoptile) encode a R2R3-MYB transcription factor TaMYB-A1 in common wheat, but the homologous genes have not been reported in Triticum urartu or Triticum monococcum, both species being sources of the ancient A genome. In this study, four alleles were discovered in 120 T. urartu accessions, and two alleles from nine T. monococcum accessions. The six alleles encoded five proteins because TuMYB-A1-F and TuMYB-A1-C encoded the same protein. All mutated sites in the amino acid sequence of these alleles were out of the R2R3 and transcript activator domains, and did not affect the conserved tryptophan (W) residues nor the “D/ELX2R/KX3LX6LX3R” motif of the MYB proteins. The alleles from T. monococcum were more similar in sequence to TaMYB-B1 and TaMYB-D1 than were those from T. urartu in the phylogenetic tree, while the alleles from T. urartu resembled TaMYB-A1 more closely than did the T. monococcum alleles. TuMYB-A1-C was the most widely distributed allele, and Turkey contained the greatest genetic variation compared with the five other countries in the geographical distribution analysis. Transient expression of TuMYB-A1-D or TuMYB-A1-F induced anthocyanin biosynthesis in white coleoptiles of wheat cultivar ‘Opata’ with the help of ZmR. RT-PCR analysis showed that the transcript level of Rc1 allele in white coleoptiles was clearly lower than in red coleoptiles, suggesting that Rc1 allele plays an important role in anthocyanin biosynthesis in coleoptiles of T. urartu. The allele isolation, phylogenetic analysis, geographical distribution and functional verification should be helpful in understanding the functions of Rc1 alleles and the molecular mechanism underlying the coleoptile color trait in Triticum.

ThMYC4E, candidate Blue aleurone 1 gene controlling the associated trait in Triticum aestivum

Blue aleurone is a useful and interesting trait in common wheat that was derived from related species. Here, transcriptomes of blue and white aleurone were compared for isolating Blue aleurone 1 (Ba1) transferred from Thinopyrum ponticum. In the genes involved in anthocyanin biosynthesis, only a basic helix-loop-helix (bHLH) transcription factor, ThMYC4E, had a higher transcript level in blue aleurone phenotype, and was homologous to the genes on chromosome 4 of Triticum aestivum. ThMYC4E carried the characteristic domains (bHLH-MYC_N, HLH and ACT-like) of a bHLH transcription factor, and clustered with genes regulating anthocyanin biosynthesis upon phylogenetic analysis. The over-expression of ThMYC4E regulated anthocyanin biosynthesis with the coexpression of the MYB transcription factor ZmC1 from maize. ThMYC4E existed in the genomes of the addition, substitution and near isogenic lines with the blue aleurone trait derived from Th. ponticum, and could not be detected in any germplasm of T. urartu, T. monococcum, T. turgidum, Aegilops tauschii or T. aestivum, with white aleurone. These results suggested that ThMYC4E was candidate Ba1 gene controlling the blue aleurone trait in T. aestivum genotypes carrying Th. ponticum introgression. The ThMYC4E isolation aids in better understanding the genetic mechanisms of the blue aleurone trait and in its more effective use during wheat breeding.

AetMYC1, the Candidate Gene Controlling the Red Coleoptile Trait in Aegilops tauschii Coss. Accession As77

The red coleoptile trait can help monocotyledonous plants withstand stresses, and key genes responsible for the trait have been isolated from Triticum aestivum, Triticum urartu, and Triticum monococcum, but no corresponding research has been reported for Aegilops tauschii. In this research, transcriptome analysis was performed to isolate the candidate gene controlling the white coleoptile trait in Ae. tauschii. There were 5348 upregulated, differentially-expressed genes (DEGs) and 4761 downregulated DEGs in red coleoptile vs. white coleoptile plants. Among these DEGs, 12 structural genes and two transcription factors involved in anthocyanin biosynthesis were identified. The majority of structural genes showed lower transcript abundance in the white coleoptile of accession ‘As77’ than in the red coleoptile of accession ‘As60’, which implied that transcription factors related to anthocyanin biosynthesis could be the candidate genes. The MYB and MYC transcription factors AetMYB7D and AetMYC1 were both isolated from Ae. tauschii accessions ‘As60’ and ‘As77’, and their transcript levels analyzed. The coding sequence and transcript level of AetMYB7D showed no difference between ‘As60’ and ‘As77’. AetMYC1p encoded a 567-amino acid polypeptide in ‘As60’ containing the entire characteristic domains, bHLH-MYC_N, HLH, and ACT-like, belonging to the gene family involved in regulating anthocyanin biosynthesis. AetMYC1w encoded a 436-amino acid polypeptide in ‘As77’ without the ACT-like domain because a single nucleotide mutation at 1310 bp caused premature termination. Transient expression of AetMYC1p induced anthocyanin biosynthesis in ‘As77’ with the co-expression of AetMYB7D, while AetMYC1w could not cause induced anthocyanin biosynthesis under the same circumstances. Moreover, the transcript abundance of AetMYC1w was lower than that of AetMYC1p. AetMYC1 appears to be the candidate gene controlling the white coleoptile trait in Ae. tauschii, which can be used for potential biotech applications, such as producing new synthetic hexaploid wheat lines with different coleoptile colors.