Haiming Han

De novo transcriptome sequencing of Agropyron cristatum to identify available gene resources for the enhancement of wheat

Agropyron cristatum is a wild grass of the tribe Triticeae that is widely grown in harsh environments. As a wild relative of wheat, A. cristatum carries many resistance genes that could be used to broaden the genetic diversity of wheat. Here, we report the transcriptome sequencing of the flag leaf and young spike tissues of a representative tetraploid A. cristatum. More than 90 million reads from the two tissues were assembled into 73,664 unigenes. All unigenes were functionally annotated against the KEGG, COG, and Gene Ontology databases and predicted long non-coding RNAs. Pfam prediction demonstrates that A. cristatum carries an abundance of stress resistance genes. The extent of specific genes and rare alleles make A. cristatum a vital genetic reservoir for the improvement of wheat. Altogether, the available gene resources in A. cristatum facilitate efforts to harness the genetic diversity of wild relatives to enhance wheat.

Genetic rearrangements of six wheat-agropyron cristatum 6P addition lines revealed by molecular markers.

Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP) not only is cultivated as pasture fodder but also could provide many desirable genes for wheat improvement. It is critical to obtain common wheat–A. cristatum alien disomic addition lines to locate the desired genes on the P genome chromosomes. Comparative analysis of the homoeologous relationships between the P genome chromosome and wheat genome chromosomes is a key step in transferring different desirable genes into common wheat and producing the desired alien translocation line while compensating for the loss of wheat chromatin. In this study, six common wheat–A. cristatum disomic addition lines were produced and analyzed by phenotypic examination, genomic in situ hybridization (GISH), SSR markers from the ABD genomes and STS markers from the P genome. Comparative maps, six in total, were generated and demonstrated that all six addition lines belonged to homoeologous group 6. However, chromosome 6P had undergone obvious rearrangements in different addition lines compared with the wheat chromosome, indicating that to obtain a genetic compensating alien translocation line, one should recombine alien chromosomal regions with homoeologous wheat chromosomes. Indeed, these addition lines were classified into four types based on the comparative mapping: 6PI, 6PII, 6PIII, and 6PIV. The different types of chromosome 6P possessed different desirable genes. For example, the 6PI type, containing three addition lines, carried genes conferring high numbers of kernels per spike and resistance to powdery mildew, important traits for wheat improvement. These results may prove valuable for promoting the development of conventional chromosome engineering techniques toward molecular chromosome engineering.

Efficient induction of Wheat-agropyron cristatum 6P translocation lines and GISH detection.

The narrow genetic background restricts wheat yield and quality improvement. The wild relatives of wheat are the huge gene pools for wheat improvement and can broaden its genetic basis. Production of wheat-alien translocation lines can transfer alien genes to wheat. So it is important to develop an efficient method to induce wheat-alien chromosome translocation. Agropyron cristatum (P genome) carries many potential genes beneficial to disease resistance, stress tolerance and high yield. Chromosome 6P possesses the desirable genes exhibiting good agronomic traits, such as high grain number per spike, powdery mildew resistance and stress tolerance. In this study, the wheat- A . cristatum disomic addition was used as bridge material to produce wheat- A . cristatum translocation lines induced by 60Co-γirradiation. The results of genomic in situ hybridization showed that 216 plants contained alien chromosome translocation among 571 self-pollinated progenies. The frequency of translocation was 37.83%, much higher than previous reports. Moreover, various alien translocation types were identified. The analysis of M2 showed that 62.5% of intergeneric translocation lines grew normally without losing the translocated chromosomes. The paper reported a high efficient technical method for inducing alien translocation between wheat and Agropyron cristatum . Additionally, these translocation lines will be valuable for not only basic research on genetic balance, interaction and expression of different chromosome segments of wheat and alien species, but also wheat breeding programs to utilize superior agronomic traits and good compensation effect from alien chromosomes.

Isolation and application of P genome-specific DNA sequences of Agropyron Gaertn. in Triticeae

AbstractMain conclusionDifferent types of P genome sequences and markers were developed, which could be used to analyze the evolution of P genome in Triticeae and identify precisely wheat-A. cristatumintrogression lines. P genome of Agropyron Gaertn. plays an important role in Triticeae and could provide many desirable genes conferring high yield, disease resistance, and stress tolerance for wheat genetic improvement. Therefore, it is significant to develop specific sequences and functional markers of P genome. In this study, 126 sequences were isolated from the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) products of microdissected chromosome 6PS. Forty-eight sequences were identified as P genome-specific sequences by dot-blot hybridization and DNA sequences analysis. Among these sequences, 22 displayed the characteristics of retrotransposons, nine and one displayed the characteristics of DNA transposons and tandem repetitive sequence, respectively. Fourteen of 48 sequences were determined to distribute on different regions of P genome chromosomes by fluorescence in situ hybridization, and the distributing regions were as following: all over P genome chromosomes, centromeres, pericentromeric regions, distal regions, and terminal regions. We compared the P genome sequences with other genome sequences of Triticeae and found that the similar sequences of the P genome sequences were widespread in Triticeae, but differentiation occurred to various extents. Additionally, thirty-four molecular markers were developed from the P genome sequences, which could be used for analyzing the evolutionary relationship among 16 genomes of 18 species in Triticeae and identifying P genome chromatin in wheat-A. cristatum introgression lines. These results will not only facilitate the study of structure and evolution of P genome chromosomes, but also provide a rapid detecting tool for effective utilization of desirable genes of P genome in wheat improvement.

P chromosomes involved in intergenomic rearrangements of Kengyilia thoroldiana affected by the environment.

1The National Key Facilities for Crop Gene Resources and Genetic Improvement, The National Key Facilities for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China 2Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences, Changchun, Jilin, People’s Republic of China

Altered expression of the TaRSL2 gene contributed to variation in root hair length during allopolyploid wheat evolution

Main conclusionAltered expression of the TaRSL2 gene was positively correlated with variation in root hair length during allopolyploid wheat evolution, and overexpression of TaRSL2 in Arabidopsis increases root hair length.Root hairs aid nutrient and water uptake and anchor the plant in the soil. Allopolyploid wheats display significant growth vigor in terms of root hair length compared to their diploid progenitors, but little is known about the molecular basis of variation in root hair length during wheat allopolyploidization. Here, we isolated three orthologs of the Arabidopsis root hair gene ROOT HAIR DEFECTIVE SIX-LIKE 2 (AtRSL2) in allohexaploid wheat, designated TaRSL2-4A, TaRSL2-4B and TaRSL2-4D. The deduced polypeptides of these three TaRSL2 homoeologous genes shared high similarity, and a conserved basic helix-loop-helix (bHLH) domain was present in their C-terminal regions. Notably, the expression of TaRSL2 was positively correlated with root hair length of wheat accessions with different ploidy levels. Moreover, ectopic overexpression of TaRSL2-4D in Arabidopsis could increase root hair length. We found that the transcript levels of TaRSL2 homoeologous genes dynamically changed during allopolyploid wheat evolution, implicating the complexity of the underlying molecular mechanism. Collectively, we propose that altered expression of the TaRSL2 gene contributed to variation in root hair length in allopolyploid wheats.

Physical Localization of a Locus from Agropyron cristatum Conferring Resistance to Stripe Rust in Common Wheat

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), one of the wild relatives of wheat, exhibits resistance to stripe rust. In this study, wheat-A. cristatum 6P disomic addition line 4844-12 also exhibited resistance to stripe rust. To identify the stripe rust resistance locus from A. cristatum 6P, ten translocation lines, five deletion lines and the BC2F2 and BC3F2 populations of two wheat-A. cristatum 6P whole-arm translocation lines were tested with a mixture of two races of Pst in two sites during 2015–2016 and 2016–2017, being genotyped with genomic in situ hybridization (GISH) and molecular markers. The result indicated that the locus conferring stripe rust resistance was located on the terminal 20% of 6P short arm’s length. Twenty-nine 6P-specific sequence-tagged-site (STS) markers mapped on the resistance locus have been acquired, which will be helpful for the fine mapping of the stripe rust resistance locus. The stripe rust-resistant translocation lines were found to carry some favorable agronomic traits, which could facilitate their use in wheat improvement. Collectively, the stripe rust resistance locus from A. cristatum 6P could be a novel resistance source and the screened stripe rust-resistant materials will be valuable for wheat disease breeding.

Three genomes differentially contribute to the seedling lateral root number in allohexaploid wheat: evidence from phenotype evolution and gene expression

Common wheat is an allohexaploid (BBAADD) that originated from the hybridization and polyploidization of the diploid Aegilops tauschii (DD) with the allotetraploid Triticum turgidum (BBAA). Phenotypic changes often arise with the formation and evolution of allopolyploid wheat, but little is known about the evolution of root traits in different wheat species with varying ploidy levels. Here, we reported that the lateral root number on the primary root (LRNPR) of synthetic and natural allohexaploid wheats (BBAADD) is significantly higher than that of their allotetraploid (BBAA) and diploid (AA and SS) progenitors, but is much lower than that of their diploid (DD) progenitors. The expression of the wheat gene TaLBD16, an ortholog of the Arabidopsis LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18 (LBD16), which is involved in lateral root development in Arabidopsis, was positively correlated with the LRNPR in diploid and allopolyploid wheats. In natural and synthetic allohexaploid wheats, the transcript of the TaLBD16 from the D genome (TaLBD16-D) was relatively more abundant compared with TaLBD16-A and TaLBD16-B. Consistent with the observed variation in LRNPR, the divergence in the expression of TaLBD16 homoeologous genes occurred before the formation of polyploidy wheat. Collectively, our observations indicate that the D genome played a crucial role in the increased lateral root number of allohexaploid wheats compared with their allotetraploid progenitors, and that TaLBD16-D was one of the key genes involved in the formation of lateral root number during wheat evolution.

Physical Mapping of a Novel Locus Conferring Leaf Rust Resistance on the Long Arm of Agropyron cristatum Chromosome 2P

Wheat leaf rust is one of the most common wheat diseases worldwide and can cause up to 40% wheat yield loss. To combat the growth and spread of leaf rust disease, continual exploration and identification of new and effective resistance genes are needed. Here, we report for the first time a locus conferring leaf rust resistance located on the long arm of Agropyron cristatum chromosome 2P in Triticum aestivum–A. cristatum 2P translocation lines. This study used 50 leaf rust races, including two Chinese major dominant leaf rust races, named by THT and PHT, and other 48 different leaf rust races collected from 11 provinces, 1autonomous region and 1 municipality of China to test the resistance to T. aestivum–A. cristatum 2P chromosome translocation lines and their backcross populations, the results indicated that the novel leaf rust resistance locus was immune or nearly immune to all tested leaf rust races. Four long arm translocation lines with different breakpoints of A. cristatum chromosome 2PL and their backcross populations were tested with leaf rust race THT at the seedling and adult stages and genotyped with 2P-specific STS markers. The results showed that the novel leaf rust resistance locus of the T. aestivum–A. cristatum 2P translocation lines was located in the chromosomal bin FL 0.66–0.86 of 2PL. Therefore, T. aestivum–A. cristatum 2P chromosome translocation lines conferring leaf rust resistance locus could provide a novel disease-resistance resource for future wheat breeding programs.