RNA-Seq Analysis Provides the First Insights into the Phylogenetic Relationship and Interspecific Variation between Agropyron cristatum and Wheat

Abstract

Agropyron cristatum, which is a wild grass of the tribe Triticeae, grows widely in harsh environments and provides many desirable genetic resources for wheat improvement. However, unclear interspecific phylogeny and genome-wide variation has limited the utilization of A. cristatum in the production of superior wheat varieties. In this study, by sequencing the transcriptome of the representative tetraploid A. cristatum Z559 and the common wheat variety Fukuhokomugi (Fukuho), which are often used as parents in a wide cross, their phylogenetic relationship and interspecific variation were dissected. First, 214,854 transcript sequences were assembled, and 3,457 orthologous genes related to traits of interest were identified in A. cristatum. Second, a total of 72 putative orthologous gene clusters were used to construct phylogenetic relationships among A. cristatum, Triticeae and other genomes. A clear division between A. cristatum and the other Triticeae species was revealed. Third, the sequence similarity of most genes related to traits of interest is greater than 95% between A. cristatum and wheat. Therefore, using the 5% mismatch parameter for A. cristatum, we mapped the transcriptome sequencing data to wheat reference sequences to discover the variations between A. cristatum and wheat and 862,340 high-quality variants were identified. Additionally, compared with the wheat A and B genomes, the P and D genomes displayed an obviously larger variant density and a longer evolutionary distance, suggesting that A. cristatum is more distantly related to the wheat D genome. Finally, by using Kompetitive Allele Specific PCR array (KASPar) technology, 37 of 53 (69.8%) SNPs were shown to be genuine in Z559, Fukuho, and additional lines with seven different P chromosomes, and function of the genes in which these SNPs are located were also determined. This study provides not only the first insights into the phylogenetic relationships between the P genome and Triticeae but also genetic resources for gene discovery and specific marker development in A. cristatum, and this information will be vital for future wheat-breeding efforts. The sequence data have been deposited in the Sequence Read Archive (SRA) database at the NCBI under accession number SRP090613.

Full-length transcriptome sequences of Agropyron cristatum facilitate the prediction of putative genes for thousand-grain weight in a wheat-A. cristatum translocation line

“…After demultiplexing, refining, clustering and polishing of FL transcripts were performed, a total of 44,372 FLNC transcripts with a maximum length of 9,468 bp, a N50 of 3,572 bp and average FL coverage of 5.1 were generated ( Table 1). As expected, the PacBio FLNC transcripts were generally longer than the transcripts assembled via the Illumina sequencing platform in a previous study [20,21] (Figure 2). Functional annotation of the FLNC transcripts was conducted using 5 different public protein databases ( Table 2 and Figure 3).…”

“…The A. cristatum FLNC transcripts, transcripts assembled using short read sequencing [21] and IWGSC wheat RefSeq V1.0 reference sequences [44] were integrated as the reference sequences in this study. To reduce redundancy, the sequences were clustered using CD-HIT-EST with sequence identity set to 100%.…”

“…High-throughput transcriptome sequencing has recently become a popular technique because it is cost-effective, does not rely on a reference genome and can contribute to transcriptional analysis, molecular marker development and gene discovery [19]. In a previous study, de novo transcriptome assembly and unigene functional annotation were conducted in A. cristatum with the Illumina sequencing technique, and gene resources that were related to traits of interest and specific to A. cristatum within the tribe Triticeae, as well as the phylogenetic relationship and interspecific variation between A. cristatum and wheat, were identified [20,21]. However, transcriptome analyses in species without reference genome sequences often encounter complicated problems, especially in the assembly of sequencing reads; thus, the assembly and annotation of A. cristatum is incomplete and error-prone, which severely impedes in-depth molecular breeding and gene functional studies of A.…”

“…cristatum [20] and has provided many new insights into the phylogenetic relationship and interspecific variation between A. cristatum and wheat [21]. However, the short sequencing reads of the Illumina platform make the assembly and annotation of the A. cristatum transcriptome incomplete and error-prone.…”

“…Then, novel and A. cristatum-specific transcripts were identified by comparison with the wheat gene model set. Furthermore, by integrating the A. cristatum transcripts, includingFLNCs and transcripts assembled in previous studies [21], and the wheat genome as reference sequences, candidate A. cristatum transcripts associated with thousand-grain weight were identified in Pubing 3035. The present study not only provides comprehensive transcriptomic insights and information for A. cristatum but also proposes a new method for the exploration of functional genes from wheat relatives under a wheat genetic background.…”