Performing Department
Biology & Microbiology
Non Technical Summary
Wheat production is facing numerous challenges from biotic and abiotic stresses. Alien gene transfer has been an effective approach for wheat germplasm enhancement. Sea wheatgrass (SWG) is a distant relative of wheat and a relatively untapped source for wheat improvement. We have identified and developed a large number of SWG-derived populations withhigh tolerance to waterlogging, manganese toxicity, salinity, heat, and low nitrogen levels as well asresistance to wheat streak mosaic virus (temperature-insensitive), Fusarium head blight, and sawflies (due to solid stem). This project seeks to facilitate simultaneous discovery and transfer of quantitative trait loci (QTL) for biotic stress resistance and abiotic stress tolerance more efficiently by combining molecular marker genotyping and molecular cytogenetics. The research team will first develop a draft SWG genome assembly and 140 SWG-specific markers. These SWG-specific markers will be used to screen the common wheat-backcrossed populations for individuals containing one or two SWG chromosomes as putative wheat-SWG addition lines, which will be further validated by genomic in-situ hybridization (GISH) by differentially painting the SWG chromosomes. This is the first and critical step in dissection of the SWG genome foruse of SWG genes to improve wheat sustainability. Resources developed by the project will also have a positive impact on effective use of wheatgrass-derived germplasm in wheat breeding.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
10%
Developmental
30%
Goals / Objectives
The goal of this project is to dissect the sea wheatgrass (SWG) genome and transfer abiotic stress tolerance and biotic stress resistance into wheat for broadening the wheat genetic basis and developing novel germplasm that will contribute to a more sustainable wheat industry.Objectives of this project include (1) to develop a draft SWG genome assembly for genome-specific markers; and (2) to construct as SWG chromosome library in wheat consisting of 14 wheat-SWG addition lines.
Project Methods
Methods for Objective 1: Develop a draft SWG genome assembly for genome-specific markersWe will sequence the genome of accession PI 414667, the parent of the wheat-SWG amphiploids and backcross populations, for draft genome assembly. To this end, three sequencing libraries, one paired end library, two mate-paired libraries with fragment length 2 kbp and 5 kbp, will be constructed from purified SWG genomic DNA, pooled and sequenced using Illumina HiSeq4000 (2 x 150-bp). For a coverage of ~20 genome equivalents, ~250 Gbp sequences with quality >Q30 are required. Therefore, 312 Gbp sequence data will be generated supposing that ~80% of data can meet this technical requirement after pretreatments. After removal of microbial contamination and trimming of low-quality bases, the clean reads will be fed into the "miraculous" assembler with k-mer = 51. Based on the results from hexaploid wheat and differentiation between the J1 and J2 genomes, the SWG genome comprises ~6 Gb 51-mer unique sequences (copy number < 1.5) that are accessible for assembly. Repeated sequences in the SWG assembly willbe identified and masked by aligning with the repeats in the Triticeae repeat sequence database (http://wheat.pw.usda.gov/ITMI/Repeats/) by BLATSN algorithm.Assembly of the 51-mer unique sequences will generate a collection of gene-rich contigs, from which SWG genes will be predicted using GENSCAN program (http://genes.mit.edu/GENSCAN.html). The predicted SWG genes will be anchored to the annotated genome of Aegilops tauschii. This will sort the SWG genes into homoeologous groups with assumption that the J and D genomes are largely collinear. The homologs from J1 and J2 genomes will be resolved using hapcut, an efficient and accurate algorithm for the haplotype assembly, which has been successfully used to resolve homoeologous transcripts of polyploid wheat.Single-copy genes define the collinearity conservation and are valuable resources for marker development. Therefore, the SWG protein data set will be compared against itself using BLASTP program, and the single copy genes will be sorted out. The single-copy gene sequences including basal promoters from SWG will be aligned with wheat homoeologous genes for detection of genic indels using the SOAPindel program. The polymorphic genic sequences of homoeologs from the A, B, D, J1 and J2 genomes will be sorted out and aligned using multiple sequence alignment program MUSCLE (http://www.drive5.com/muscle/) and the outputs will be fed into the GSP, a program for designing genome specific primers in polyploid species (https://github.com/bioinfogenome/GSP), by targeting the indels. These genome-specific primers will be used for STARP marker development by tailing the 5' ends to overlap with the universal primers. For each homoeologous chromosome arm, 10 markers will be developed more or less evenly distributed along the arm, 140 markers for the seven homoeologous groups. For the STARP markers developed, the SWG-specific alleles will be first validated using Tt139, SWG and the two amphiploids, and wheat chromosome-specific alleles will be validated using CS NT stocks 30 for chromosome specificity. Marker validation will be performed by Xu Lab, who have successfully applied the STARP genotyping system to detect alien introgressions in wheat 90. At the same time, the technical system of STARP genotyping will be transferred to Li Lab, and the validated STARP markers will be used to genotype the SWG-derived populations.Methods for Objective 2: Construct as SWG chromosome library in wheat consisting of 14 wheat-SWG addition lines. The SWG-derived populations of advanced generations will be screened by the SWG-specific STARP markers to identify individuals containing one or two SWG chromosomes, i.e. addition lines. To do so, a fully expended leaf is collected from each plant at 3-leaf stage for DNA extraction, and three SWG-specific STARP markers will be first selected from each chromosome, one close to centromere (Mc) and remaining two close to telomeres (Mt), to genotype all the populations and distinguish J1 and J2 alleles. A total of 42 markers will be used. The plants positive for markers from one or two chromosomes will be sorted out for further characterization by four more markers from the same chromosome. The chromosome arm location of the markers will be aligned with the wheat chromosomes to detect potential chromosome rearrangements. We plan to initially genotype 960 individuals from the common wheat-backcrossed populations. More plants will be screened until addition lines for all 14 chromosomes of the seven homoeologous groups are identified.We will combine genotyping and cytological data to determine the homoeologous relationship between the added SWG chromosomes and wheat chromosomes. To this end, root tips of 1 to 2 cm in length will be collected from the fresh roots, pretreated with N2O for 2 h, fixed in 90% acetic acid and then shipped to Xu Lab for determination of chromosome numbers and GISH. Parallel to marker and GISH characterization, the addition lines identified will be compared to their recurrent parents, CS, Louis and Lloyd, for assignment of morphological traits, such as awnless spike and solid stem, to specific SWG chromosomes. We are also developing separate backcrossing populations for transferring waterlogging tolerance and WSMV resistance because the phenotyping of these traits is destructive.