Current efforts in sorghum breeding programs are exploiting genotyping-by-sequencing (GBS) data to provide full-genome scans for desired traits. The aim of this study was to utilize GBS approach for the identification of genomic regions associated with stress response in sorghum ( Sorghum bicolor L. Monech) accessions. DNA samples of twenty sorghum accessions, having different response to drought, were used to prepare GBS libraries for sequencing. SNPs were called using the TASSELGBS pipeline and the tags that present at least 10 times in the dataset were considered and aligned to the reference genome of Sorghum bicolor. The identified SNPs were all compared with the published sorghum transcript related to stress response gene activity. Overall; 94.40% tags were aligned and 69,736 putative SNPs positions were identified. Blast search revealed homology to annotated heat and drought–tolerance associated genes which code for ATPases, Peroxidase, Hydrophobic protein LTI6A, Aquaporin SIP2-1, Aconitate hydratase and phosphatidylinositol-4-phosphate-5-kinase. The phylogeny of the 20 accessions was constructed using the generated SNPs data . Phylogenetic analysis data showed that the phenotypically tolerant line (El9) makes a separate cluster and the same for the accessions HSD8653 and HSD5612 near to the cluster that includes most accessions with known post-flowering drought tolerance (HSD7410, HDS10033, HSD8552, GESHEISH and HSD8849). Post-flowering drought sensitive accessions (Tabat, Wadahmed, HSD6468 and HSD6478) formed a separate cluster while the sensitive accession HSD9959 and the tolerant accessions HSD8511 and HSD9566 were distributed between the two clusters. Thus, cluster analysis confirmed the variation among accessions in post-flowering drought tolerance. With further validation, these markers may be used for marker assisted selection for breeding new sorghum genotypes with stress adaptation.
Sorghum is the dietary staple food for about 500 million people over 30 countries and is especially important in semi-arid regions of Africa, along with pearl millet and cassava [
Marker-assisted selection was employed to improve the stay green trait involved in the drought tolerance of sorghum [
GBS is a simple highly multiplexed system for constructing reduced representation libraries for the Illumina NGS platform developed in the Buckler lab [
Current efforts in sorghum are exploiting genotyping-by-sequencing data to provide full-genome scans across >100,000 SNP (single-nucleotide polymorphism) loci in each member of a portion of a global reference collection of sorghum germplasm that has been phenotyped with a lysimetric system to explore the allelic variation available for key components of the drought resistance phenotype.
The objective of the present study was to identify significant SNPs of genomic regions associated with stress response in 20 sorghum accessions collected from Sudan, using GBS.
Seeds of twenty sorghum accessions, for this study, were obtained from Agricultural Plant Genetic Resources Conservation and Research Center, Agricultural Research Corporation (ARC), Wad Medani, Sudan, as well as from Department of Agronomy, Faculty of Agriculture, University of Khartoum, Sudan. The accessions were classified by Agricultural Research Corporation into three groups (tolerant, intermediate and sensitive) depending on morphological characters previously measured in the field (
Genomic DNA was isolated from leaf tissues using CTAB method [
To 100 ng of gDNA, barcoded adapters were added and digested with Apek1 enzyme for 2 h at 75˚C. The barcoded and common adapters were ligated to the sticky ends of the digested DNA with T4 DNA Ligase enzyme at 22˚C for 60 mins, followed by heat inactivation. The ligated products were pooled into two groups of 10 samples each. The pool of ligated products was size-selected to 700 bp - 1 KB on 2% gel. The pool was PCR amplified to generate the final library pool.
The library pool was analyzed in Bioanalyzer 2100 (Agilent Technologies) using High Sensitivity (HS) DNA chip as per manufacturer’s instructions. The library pool was sequenced on illumina NextSeq platform.
Sorghum accession | Post flowering drought tolerance | Sorghum accession | Post flowering drought tolerance |
---|---|---|---|
HSD5612 | Intermediate tolerance | TABAT | Sensitive |
HSD7410 | Tolerant | E19 | Tolerant |
HSD8511 | Tolerant | GESHEISH | Intermediate tolerance |
HSD6478 | Sensitive | WADAHMED | Sensitive |
HSD9566 | Tolerant | HSD8266 | Tolerant |
HSD9959 | Sensitive | HSD10033 | Tolerant |
HSD8653 | Tolerant | HSD8163 | Tolerant |
HSD6468 | Sensitive | HSD8552 | Intermediate tolerance |
HSD8228 | Tolerant | HSD8849 | Tolerant |
HSD8150 | Intermediate tolerance | HSD7507 | Tolerant |
SNPs were called from illumina FASTq files using the TASSELGBS pipeline. Only 75 bp tags present at least 10 times in the dataset were considered. Reads were aligned to the reference genome by Bowtie 2 tool with only the best hit alignment used. SNPs with >95% missing data were discarded. SNPs were filtered by minor allele frequency, as rare SNPs are especially useful for inferring differences between accessions after aligned with reference genome.
To identify stress response genes, the published stress response related genes from a reference sorghum plant were used. A total of 587 genes were obtained from the published sorghum transcriptome [
The accessions data were used for phylogenetic analysis using the cladogram function in TASSELGBS.
The gDNA extracted and purified from the 20 sorghum accessions was digested using ApeK1 enzyme to prepare the GBS library; the library pool was sequenced independently on Illumina Nextseq platform. The next generation sequencing for the accessions’ samples was performed using 2 × 150 bp chemistry; the reads statistics for the generated data is shown in
Accession | Raw Reads | Accession | Raw Reads |
---|---|---|---|
HSD5612 | 4,198,996 | TABAT | 3,436,844 |
HSD7410 | 1,687,323 | E19 | 3,191,451 |
HSD8511 | 4,320,193 | GESHEISH | 1,777,488 |
HSD6478 | 1,870,329 | WADAHMED | 2,571,771 |
HSD9566 | 3,605,439 | HSD8266 | 4,342,633 |
HSD9959 | 3,851,744 | HSD10033 | 965,338 |
HSD8653 | 3,522,793 | HSD8163 | 4,065,312 |
HSD6468 | 4,264,344 | HSD8552 | 1,564,602 |
HSD8228 | 3,966,240 | HSD8849 | 1,607,044 |
HSD8150 | 4,486,288 | HSD7507 | 2,360,612 |
Total reads | 35,773,689 | Total reads | 25,883,095 |
Three millions quality reads data per sample were generated; the TASSEL- GBS pipeline was used for identifying and calling SNPs from next generation sequenced accessions with a reference genome, as a result; a total of 814859 distinct tags were identified for all 20 sorghum accessions (
Blast search using the flanking sequence against NCBI databases against the reference genome B35 (BT × 623) was considered significant and revealed homology to annotated genes with function in stress adaptation. This genomic regions is found to be coding for the AAA+ ATPases, Peroxidase, Hydrophobic protein LTI6A, Aquaporin SIP2-1, Aconitate hydratase and phosphatidylinositol-4- phosphate-5-kinase.
According to [
Total Tags | 814,859 (100%) |
---|---|
Aligned tags > 1 times | 194,203 (23.83%) |
Aligned exactly 1 time | 566,877 (69.57%) |
Aligned 0 times | 53,779 (6.60%) |
Overall alignment rate | 93.40% |
have identified genomic regions involved in tolerance to drought stress and drought stress induced leaf senescence in juvenile barley. They found that the major QTLs for drought stress and leaf senescence were located on chromosome 5H and 2H. Blast X search for associated marker sequences revealed that respective SNPs are in some cases located in proteins related to drought stress or leaf senescence.
To investigate genetic relationships among these accessions, a phylogenic analysis using the marker data generated and developed by GBS was performed (
The population structure and genome-wide linkage disequilibrium in 478 spring wheat cultivars from 17 populations across the United States and Mexico, have been studied by [
Blast search using the flanking sequence revealed homology to annotated gene with function in stress adaptation. With further validation, these markers may be used for marker assisted selection for breeding new sorghum genotypes with stress adaptation.
We kindly acknowledge the Ministry of Higher Education, Sudan and The Deutscher Akademischer Austauschdienst, DAAD, for financial support.
Fadoul, H.E., El Siddig, M.A., Abdalla, A.W.H. and El Hussein, A.A. (2017) Genome-Wide SNPs Identification and Determination of Proteins Associated with Stress Response in Sorghum (Sorghum bicolor L. Monech) Accessions. American Journal of Plant Sci- ences, 8, 1624-1631. https://doi.org/10.4236/ajps.2017.87112