Project description:Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.
Project description:In sorghum [Sorghum bicolor (L.) Moench] the Maturity (Ma1, Ma2, Ma3, Ma4, Ma5, Ma6) and Dwarf (Dw1, Dw2, Dw3, Dw4) loci, encode genes controlling flowering time and plant height, respectively, which are critical for designing sorghum ideotypes for a maturity timeframe and a harvest method. Publicly available whole-genome resequencing data from 860 sorghum accessions was analyzed in silico to identify genomic variants at 8 of these loci (Ma1, Ma2, Ma3, Ma5, Ma6, Dw1, Dw2, Dw3) to identify novel loss of function alleles and previously characterized ones in sorghum germplasm. From ~ 33 million SNPs and ~ 4.4 million InDels, 1445 gene variants were identified within these 8 genes then evaluated for predicted effect on the corresponding encoded proteins, which included newly identified mutations (4 nonsense, 15 frameshift, 28 missense). Likewise, most accessions analyzed contained predicted loss of function alleles (425 ma1, 22 ma2, 40 ma3, 74 ma5, 414 ma6, 289 dw1, 268 dw2 and 45 dw3) at multiple loci, but 146 and 463 accessions had no predicted ma or dw mutant alleles, respectively. The ma and dw alleles within these sorghum accessions represent a valuable source for manipulating flowering time and plant height to develop the full range of sorghum types: grain, sweet and forage/biomass.
Project description:In the last decades, deficiency of macro- and micronutrients was considered as a serious problem associated with the increase in the human population. To meet the increased demand for food consumption, the wild relative plant might serve as an important source of new genetic material for increasing macro- and micronutrients. To investigate this, the variations in protein content, in vitro protein digestibility, tannin content, phytic acid content, total polyphenol content, and total and bioavailability of minerals were studied in grains of ten wild sorghums and two released sorghum cultivars. The results showed significant differences (p ≤ 0.05) in all quality tests among the genotypes. The highest percentage of total protein contents and in vitro protein digestibility were encountered in the grains of PQ-434 (14.6%) and the released cultivar AG8 (49.8%), respectively, while the highest concentrations of total and bioavailable iron were found in the grains of Almahkara (3.17 mg/100 g) and Abusabiba (92.8 mg/100 g), respectively. The grains of wild sorghum genotype Adar Umbatikh grains were found to possess higher total zinc contents. The PCA identified only five components of eigenvalues greater than one and cumulatively accounted for 88% of the total variation. It could be concluded that Almahkara and PQ-434 could be used as potential sources for iron and protein sorghum biofortification, respectively. Results from this study might be used in the development of new value-added products from wild sorghum grains by-products.
Project description:Fast neutron radiation has been used as a mutagen to develop extensive mutant collections. However, the genome-wide structural consequences of fast neutron radiation are not well understood. Here, we examine the genome-wide structural variants observed among 264 soybean [Glycine max (L.) Merrill] plants sampled from a large fast neutron-mutagenized population. While deletion rates were similar to previous reports, surprisingly high rates of segmental duplication were also found throughout the genome. Duplication coverage extended across entire chromosomes and often prevailed at chromosome ends. High-throughput resequencing analysis of selected mutants resolved specific chromosomal events, including the rearrangement junctions for a large deletion, a tandem duplication, and a translocation. Genetic mapping associated a large deletion on chromosome 10 with a quantitative change in seed composition for one mutant. A tandem duplication event, located on chromosome 17 in a second mutant, was found to cosegregate with a short petiole mutant phenotype, and thus may serve as an example of a morphological change attributable to a DNA copy number gain. Overall, this study provides insight into the resilience of the soybean genome, the patterns of structural variation resulting from fast neutron mutagenesis, and the utility of fast neutron-irradiated mutants as a source of novel genetic losses and gains.
Project description:Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium's efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C) of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor) mutant with a low δ13C characteristic. A mutant (named Mut33) with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT). The back-cross (BC1F1) progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 μmol CO2.mol-1air) and the maximum rate of photosynthesis was less than 5μmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs) between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used in the transformation of C3 to C4 plants.
Project description:Sorghum (Sorghum bicolor Moench, L.) plant accumulates copious layers of epi-cuticular wax (EW) on its aerial surfaces, to a greater extent than most other crops. EW provides a vapor barrier that reduces water loss, and is therefore considered to be a major determinant of sorghum's drought tolerance. However, little is known about the genes responsible for wax accumulation in sorghum. We isolated two allelic mutants, bloomless40-1 (bm40-1) and bm40-2, from a mutant library constructed from ethyl methane sulfonate (EMS) treated seeds of an inbred, BTx623. Both mutants were nearly devoid of the EW layer. Each bm mutant was crossed to the un-mutated BTx623 to generated F2 populations that segregated for the bm phenotype. Genomic DNA from 20 bm F2 plants from each population was bulked for whole genome sequencing. A single gene, Sobic.001G228100, encoding a GDSL-like lipase/acylhydrolase, had unique homozygous mutations in each bulked F2 population. Mutant bm40-1 harbored a missense mutation in the gene, whereas bm40-2 had a splice donor site mutation. Our findings thus provide strong evidence that mutation in this GDSL-like lipase gene causes the bm phenotype, and further demonstrate that this approach of sequencing two independent allelic mutant populations is an efficient method for identifying causal mutations. Combined with allelic mutants, MutMap provides powerful method to identify all causal genes for the large collection of bm mutants in sorghum, which will provide insight into how sorghum plants accumulate such abundant EW on their aerial surface. This knowledge may facilitate the development of tools for engineering drought-tolerant crops with reduced water loss.
Project description:Sorghum is one of the most promising bioenergy crops. Stem juice yield, together with stem sugar concentration, determines sugar yield in sweet sorghum. Bulked segregant analysis (BSA) is a gene mapping technique for identifying genomic regions containing genetic loci affecting a trait of interest that when combined with deep sequencing could effectively accelerate the gene mapping process. In this study, a dry stem sorghum landrace was characterized and the stem water controlling locus, qSW6, was fine mapped using QTL analysis and the combined BSA and deep sequencing technologies. Results showed that: (i) In sorghum variety Jiliang 2, stem water content was around 80% before flowering stage. It dropped to 75% during grain filling with little difference between different internodes. In landrace G21, stem water content keeps dropping after the flag leaf stage. The drop from 71% at flowering time progressed to 60% at grain filling time. Large differences exist between different internodes with the lowest (51%) at the 7th and 8th internodes at dough stage. (ii) A quantitative trait locus (QTL) controlling stem water content mapped on chromosome 6 between SSR markers Ch6-2 and gpsb069 explained about 34.7-56.9% of the phenotypic variation for the 5th to 10th internodes, respectively. (iii) BSA and deep sequencing analysis narrowed the associated region to 339 kb containing 38 putative genes. The results could help reveal molecular mechanisms underlying juice yield of sorghum and thus to improve total sugar yield.
Project description:The objective of this investigation was to characterize, at individual level, the transcriptional response and the onset of regenerative processes in mouse skin irradiated with different doses of fast neutrons. We performed a high-throughput gene expression analysis, by DNA oligonucleotide microarray on 24 three months old C57Bl/6 mice irradiated with 0, 0,2 and 1 Gy of mono-energetic 14 MeV neutron. The results, partially validated by quantitative real time RT-PCR, showed an up-regulation of a sub-class of keratin and keratin associated proteins, and of components of the S100 family of Ca2+-binding proteins which was limited to the lower dose. We conclude that the dose-dependent differential gene expression, reminiscent of the onset of re-epithelialization and wound healing, depends upon the proportion of cells carrying multiple lesions at chromosomal level post-irradiation, and it represents an in vivo evidence of a skin regenerative program exerted independently from DNA repair-associated pathways.
Project description:Polyphenols and other potential health-promoting components of sorghum (Sorghum bicolor (L.) Moench) drove its recent growth in the U.S. consumer food industry. Linear sweep (cyclic voltammetry, CV) and differential (cyclic differential pulse) voltammetry methods were developed to detect target polyphenols and amino acids in sweet sorghum juice without interference from the dominant secondary (trans-aconitic acid) and primary (sucrose) metabolites. Of 24 cultivars investigated, No.5 Gambela showed the highest electron-donating capacity, as indicated by the highest peak area, height, and peak anodic potential. Pearson's correlation analysis indicated the contribution of polyphenols (rather than amino acids) on CV voltammograms of juice samples. The Eh-pH values of 173 sweet sorghum juice samples collected in 2017 aligned with quercetin model polyphenol. Accumulation of quercetin-like polyphenols in No.5 Gambela could offer antioxidant-rich juice for conversion to edible syrup as well as an increased tolerance against a recently emerged pest, sugarcane aphid [(Melanaphis sacchari (Zehntner)].
Project description:Multi-parent advanced generation intercross (MAGIC) populations are powerful next-generation mapping resources. We describe here the mating design and structure of the first MAGIC population in sorghum, and test its utility for mapping. The population was developed by intercrossing 19 diverse founder lines through a series of paired crosses with a genetic male sterile (MS) source, followed by 10 generations of random mating. At the final stage of random mating, 1000 random fertile plants in the population were identified and subjected to six generations of selfing to produce 1000 immortal MAGIC inbred lines. The development of this sorghum MAGIC population took over 15 yr. Genotyping-by-sequencing (GBS) of a subset of 200 MAGIC lines identified 79,728 SNPs, spanning high gene-rich regions. Proportion of SNPs per chromosome ranged from 6 to 15%. Structure analyses produced no evidence of population stratification, portraying the desirability of this population for genome-wide association studies (GWAS). The 19 founders formed three clusters, each with considerable genetic diversity. Further analysis showed that 73% of founder alleles segregated in the MAGIC population. Linkage disequilibrium (LD) patterns depicted the MAGIC population to be highly recombined, with LD decaying to r2 [Formula: see text] 0.2 at 40 kb and down to r2 [Formula: see text] 0.1 at 220 kb. GWAS detected two known plant height genes, DWARF1 (chromosome 9) and DWARF3 (chromosome 7), and a potentially new plant height quantitative trait locus (QTL) (QTL-6) on chromosome 6. The MAGIC population was found to be rich in allelic content with high fragmentation of its genome, making it fit for both gene mapping and effective marker-assisted breeding.