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Characterization of novel Sorghum brown midrib mutants from an EMS-mutagenized population.

ABSTRACT: Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles.

SUBMITTER: Sattler SE

PROVIDER: S-EPMC4232537 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Characterization of novel Sorghum brown midrib mutants from an EMS-mutagenized population.

Sattler Scott E SE Saballos Ana A Xin Zhanguo Z Funnell-Harris Deanna L DL Vermerris Wilfred W Pedersen Jeffrey F JF

G3 (Bethesda, Md.) 20140902 11

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described s ...[more]

PMID: 25187038

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Project description:The precise detection of causal DNA mutations (deoxyribonucleic acid) is very crucial for forward genetic studies. Several sources of errors contribute to false-positive detections by current variant-calling algorithms, which impact associating phenotypes with genotypes. To improve the accuracy of mutation detection, we implemented a binning method for the accurate detection of likely ethyl methanesulfonate (EMS)-induced mutations in a sequenced mutant population. We also implemented a clustering algorithm for detecting likely false negatives with high accuracy. Sorghum bicolor is a very valuable crop species with tremendous potential for uncovering novel gene functions associated with highly desirable agronomical traits. We demonstrate the precision of the described approach in the detection of likely EMS-induced mutations from the publicly available low-cost sequencing of the M3 generation from 600 sorghum BTx623 mutants. The approach detected 3,274,606 single nucleotide polymorphisms (SNPs), of which 96% (3,141,908) were G/C to A/T DNA substitutions, as expected by EMS-mutagenesis mode of action. We demonstrated the general applicability of the described method and showed a high concordance, 94% (3,074,759) SNPs overlap between SAMtools-based and GATK-based variant-calling algorithms. Our clustering algorithm uncovered evidence for an additional 223,048 likely false-negative shared EMS-induced mutations. The final 3,497,654 SNPs represent an 87% increase in SNPs detected from the previous analysis of the mutant population, with an average of one SNP per 125 kb in the sorghum genome. Annotation of the final SNPs revealed 10,263 high-impact and 136,639 moderate-impact SNPs, including 7217 stop-gained mutations, which averages 12 stop-gained mutations per mutant, and four high- or medium-impact SNPs per sorghum gene. We have implemented a public search database for this new genetic resource of 30,285 distinct sorghum genes containing medium- or high-impact EMS-induced mutations. Seedstock for a select 486 of the 600 described mutants are publicly available in the Germplasm Resources Information Network (GRIN) database.

Project description:The accurate detection of induced mutations is critical for both forward and reverse genetics studies. Experimental chemical mutagenesis induces relatively few single base changes per individual. In a complex eukaryotic genome, false positive detection of mutations can occur at or above this mutagenesis rate. We demonstrate here, using a population of ethyl methanesulfonate (EMS)-treated Sorghum bicolor BTx623 individuals, that using replication to detect false positive-induced variants in next-generation sequencing (NGS) data permits higher throughput variant detection with greater accuracy. We used a lower sequence coverage depth (average of 7×) from 586 independently mutagenized individuals and detected 5,399,493 homozygous single nucleotide polymorphisms (SNPs). Of these, 76% originated from only 57,872 genomic positions prone to false positive variant calling. These positions are characterized by high copy number paralogs where the error-prone SNP positions are at copies containing a variant at the SNP position. The ability of short stretches of homology to generate these error-prone positions suggests that incompletely assembled or poorly mapped repeated sequences are one driver of these error-prone positions. Removal of these false positives left 1,275,872 homozygous and 477,531 heterozygous EMS-induced SNPs, which, congruent with the mutagenic mechanism of EMS, were >98% G:C to A:T transitions. Through this analysis, we generated a collection of sequence indexed mutants of sorghum. This collection contains 4035 high-impact homozygous mutations in 3637 genes and 56,514 homozygous missense mutations in 23,227 genes. Each line contains, on average, 2177 annotated homozygous SNPs per genome, including seven likely gene knockouts and 96 missense mutations. The number of mutations in a transcript was linearly correlated with the transcript length and also the G+C count, but not with the GC/AT ratio. Analysis of the detected mutagenized positions identified CG-rich patches, and flanking sequences strongly influenced EMS-induced mutation rates. This method for detecting false positive-induced mutations is generally applicable to any organism, is independent of the choice of in silico variant-calling algorithm, and is most valuable when the true mutation rate is likely to be low, such as in laboratory-induced mutations or somatic mutation detection in medicine.

Project description:The hydrophobic cell wall polymer lignin is deposited in specialized cells to make them impermeable to water and prevent cell collapse as negative pressure or gravitational force is exerted. The variation in lignin subunit composition that exists among different species, and among different tissues within the same species suggests that lignin subunit composition varies depending on its precise function. In order to gain a better understanding of the relationship between lignin subunit composition and the physico-chemical properties of lignified tissues, detailed analyses were performed of near-isogenic brown midrib2 (bm2), bm4, bm2-bm4, and bm1-bm2-bm4 mutants of maize. This investigation was motivated by the fact that the bm2-bm4 double mutant is substantially shorter, displays drought symptoms even when well watered, and will often not develop reproductive organs, whereas the phenotypes of the individual bm single mutants and double mutant combinations other than bm2-bm4 are only subtly different from the wild-type control. Detailed cell wall compositional analyses revealed midrib-specific reductions in Klason lignin content in the bm2, bm4, and bm2-bm4 mutants relative to the wild-type control, with reductions in both guaiacyl (G)- and syringyl (S)-residues. The cellulose content was not different, but the reduction in lignin content was compensated by an increase in hemicellulosic polysaccharides. Linear discriminant analysis performed on the compositional data indicated that the bm2 and bm4 mutations act independently of each other on common cell wall biosynthetic steps. After quantitative analysis of scanning electron micrographs of midrib sections, the variation in chemical composition of the cell walls was shown to be correlated with the thickness of the sclerenchyma cell walls, but not with xylem vessel surface area. The bm2-bm4 double mutant represents the limit of phenotypic plasticity in cell wall composition, as the bm1-bm2-bm4 and bm2-bm3-bm4 mutants did not develop into mature plants, unlike the triple mutants bm1-bm2-bm3 and bm1-bm3-bm4.

Dataset Information

Characterization of novel Sorghum brown midrib mutants from an EMS-mutagenized population.

Publications

Characterization of novel Sorghum brown midrib mutants from an EMS-mutagenized population.

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