Mitchell R. Tuinstra

Heterogeneous inbred family (HIF) analysis: a method for developing near-isogenic lines that differ at quantitative trait loci

Abtract Analysis of near-isogenic lines (NILs) that differ at quantitative trait loci (QTL) can be an effective approach for the detailed mapping and characterization of individual loci. Although NILs are useful for genetic and physiological studies, the time and effort required to develop these lines have limited their use. Here we describe a procedure to identify NILs for any region of the genome that can be analyzed with molecular or other genetic markers. The procedure utilizes molecular markers to identify heterogeneous inbred families (HIFs) that segregate for a genomic region of interest. Each HIF is isogenic at the majority of loci in the genome, but NILs differing for markers linked to QTL of interest can be extracted from segregating families. The application of this procedure is described for two QTL associated with seed weight in sorghum. A population of 98 HIFs was screened with two RAPD markers from different linkage groups that were associated with seed weight. Three segregating families were identified for each marker. The progeny of these HIFs were characterized for the segregation of seed weight and other yield components and for markers flanking each QTL. NILs derived from each HIF had significantly different seed weights confirming the presence of at least two loci that influence seed weight in sorghum.

Parallel domestication of the Shattering1 genes in cereals

A key step during crop domestication is the loss of seed shattering. Here, we show that seed shattering in sorghum is controlled by a single gene, Shattering1 (Sh1), which encodes a YABBY transcription factor. Domesticated sorghums harbor three different mutations at the Sh1 locus. Variants at regulatory sites in the promoter and intronic regions lead to a low level of expression, a 2.2-kb deletion causes a truncated transcript that lacks exons 2 and 3, and a GT-to-GG splice-site variant in the intron 4 results in removal of the exon 4. The distributions of these non-shattering haplotypes among sorghum landraces suggest three independent origins. The function of the rice ortholog (OsSh1) was subsequently validated with a shattering-resistant mutant, and two maize orthologs (ZmSh1-1 and ZmSh1-5.1+ZmSh1-5.2) were verified with a large mapping population. Our results indicate that Sh1 genes for seed shattering were under parallel selection during sorghum, rice and maize domestication.

Genetic diversity and population structure analysis of accessions in the US historic sweet sorghum collection

Sweet sorghum has the potential to become a versatile feedstock for large-scale bioenergy production given its sugar from stem juice, cellulose/hemicellulose from stalks, and starch from grain. However, for researchers to maximize its feedstock potential a first step includes additional evaluations of the 2,180 accessions with varied origins in the US historic sweet sorghum collection. To assess genetic diversity of this collection for bioenergy breeding and population structure for association mapping, we selected 96 accessions and genotyped them with 95 simple sequence repeat markers. Subsequent genetic diversity and population structure analysis methods identified four subpopulations in this panel, which correlated well with the geographic locations where these accessions originated or were collected. Model comparisons for three quantitative traits revealed different levels of population structure effects on flowering time, plant height, and brix. Our results suggest that diverse germplasm accessions curated from different geographical regions should be considered for plant breeding programs to develop sweet sorghum cultivars or hybrids, and that this sweet sorghum panel can be further explored for association mapping.

Factors impacting ethanol production from grain sorghum in the dry-grind process.

ABSTRACT The goal of this research is to understand the key factors affecting ethanol production from grain sorghum. Seventy genotypes and elite hybrids with a range of chemical compositions and physical properties selected from ≈1,200 sorghum lines were evaluated for ethanol production and were used to study the relationships of composition, grain structure, and physical features that affect ethanol yield and fermentation efficiency. Variations of 22% in ethanol yield and 9% in fermentation efficiency were observed among the 70 sorghum samples. Genotypes with high and low conversion efficiencies were associated with attributes that may be manipulated to improve fermentation efficiency. Major characteristics of the elite sorghum genotypes for ethanol production by the dry-grind method include high starch content, rapid liquefaction, low viscosity during liquefaction, high fermentation speed, and high fermentation efficiency. Major factors adversely affecting the bioconversion process are tannin content, l...

Ethanol and lactic acid production as affected by sorghum genotype and location

Genotype, environment, location, and their interactions have a significant effect on end-use quality characteristics of grain sorghum (Sorghum biocolor (L.) Moench ). The objective of this research was to study the effect of sorghum genotype and production environment on ethanol and lactic acid production. Eight sorghum varieties from two locations were used. Whole sorghum grain was ground, liquefied, saccharified, and fermented to ethanol using Saccharomyces cerevisiae (S. cerevisae , ATCC 24860). For lactic acid fermentation, whole ground sorghum grain was liquefied and fermented to lactic acid with Rhizopus oryzae NRRL 395; saccharification depended upon native gluco- amylase. Results with this limited number of sorghum varieties and locations showed that both sorghum genotype and location had a significant effect on ethanol and lactic acid yields. Variations of 5 and 15% in ethanol and lactic acid yields were observed among the 16 sorghum samples. The effect of location on the fermentation yields was as much as 5% for ethanol and 10% for lactic acid. The effects of variety and location on ethanol and lactic acid production are strongly related to chemical composition and physical properties of grain sorghum samples. Ethanol and lactic acid production increased as starch content increased, whereas the ethanol and lactic acid production decreased as protein content increased. Chemical composition had a greater effect on the ethanol and lactic acid yields than physical properties of the sorghum kernels. The effect of physical properties on ethanol and lactic acid yields was not significant (P /0.05).

Effects of amylose, corn protein, and corn fiber contents on production of ethanol from starch-rich media.

ABSTRACT The effects of amylose, protein, and fiber contents on ethanol yields were evaluated using artificially formulated media made from commercial corn starches with different contents of amylose, corn protein, and corn fiber, as well as media made from different cereal sources including corn, sorghum, and wheat with different amylose contents. Second-order response-surface regression models were used to study the effects and interactions of amylose, protein, and fiber contents on ethanol yield and conversion efficiency. The results showed that the amylose content of starches had a significant (P 35%. The reduced quadratic model fits the conversion efficiency data better than the full quadratic mode...

Simulation Appraisal of the Adequacy of Number of Background Markers for Relationship Estimation in Association Mapping

Complex trait dissection through association mapping provides a powerful complement to traditional linkage analysis. The genetic structure of an association mapping panel can be estimated by genomewide background markers and subsequently accounted for in association analysis. Deciding the number of background markers is a common issue that needs to be addressed in many association mapping studies. We first showed that the adequacy of markers in relationship estimation influences the maximum likelihood of the model explaining phenotypic variation and demonstrated this influence with a series of computer simulations with different trait architectures. Analyses and computer simulations were then conducted using two different data sets: one from a diverse set of maize (Zea mays L.) inbred lines with a complex population structure and familial relatedness, and the other from a group of crossbred dogs. Our results showed that the likelihood‐based model‐fitting approach can be used to quantify the robustness of genetic relationships derived from molecular marker data. We also found that kinship estimation was more sensitive to the number of markers used than population structure estimation in terms of model fitting, and a robust estimate of kinship for association mapping with diverse germplasm requires a certain amount of background markers (e.g., 300–600 biallelic markers for the simulated pedigree materials, >1000 single nucleotide polymorphisms or 100 simple sequence repeats [SSRs] for the diverse maize panel, and about 100 SSRs for the canine panel). Kinship construction with subsets of the whole marker panel and subsequent model testing with multiple phenotypic traits could provide ad hoc information on whether the number of markers is sufficient to quantify genetic relationships among individuals.

Presence of tannins in sorghum grains is conditioned by different natural alleles of Tannin1

Sorghum, an ancient old-world cereal grass, is the dietary staple of over 500 million people in more than 30 countries in the tropics and semitropics. Its C4 photosynthesis, drought resistance, wide adaptation, and high nutritional value hold the promise to alleviate hunger in Africa. Not present in other major cereals, such as rice, wheat, and maize, condensed tannins (proanthocyanidins) in the pigmented testa of some sorghum cultivars have been implicated in reducing protein digestibility but recently have been shown to promote human health because of their high antioxidant capacity and ability to fight obesity through reduced digestion. Combining quantitative trait locus mapping, meta-quantitative trait locus fine-mapping, and association mapping, we showed that the nucleotide polymorphisms in the Tan1 gene, coding a WD40 protein, control the tannin biosynthesis in sorghum. A 1-bp G deletion in the coding region, causing a frame shift and a premature stop codon, led to a nonfunctional allele, tan1-a. Likewise, a different 10-bp insertion resulted in a second nonfunctional allele, tan1-b. Transforming the sorghum Tan1 ORF into a nontannin Arabidopsis mutant restored the tannin phenotype. In addition, reduction in nucleotide diversity from wild sorghum accessions to landraces and cultivars was found at the region that codes the highly conserved WD40 repeat domains and the C-terminal region of the protein. Genetic research in crops, coupled with nutritional and medical research, could open the possibility of producing different levels and combinations of phenolic compounds to promote human health.

Association Mapping for Grain Quality in a Diverse Sorghum Collection

Knowledge of the genetic bases of grain quality traits will complement plant breeding efforts to improve the end‐use value of sorghum [Sorghum bicolor (L.) Moench]. Candidate gene association mapping was used on a diverse panel of 300 sorghum accessions to assess marker–trait associations for 10 grain quality traits measured using the single kernel characterization system (SKCS) and near‐infrared reflectance spectroscopy (NIRS). The analysis of the accessions through 1290 genomewide single nucleotide polymorphisms (SNPs) separated the panel into five subpopulations that corresponded to three major sorghum races (durra, kafir, and caudatum), one intermediate race (guinea‐caudatum), and one working group (zerazera‐caudatum). These subpopulations differed in kernel hardness, acid detergent fiber, and total digestible nutrients. After model testing, association analysis between 333 SNPs in candidate genes and/or loci and grain quality traits resulted in eight significant marker–trait associations. A SNP in starch synthase IIa (SSIIa) gene was associated with kernel hardness (KH) with a likelihood ratio‐based R2 (RLR2) value of 0.08, a SNP in starch synthase (SSIIb) gene was associated with starch content with an RLR2 value of 0.10, and a SNP in loci pSB1120 was associated with starch content with an RLR2 value of 0.09.

Evaluation of the single kernel characterization system (SKCS) for measurement of sorghum grain attributes

ABSTRACT The single kernel characterization system (SKCS) has been widely used in the wheat industry, and SKCS parameters have been linked to end-use quality in wheat. The SKCS has promise as a tool for evaluating sorghum grain quality. However, the SKCS was designed to analyze wheat, which has a different kernel structure from sorghum. To gain a better understanding of the meaning of SKCS predictions for grain sorghum, individual sorghum grains were measured for length, width, thickness (diameter), and weight by laboratory methods and by the SKCS. SKCS predictions for kernel weight and thickness were highly correlated to laboratory measurements. However, SKCS predictions for kernel thickness were underestimated by ≈20%. The SKCS moisture prediction for sorghum was evaluated by tempering seven samples with varying hardness values to four moisture levels. The moisture contents predicted by SKCS were compared with a standard oven method and, while correlated, SKCS moisture predictions were less than moistur...