Abstract Although sorghum [Sorghum bicolor (L.) Moench ssp. bicolor] is the fifth most important grain crop in terms of global production, no commercial hybrids carry genetically engineered (GE) traits for resistance to insect pests or herbicides due to regulatory concerns about gene flow to weedy relatives. However, non-GE herbicide resistance currently is being developed in grain sorghum and will likely transfer to related weeds. Monitoring the impact of this new nuclear technology on the evolution and invasiveness of related weeds requires a baseline understanding of the population biology of grain sorghum genes once they transfer to in situ weed populations. We previously characterized the rate of gene flow from grain sorghum to shattercane [Sorghum bicolor (L.) Moench nothosubsp. drummondii (Steud.) de Wet ex. Davidse], a conspecific weed relatively common in North America; as well as the ecological fitness of an F1 population when S. bicolor nothosubsp. drummondii was the maternal parent. Here we report the ecological fitness of a S. bicolor nothosubsp. drummondii × S. bicolor ssp. bicolor F2 population relative to its crop and weed parents. Parental and F2 populations were grown in two Nebraska environments in 2012 and 2013. Traits evaluated included overwinter survival, field emergence, biomass production and partitioning at anthesis, total seed production, and 100-seed weight. Results indicated that F2 traits were generally intermediate between the parents, but more similar to S. bicolor nothosubsp. drummondii than to grain sorghum. The one exception was overwinter survival, which was nearly 0% for both the F2 and the grain sorghum parent in these northern environments. Thus, the frequency of crop alleles stably introgressed into S. bicolor nothosubsp. drummondii populations appears to primarily depend on overwinter survival of the F2 and which selective pressures are imposed upon it by the cropping system. These data provide needed baseline information about the environmental fate of nuclear genetic technologies deployed in this important global crop.

Sorghum [Sorghum bicolor (L.) Moench] has drawn attention as potential feedstock for lignocellulosic biofuels production, and reducing lignin is one way to increase conversion efficiency. Little research has been previously conducted to assess the response of reduced lignin sorghum lines to the Fusarium stalk rot pathogens Fusarium verticillioides and Fusarium proliferatum and the charcoal rot pathogen, Macrophomina phaseolina. Loss of function mutations in either the Brown midrib (Bmr) 6 or 12 gene that both encode a monolignol biosynthetic enzyme in the pathway that produces subunits of the lignin polymer, results in reduced lignin content. Near-isogenic bmr6, bmr12, and bmr6 bmr12 lines had previously been developed, which were shown to have significantly reduced lignin content and increased levels of soluble phenolics. In the current study, these lines in two backgrounds were shown to not be more susceptible to F. verticillioides, F. proliferatum and M. phaseolina inoculations, and some bmr lines exhibited increased resistance to F. proliferatum and M. phaseolina, compared to wild-type lines. When the Fusarium stalk rot pathogen, Fusarium thapsinum, was grown on methanol soluble stalk extracts from bmr6 and wild-type plants, it grew significantly faster on medium with bmr6 extract than on wild-type extract or controls. This result suggested that factors other than soluble phenolics from the extract, such as cell wall bound phenolics or inducible defense compounds, contributed to increased resistance observed in bmr6 plants.

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright’s F statistic (FST) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. FST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.

ABSTRACTGrain sorghum [Sorghum bicolor (L.) Moench] is an important crop for rainfed production systems with 2.7 million ha grown in the United States in 2013. The brown‐midrib (bmr) mutations, especially bmr‐12, have resulted in low stover lignin and high fiber digestibility without reducing grain yield in some sorghum lines. However, the effect of the bmr trait on beef cattle (Bos taurus) performance when grazing crop residue is unknown. Our objectives were to validate previous small‐plot results reporting no grain yield difference between near‐isogenic bmr‐12 (BMR) and wild‐type control (CON) A Wheatland × R Tx430 sorghum hybrids in a field‐scale experiment and to determine if BMR stover enhances beef production in a grazing experiment. Four replicated paddocks (2.3 ha) were planted in 2006 and 2008 near Mead, NE. Crossbred yearling steers (240 ± 17 kg hd−1) grazed (2.6 steers ha−1) paddocks following grain harvest for 72 d in 2006 and 61 d in 2008. Forage was sampled 4, 30, and 60 d after grazing began. Grain yield of BMR was 6% less (P = 0.01) than CON with no difference in stover neutral detergent fiber (NDF) content, but BMR stover had higher in vitro NDF digestibility (IVNDFD) (31%; P < 0.0001), steer average daily gain (ADG; 0.18 kg hd−1 d−1; P = 0.001), and body weight (BW) gain (29 kg ha−1; P = 0.002), resulting in an estimated increase in net return of $133.84 ha−1 due to BMR. Results suggest that the A Wheatland × R Tx430 bmr‐12 hybrid is an effective dual‐purpose sorghum crop for both grain and beef production.

Loss-of-function mutations in the granule-bound starch synthase gene result in an endosperm with a waxy appearance and a near absence of starch amylose. Three waxy grain sorghum [Sorghum bicolor (L.) Moench] lines, AN641 (Reg. No. GS-741, PI 672150), BN641 (Reg. No. GS-742, PI 672151), and RN642 (Reg. No. GS-743, PI 672152) were developed jointly by the USDA-ARS and the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska, and were released in June 2014. AN641 and BN641 have the waxy(b) (wx(b)) allele and are near-isogenic to 'Wheatland'. RN642 has the waxy(a) (wx(a)) allele and is near-isogenic to 'Tx430'. Release of these lines with cytoplasmic male-sterile (A), maintainer (B), and fertility restorer (R) fertility reactions to A 1 cytoplasm facilitates the production and evaluation of interallelic (wx(b) x wx(a)) waxy and heterowaxy [wx(b) x wild-type (WT) and WT x wx(a)] hybrids as a source of low-amylose starch for the ethanol and food industries.

ABSTRACTFour near‐isogenic Wheatland × Tx430 grain sorghum [Sorghum bicolor (L.) Moench] hybrids differing in allelic status at the Waxy locus were grown in yield trials to determine their potential to expand existing sources of low‐amylose starch. The hypothesis tested was that agronomic performance and grain yield do not differ among hybrid genotypes. Hybrids were generated in a two‐by‐two factorial design using wxb and wild‐type (WT) Wheatland as female parents with wxa and WT Tx430 as male parents. Yield trials were conducted at two Nebraska locations in 2009 and 2010. No differences were observed for field emergence, but grain yield of the interallelic waxy (wxb × wxa) hybrid was 330 kg ha−1 greater than the WT × WT hybrid (P = 0.0482). The wxb × Wx hybrid had the highest grain yield, 633 kg ha−1 greater than the WT (P = 0.0003). Amylose starch content was lowest for wxb × wxa (7.66 g kg−1), intermediate for wxb × Wx and Wx × wxa (25.06 and 27.20 g kg−1, respectively); and highest for WT × WT (34.80 g kg−1) (n = 4, P < 0.0001). The waxy and heterowaxy hybrids evaluated in this study are promising options for commercial production of starches with reduced amylose contents in a drought‐tolerant crop.

Sorghum [Sorghum bicolor (L.) Moench] lines N619 to N636 (A lines; Reg. No. GS-699 to GS-716, PI 670134 to PI 670151); N619 to N636 (B lines; Reg. No. GS-721 to GS-738, PI 671777 to PI 671794); and N637 to N640 (R lines; Reg. No. GS-717 to GS-720, PI 670152 to PI 670155) comprise nine pairs of seed parent (A/B) lines, and two pairs of pollinator (R) lines (11 pairs total) that are near-isogenic for waxy (low-amylose) or wildtype endosperm. Breeding work was conducted jointly by the USDA-ARS and the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska, and the lines were released in May 2014. Release of these lines makes available two different waxy (wx) alleles (wx(a) and wx(b)) for development of grain sorghum as a source of low-amylose starch, whose end use is targeted to the ethanol and food industries. In particular, the release of wx and wild-type near-isogenic pairs facilitates the evaluation of agronomic performance of wx genotypes, and the release of both A/B and R lines facilitates the production of waxy grain hybrids.

Sorghum lines were bred for reduced lignin for cellulosic bioenergy uses, through the incorporation of brown midrib (bmr)6 or -12 into two backgrounds (RTx430 and Wheatland) as either single or double-mutant lines. When these lines were assessed for resistance to Fusarium thapsinum stalk rot, a cause of lodging, they were as resistant to F. thapsinum as the near-isogenic wild type. Peduncles of newly identified bmr lines from an ethyl-methanesulfonate-mutagenized population, inoculated with F. thapsinum, were as resistant as the wild-type line, BTx623. One bmr line (1107) had significantly smaller mean lesion lengths than BTx623, suggesting that a mutation is associated with reduced susceptibility. Growing F. thapsinum on medium with ferulic, vanillic, sinapic, syringic, and caffeic acids (phenolic compounds derived from the lignin pathway and elevated in different bmr lines) indicated that F. thapsinum was tolerant to these compounds. When eight other sorghum fungi were tested for response to the presence of these compounds, ferulic acid inhibited these fungi. Most of the phenolics inhibited F. verticillioides and F. proliferatum. Accumulation of phenolic metabolites in bmr plants may inhibit growth of some sorghum pathogens, while other factors such as aromatic phytoalexins or salicylic acid may be involved in resistance to F. thapsinum.

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.

A fundamental need for commercialization of sweet sorghum [Sorghum bicolor (L.) Moench] as a bioenergy crop is an adequate seed supply, which will require development of hybrid varieties using dwarf seed-parent lines. A set of six public sweet sorghum A-lines (Dwarf Kansas Sourless, KS9, N36, N38, N39, and N4692) were crossed with a set of six public sweet sorghum cultivars (Brawley, Kansas Collier, Dale, Sugar Drip, Waconia, and Wray). Grain, fiber, and sugar yields were determined, and conversion formulas were applied to estimate ethanol yields. Hybrids were grown in fields at Ithaca, NE, USA, in 1983–1984 fertilized with 112 kg ha−1 N. In terms of yield components and overall ethanol yields, one A-line, N38, was inferior. Average total ethanol yields from hybrids made on the other A-lines were not significantly different, suggesting that any of those five A-lines could be useful seed-parents. With the exception of grain yield, cultivars used as pollen parents were among the highest-performing entries for all traits. For all traits directly contributing to total ethanol yield (grain yield, juice yield, % soluble solids, sugar yield, fiber yield), hybrids were also among the highest-performing entries. Results of this study demonstrate that hybrid sweet sorghum with performance criteria equivalent to existing sweet sorghum cultivars can be produced on the sweet sorghum seed-parent lines A-Dwarf Kansas Sourless, A-KS9, A-N36, A-N39, and A-N4692. Identification of specific seed-parent × pollen parent lines with characteristics best suited for particular growing regions and end-user needs will be critical for commercial hybrid development.

Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum.

Four shattercane [Sorghum bicolor subsp. drummondii (Nees ex Steud.) de Wet ex Davidse] genetic stocks—N614 (Reg. No. GS‐652, PI 665684), A3N615 (Reg. No. GS‐651, PI 665683), N616 (Reg. No. GS‐653, PI 665685), and N617 (Reg. No. GS‐654, PI 665686)—with A3 cytoplasmic male sterility or the nuclear male sterility gene ms3 containing either juicy (dd) or dry (DD) culms were developed jointly by the USDA‐ARS; the Iowa Agricultural and Home Economics Experiment Station, College of Agriculture and Life Sciences, Iowa State University; and the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska. The stocks were released in July 2011. The source material for these genetic stocks was isolated from an archetypical shattercane population found near Lincoln, NE. Release of these genetic stocks makes available shattercane lines with both A3 cytoplasmic male sterility, and ms3 genetic (nuclear) male sterility to facilitate crossing. These genetic stocks also contain juicy (dd) or dry (DD) culms, a visible genetic marker to facilitate screening progeny resulting from crosses. The genetic stocks have immediate application for basic research involving gene flow from cultivated sorghum [Sorghum bicolor (L.) Moench] to shattercane and on the fitness of offspring resulting from such crosses.

Sorghum is used as bioenergy feedstock, animal feed, and food. Economical methods for disease prevention and control are valuable for producers. Fluorescent Pseudomonas spp. were isolated from sorghum roots and surrounding soil with the goal of finding isolates that significantly inhibited sorghum fungal pathogens. Fluorescent pseudomonads were collected from seedlings of sorghum cultivars RTx433 and Redlan and wheat cultivar Lewjain, grown in two soils. Lewjain is known to support growth of producers of the antibiotic, 2,4-diacetylphloroglucinol (2,4-DAPG). Isolates from all three plants were assessed for hydrogen cyanide (HCN) and extracellular protease production, and for a 2,4-DAPG gene, phlD. Both soil type and plant type affected HCN- and protease-production, but phlD was not affected. Subsets of phlD (+) isolates were chosen to determine phlD genotypes and to conduct in vitro inhibition assays against sorghum pathogens. Most isolates from sorghum and wheat were genotype D, previously associated with superior root colonization. phlD (+) sorghum isolates were co-cultured with five sorghum pathogens. One isolate from each sorghum line exhibited inhibition to all five pathogens but more Redlan isolates were inhibitory to the virulent pathogen, Fusarium thapsinum, than RTx433 isolates. Nearly all inhibitory isolates from either sorghum cultivar were from one soil type. This is consistent with what had been previously observed in field studies: that soil type played a significant role in determining characteristics of fluorescent Pseudomonas spp. isolated from roots or soil, but sorghum genotype also had a considerable effect.

ABSTRACTCultivated sorghum [Sorghum bicolor (L.) Moench subsp. bicolor] can interbreed with a feral weedy relative shattercane [S. bicolor nothosubsp. drummondii (Steud.) de Wet ex Davidse]. Traits introduced from cultivated sorghum could contribute to the invasiveness of a shattercane population. An experiment was conducted to determine the potential for pollen‐mediated gene flow from grain sorghum to shattercane. Shattercane with juicy midrib (dd) was planted in soybean [Glycine max (L.) Merr.] fields during 2 yr in concentric arcs at varying distances from a 0.39 ha sorghum pollen source with dry midrib (DD). The arcs were placed so that prevailing winds would carry sorghum pollen to the shattercane. Seven hundred twenty seeds from each of over 300 shattercane panicles in anthesis during sorghum pollen shed each year were collected. Progeny were evaluated by phenotype to determine rate of hybridization. Hybridization averaged 3.6% within the source in 2008 and 16.0% in 2009 and declined as distance increased. Hybridization as high as 2.6% for an individual panicle was measured at the farthest distance evaluated (200 m). Wind direction and speed were also measured and their product affected hybridization rate for all pollination periods. Results indicate that genes from cultivated sorghum will likely be introduced into shattercane populations at distances of at least 200 m and that rate of hybridization is dependent on weather factors such as wind. Source size is also important in determining hybridization rate but was not studied here.

Background There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. Results We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans , incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow ( Salix spp.). Conclusion Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.

Digestibility of cereal grain protein is conventionally expressed as percentage of protein digested by pepsin in a defined period. Sorghum has the lowest protein digestibility among cereals especially after wet cooking. If additional details were known as to which proteins are resistant to digestion, it should be possible to find means to improve this property. In this paper, two approaches are described to extract and identify proteins undigested by pepsin in uncooked and cooked meal from numerous sorghum and corn varieties. The first, one-solvent approach involves extracting residues undigested after 2 h with Borate-SDS-ME and separating by PAGE. Improved separation of undigested sorghum proteins was achieved using NuPAGE Bis-Tris gels. Western blots with antibodies against particular zeins and kafirins, used to monitor fates of different kafirins, revealed differential digestion rates. A second approach involved extracting undigested residues sequentially with 60% t-butanol, 60% t-butanol-ME and Borate-SDS-ME. The second, three-solvent approach, coupled with western analysis, revealed the following. (i) Oligomeric forms of certain kafirins exist that differ in the degree of their susceptibility to pepsin digestion. (ii) Effect of cooking on the formation and digestion of the oligomers. (iii) Cross-linked forms of most α-kafirins became more resistant to digestion after cooking. (iv) Most α-kafirins are preferentially extracted in 60% t-butanol-ME while most γ-kafirins are extracted in Borate-SDS-ME buffer. (v) Monomeric γ-kafirins are resistant to pepsin digestion. (vi) γ-Kafirins form a series of oligomers that exhibit differential resistance to digestion. Our results suggest that the presently described systematic approach to analyzing the digestion by pepsin of sorghum prolamins should lead to greater insights into the digestion of specific types of sorghum grain proteins.

Mold diseases, caused by fungal complexes including Alternaria, Cochliobolus, and Fusarium species, limit sorghum grain production. Media were tested by plating Fusarium thapsinum, Alternaria sp., and Curvularia lunata, individually and competitively. Dichloran chloramphenicol rose bengal (DRBC) and modified V8 juice (ModV8) agars, found to be useful, were compared with commonly used agar media, dichloran chloramphenicol peptone (DCPA) and pentachloronitrobenzene (PCNB). Radial growth, starting with mycelia or single-conidia and hyphal tips, demonstrated an effect of media. For isolation of grain fungi, DRBC and ModV8 were similar or superior to DCPA and PCNB. When seedlings were inoculated with conidia of C. lunata, Alternaria sp., F. thapsinum, or mixtures, the percentage of root infection ranged from 28% to 77%. For mixed inoculations, shoot weights, lesion lengths, and percentage of root infections were similar to F. thapsinum inoculations; most colonies recovered from roots were F. thapsinum. For Alternaria grain isolates, 5 morphological types, including Alternaria alternata, were distinguished by colony morphologies and conidial dimensions. Sequence analysis using a portion of the endo-polygalacturonase gene was able to further distinguish isolates. Cochliobolus isolates were identified morphologically as C. lunata, Curvularia sorghina, and Bipolaris sorghicola. Multiple molecular genotypes were apparent from rRNA internal transcribed spacer region sequences from Cochliobolus grain isolates.