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Main conclusion SorghumBase provides a community portal that integrates genetic, genomic, and breeding resources for sorghum germplasm improvement. Abstract Public research and development in agriculture rely on proper data and resource sharing within stakeholder communities. For plant breeders, agronomists, molecular biologists, geneticists, and bioinformaticians, centralizing desirable data into a user-friendly hub for crop systems is essential for successful collaborations and breakthroughs in germplasm development. Here, we present the SorghumBase web portal (https://www.sorghumbase.org), a resource for the sorghum research community. SorghumBase hosts a wide range of sorghum genomic information in a modular framework, built with open-source software, to provide a sustainable platform. This initial release of SorghumBase includes: (1) five sorghum reference genome assemblies in a pan-genome browser; (2) genetic variant information for natural diversity panels and ethyl methanesulfonate (EMS)-induced mutant populations; (3) search interface and integrated views of various data types; (4) links supporting interconnectivity with other repositories including genebank, QTL, and gene expression databases; and (5) a content management system to support access to community news and training materials. SorghumBase offers sorghum investigators improved data collation and access that will facilitate the growth of a robust research community to support genomics-assisted breeding.

Grain number per panicle (GNP) is a major determinant of grain yield in cereals. However, the mechanisms that regulate GNP remain unclear. To address this issue, we isolate a series of sorghum [Sorghum bicolor (L.) Moench] multiseeded (msd) mutants that can double GNP by increasing panicle size and altering floral development so that all spikelets are fertile and set grain. Through bulk segregant analysis by next-generation sequencing, we identify MSD1 as a TCP (Teosinte branched/Cycloidea/PCF) transcription factor. Whole-genome expression profiling reveals that jasmonic acid (JA) biosynthetic enzymes are transiently activated in pedicellate spikelets. Young msd1 panicles have 50% less JA than wild-type (WT) panicles, and application of exogenous JA can rescue the msd1 phenotype. Our results reveal a new mechanism for increasing GNP, with the potential to boost grain yield, and provide insight into the regulation of plant inflorescence architecture and development., Inflorescence architecture affects crop grain yield. Here, the authors deploy whole-genome sequencing-based bulk segregant analysis to identify the causal gene of a sorghum multi-seeded (msd) mutant and suggest MSD1 regulating the fertility of the pedicellate spikelets through jasmonic acid pathway.

The USDA–ARS has released a new nuclear male sterile sorghum [Sorghum bicolor (L.) Moench] mutant BTx623ms8 (Reg. No. GS-786, PI 682727). Previously, seven sorghum nuclear male sterile mutants have been reported, but three are no longer available. Here we register a new nuclear male sterile mutant isolated from a mutagenized BTx623 mutant library that is genetically different from any of the four male sterile mutants currently available. The ms8 mutant had microscopic transparent anthers that abort early during microsporogenesis. It can be easily identified at early stages of anthesis by the appearance of prominent white, hairy stigma, rather than yellow anthers as seen in BTx623 and all other fertile sorghum accessions. We have developed BC6F2 seeds that produce approximately 50% male sterile plants by pollinating the homozygous ms8 mutants with the heterozygous fertile plants. The ms8 is a single recessive nuclear gene mutation and can be easily bred into other sorghum lines through recurrent backcrossing, broadening the utility of this easily recognizable nuclear male sterility. © Crop Science Society of America. All rights reserved.

Grain number per panicle is an important component of grain yield in sorghum (Sorghum bicolor (L.)) and other cereal crops. Previously, we reported that mutations in multi-seeded 1 (MSD1) and MSD2 genes result in a two-fold increase in grain number per panicle due to the restoration of the fertility of the pedicellate spikelets, which invariably abort in natural sorghum accessions. Here, we report the identification of another gene, MSD3, which is also involved in the regulation of grain numbers in sorghum. Four bulked F2 populations from crosses between BTx623 and each of the independent msd mutants p6, p14, p21, and p24 were sequenced to 20&times, coverage of the whole genome on a HiSeq 2000 system. Bioinformatic analyses of the sequence data showed that one gene, Sorbi_3001G407600, harbored homozygous mutations in all four populations. This gene encodes a plastidial &omega, 3 fatty acid desaturase that catalyzes the conversion of linoleic acid (18:2) to linolenic acid (18:3), a substrate for jasmonic acid (JA) biosynthesis. The msd3 mutants had reduced levels of linolenic acid in both leaves and developing panicles that in turn decreased the levels of JA. Furthermore, the msd3 panicle phenotype was reversed by treatment with methyl-JA (MeJA). Our characterization of MSD1, MSD2, and now MSD3 demonstrates that JA-regulated processes are critical to the msd phenotype. The identification of the MSD3 gene reveals a new target that could be manipulated to increase grain number per panicle in sorghum, and potentially other cereal crops, through the genomic editing of MSD3 functional orthologs.

As in other cereal crops, the panicles of sorghum (Sorghum bicolor (L.) Moench) comprise two types of floral spikelets (grass flowers). Only sessile spikelets (SSs) are capable of producing viable grains, whereas pedicellate spikelets (PSs) cease development after initiation and eventually abort. Consequently, grain number per panicle (GNP) is lower than the total number of flowers produced per panicle. The mechanism underlying this differential fertility is not well understood. To investigate this issue, we isolated a series of EMS-induced multiseeded (msd) mutants that result in full spikelet fertility, effectively doubling GNP. Previously, we showed that MSD1 is a TCP (Teosinte branched/Cycloidea/PCF) transcription factor that regulates jasmonic acid (JA) biosynthesis, and ultimately floral sex organ development. Here, we show that MSD2 encodes a lipoxygenase (LOX) that catalyzes the first committed step of JA biosynthesis. Further, we demonstrate that MSD1 binds to the promoters of MSD2 and other JA pathway genes. Together, these results show that a JA-induced module regulates sorghum panicle development and spikelet fertility. The findings advance our understanding of inflorescence development and could lead to new strategies for increasing GNP and grain yield in sorghum and other cereal crops.SignificanceThrough a single base pair mutation, grain number can be increased by ~200% in the globally important crop Sorghum bicolor. This mutation affects the expression of an enzyme, MSD2, that catalyzes the jasmonic acid pathway in developing floral meristems. The global gene expression profile in this enzymatic mutant is similar to that of a transcription factor mutant, msd1, indicating that disturbing any component of this regulatory module disrupts a positive feedback loop that occurs normally due to regular developmental perception of jasmonic acid. Additionally, the MSD1 transcription factor is able to regulate MSD2 in addition to other jasmonic acid pathway genes, suggesting that it is a primary transcriptional regulator of this hormone signaling pathway in floral meristems.

The brown midrib (bmr) phenotype is a recessive trait of sorghum (Sorghum bicolor L. Moench) that results in overall lignin reduction and is associated with enhanced ruminant digestibility and increased feedstock to ethanol conversion efficiency. The three cloned Bmr loci encode genes involved in the monolignol biosynthetic pathway. There are eight known bmr loci in sorghum, but the two widely known are bmr6 and bmr12. A number of sorghum cultivars are referred to as bmr, but there is limited information as to their specific grouping, and it is important that new bmr lines in the pipeline can be easily genetically characterized. Hence, this study was performed to translate the genetic and DNA sequence information for bmr6 and bmr12, for use in the biomarker technology of single-nucleotide polymorphisms (SNPs). Through an analysis of variation for the two bmr genes, DNA markers in the form of cleave amplified polymorphic sequence (CAPS) and Kompetitive Allelele Specific Polymerase chain reaction (KASP) SNP markers were developed. The utility of the DNA markers for rapid and accurate identification of bmr6 or 12 individuals at the seedling stage were validated in a group of sorghum germplasm and a genetic population through marker-assisted selection. Two CAPS DNA markers (one each for bmr6 and 12) were evaluated and found to positively identify lines that harbor the different bmr genes. Fifteen KASP SNP markers (six for bmr6 and nine for bmr12) were developed and utilized for allelic discrimination and selection of bmr individuals at the seedling stage. The KASP marker bmr6-132 positively identified bmr6-ref allele among materials evaluated and individuals of segregating F2 population at the seedling stage. The KASP DNA marker bmr12-129, positively identified germplasm among materials evaluated and individuals of the F2 population at the seedling stage to select plants that carry the bmr12-ref allele . Results from this study also classified two sorghum germplasm as bmr12 and these lines can be utilized as specific donor of this bmr class using marker-assisted selection. This work demonstrated the successful translation and deployment of molecular information into specific CAPS and KASP DNA markers that are easy to access and can be applied in a breeding program, for use in efficient marker-assisted selection of bmr in sorghum.

Dissecting the genetic architecture of stress tolerance in crops is critical to understand and improve adaptation. In temperate climates, early planting of chilling-tolerant varieties could provide longer growing seasons and drought escape, but chilling tolerance ((Sorghum bicolor[L.] Moench). The NAM resource, developed from reference line BTx623 and three chilling-tolerant Chinese lines, is comprised of 771 recombinant inbred lines genotyped by sequencing at 43,320 single nucleotide polymorphisms. We phenotyped the NAM population for emergence, seedling vigor, and agronomic traits (>75,000 data points from ∼16,000 plots) in multi-environment field trials in Kansas under natural chilling stress (sown 30–45 days early) and normal growing conditions. Joint linkage mapping with early-planted field phenotypes revealed an oligogenic architecture, with 5–10 chilling tolerance loci explaining 20–41% of variation. Surprisingly, several of the major chilling tolerance loci co-localize precisely with the classical grain tannin (Tan1andTan2) and dwarfing genes (Dw1andDw3) that were under strong directional selection in the US during the 20th century. These findings suggest that chilling sensitivity was inadvertently selected due to coinheritance with desired nontannin and dwarfing alleles. The characterization of genetic architecture with NAM reveals why past chilling tolerance breeding was stymied and provides a path for genomics-enabled breeding of chilling tolerance.Article SummaryChilling sensitivity limits productivity of tropical-origin crops in temperate climates, and remains poorly understood at a genetic level. We developed a nested association mapping resource in sorghum, a tropical-origin cereal, to understand the genetic architecture of chilling tolerance. Linkage mapping of growth traits from early-planted field trials revealed several major chilling tolerance loci, including some colocalized with genes that were selected in the origin of US grain sorghum. These findings suggest chilling sensitivity was inadvertently selected during 20th century breeding, but can be bypassed using a better understanding of the underlying genetic architecture.DisclaimerMention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the USDA and does not imply approval or recommendation of the product to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.

To test the hypothesis that the cultivated peanut species possesses almost no molecular variability, we sequenced a diverse panel of 22 Arachis accessions representing Arachis hypogaea botanical classes, A-, B-, and K- genome diploids, a synthetic amphidiploid, and a tetraploid wild species. RNASeq was performed on pools of three tissues, and de novo assembly was performed. Realignment of individual accession reads to transcripts of the cultivar OLin identified 306,820 biallelic SNPs. Among 10 naturally occurring tetraploid accessions, 40,382 unique homozygous SNPs were identified in 14,719 contigs. In eight diploid accessions, 291,115 unique SNPs were identified in 26,320 contigs. The average SNP rate among the 10 cultivated tetraploids was 0.5, and among eight diploids was 9.2 per 1000 bp. Diversity analysis indicated grouping of diploids according to genome classification, and cultivated tetraploids by subspecies. Cluster analysis of variants indicated that sequences of B genome species were the most similar to the tetraploids, and the next closest diploid accession belonged to the A genome species. A subset of 66 SNPs selected from the dataset was validated; of 782 SNP calls, 636 (81.32%) were confirmed using an allele-specific discrimination assay. We conclude that substantial genetic variability exists among wild species. Additionally, significant but lesser variability at the molecular level occurs among accessions of the cultivated species. This survey is the first to report significant SNP level diversity among transcripts, and may explain some of the phenotypic differences observed in germplasm surveys. Understanding SNP variants in the Arachis accessions will benefit in developing markers for selection.

Marker assisted selection (MAS), an advance tool in plant breeding that allows accurate and efficient introgression of important agronomic trait(s) from a germplasm source to desired elite lines, has been applied to sorghum recently. Here, we report the methods for the deployment of MAS for trait introgression using endpoint genotyping technology for single nucleotide polymorphism (SNP)/insertion deletion (InDel) coupled with an application of KASP (Kompetitive Allele Specific Polymerase Chain Reaction [PCR]) chemistry allowing for the selection of parents for generational advancement without going through the laborious and time consuming phenotypic selection and additional generations for selection of desired individuals. This MAS-SNP marker assisted backcrossing scheme can be applied to accurately select heterozygotes for use as an allele donor parent in each backcross generation, thus expediting the backcrossing scheme and resulting in time savings of 3 years compared to conventional methods of introgression practiced in sorghum breeding and improvement.

Marker assisted selection (MAS), an advance tool in plant breeding that allows accurate and efficient introgression of important agronomic trait(s) from a germplasm source to desired elite lines, has been applied to sorghum recently. Here, we report the methods for the deployment of MAS for trait introgression using endpoint genotyping technology for single nucleotide polymorphism (SNP)/insertion deletion (InDel) coupled with an application of KASP (Kompetitive Allele Specific Polymerase Chain Reaction [PCR]) chemistry allowing for the selection of parents for generational advancement without going through the laborious and time consuming phenotypic selection and additional generations for selection of desired individuals. This MAS-SNP marker assisted backcrossing scheme can be applied to accurately select heterozygotes for use as an allele donor parent in each backcross generation, thus expediting the backcrossing scheme and resulting in time savings of 3 years compared to conventional methods of introgression practiced in sorghum breeding and improvement.

Two allelic sorghum [Sorghum bicolor (L.) Moench] erect leaf (erl) mutants were isolated from an Annotated Individually pedigreed Mutagenized Sorghum (AIMS) mutant library developed at the Plant Stress and Germplasm Development Unit at Lubbock, TX. The two mutants, erl1-1 (Reg. No. GS- 739, PI 673426) and erl1-2 (Reg. No. GS-740, PI 673427), were isolated from two independently pedigreed M3 families mutagenized with ethyl methanesulfonate in a prominent sorghum inbred line, BTx623, which was used to sequence the sorghum genome. Both mutants were backcrossed three times to the wild-type (WT) BTx623 and shown to be a stable recessive mutation on a nuclear gene. The F1 plants of a cross between the two mutants displayed the erect leaf phenotype, indicating the mutations that result in the erect leaf phenotype in the two mutants are allelic. Both mutants have upright and slightly short leaves and reduced height. The panicles from both mutants are smaller than WT even after three backcrosses. As a highly penetrant trait mediated by a single gene, these mutants may serve as a novel resource to design canopy architecture for optimizing radiation capture and use efficiency to increase biomass and grain yield.

Water-deficit stress responses in sorghum [ Sor-ghum bicolor (L.) Moench] have been described in the literature as preflowering drought toler-ant (postflowering senescent) or postflowering drought tolerant (preflowering drought sensi-tive). The underlying physiological mechanisms associated with these drought traits remain unclear. It was hypothesized that the preflower-ing drought sensitivity of stay-green lines could be related to reported differences in osmotic potential among stay-green and senescent lines resulting in an inability of the cultivars to either sense or respond the soil drying until the rate of drying is too great for the stay-green lines to compensate. The objective of this study was to measure stress-induced changes in rela-tive water content, abscisic acid (ABA), proline, dhurrin, sucrose, and carbon assimilation dur-ing the onset of water-deficit stress in the pre-flowering drought-tolerant line SC1211–11E and the postflowering drought-tolerant line BTx642 to determine if there were differential responses to the onset of soil drying. In both greenhouse and field studies, it was found that SC1211–11E had lower relative water contents and accumu-lated higher levels of ABA and proline than the BTx642. The SC1211–11E also showed increases in carbon assimilation shortly after the cessa-tion of irrigation that declined with prolonged stress. These results provide new insights into the differential responses of pre and postflower -ing drought-tolerant sorghum lines.USDA-ARS, Cropping Systems Research Lab., 3810 4th Street, Lub-bock, TX 79415. Mention of a trademark, warranty, proprietary prod-uct, or vendor does not constitute a guarantee by the USDA and does not imply approval or recommendation of the product to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer. Received 25 April 2014. *Corresponding author (john.burke@ars.usda.gov).

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.

Sorghum (Sorghum bicolor (L.) Moench) has branched panicles with primary, secondary, and tertiary branches. These in�耀orescence branches are composed of a terminal triplet spikelet, con- sisting of one sessile (directly attached to the branch) and two pedicellate (spikelets attached to the branch through a short pedicel), followed by one or more spikelet pairs (one sessile and one pedicellate). In BTx623, and most sorghum lines, only the sessile spikelets can develop into seeds and the pedicellate spikelets, occasion- ally developing anthers, eventually abort. Here, we report the isolation and characterization of a stable multiseeded (msd1) mutant of sorghum (in BTx623 background, referred to as wild-type (WT)) in which both sessile and pedicellate spike- lets are fertile. In addition, this mutant displayed increased length and total number of primary and secondary in�耀orescence branches. Genetic analysis showed that this multiseeded mutation is a recessive trait. Pedicellate spikelets in msd1 mutants exhibited complete �耀ower with func - tional ovary and anthers, of which 75 to 95% can develop into viable seeds. The individual msd1 seeds are smaller than WT; however, the results indicated that this reduction in seed weight can be compensated by the increase in seed num- ber. The total seed weight per panicle in msd1 mutants was increased by 30 to 40% as com- pared to the WT. Further experiments are needed to demonstrate whether the msd1 mutation can increase grain yield under �耀eld conditions. Plant Stress and Germplasm Development Unit, Cropping Systems Research Lab., USDA-ARS, 3810 4th St., Lubbock, TX 79415. Mention of trade names or commercial products in this publication is solely for the purpose of providing speci�耀c information and does not imply recommen - dation or endorsement by the U.S. Department of Agriculture. Received 26 Aug. 2013. *Corresponding author (zhanguo.xin@ars.usda.gov). Abbreviation: WT, wild-type.

Aim: The relationships between early -season cold temperature germination and pre flowering drought stress in eight grain sorghum inbreds were assessed using morpho physiological traits. Study Design: Field was laid out in a randomized complete block design. Place and Duration: The experiment was conducted at Vernon, Texas (34∫ 9'4"N 99∞17'26"W) during the month of April to August, 2013. Methodology: Lines were sown in the field in early April to deter mine early-season cold temperature germination. At the pre -booting stage, lines were subjected to limited and no irrigation treatments, to establish a correlation between cold temperature germination and pre-flowering drought stress tolerance. Pre -flowering drought tolerance was assessed at five defined phenological phases of the reproductive growth stage, using morpho physiological traits. Results:Final germination percent (FGP) differed among lines (range 30

Changes in plant architecture, specifically conversion to compact canopy for cereal crops, have resulted in significant increases in grain yield for wheat (Triticum aestivum) and rice (Oryza sativa). For sorghum (Sorghum bicolor L. Moench.) a versatile crop with an open canopy, plant architecture is an important feature that merits strong consideration for modification. Here, we report the genetic, developmental, and physiological characterization of a sorghum genetic stock, KFS2061, a stable mutant (in the Western Black Hull Kafir background) which exhibit short and erect leaves resulting in compact plant architecture. Genetic study of an F2 population derived from the cross of KFS2061 to BTx623 showed that the short leaf is recessive and appeared to be controlled by a single gene. The expression of the short leaf trait commenced with the 3rd leaf and is propagated through the entire leaf hierarchy of the canopy. The short leaf mutant exhibited consistent steep leaf angle, 43° (with the main culm as reference), and greener leaves than wild type. Biochemical analyses indicated significantly higher chlorophyll and cellulose content per leaf area in the mutant than wild type. Histological studies revealed reduction in cell length along the longitudinal axis and enlargement of bulliform cells in the adaxial surface of the mutant leaf. Further evaluation of agronomic traits indicated that this mutation could increase harvest index. This study provides information on a short leaf genetic stock that could serve as a vital resource in understanding how to manipulate plant canopy architecture of sorghum.

The presence of pre�耀owering or post�耀ower ing drought tolerance has been described in sorghum [Sorghum bicolor (L.) Moench]. Sorghum lines with pre�耀owering drought tolerance tend to senesce under post�耀owering drought stress while sorghum lines with post�耀owering drought tolerance (the stay-green trait) tend to be sensitive to pre�耀owering drought stress. Assessments of these phenotypes have been dependent on the incidence of drought stress conditions at speci�耀c developmental stages to achieve meaningful evaluation. Therefore, �耀eldbased evaluations, most of which are visual, are notoriously dif�耀cult to perform and require growing lines in multiple locations across several years to achieve ef�耀cient selection for these traits. Here we report and demonstrate a correlation between leaf dhurrin [(S)-p-hydroxymandelonitrile-C-D-glucopyranoside] contents and the level of stay-green based on leaf and plant death ratings during post�耀owering drought stresses. Post�耀owering drought-tolerant staygreen sorghums had elevated dhurrin contents while pre�耀owering drought-tolerant sorghum lines exhibited lower dhurrin contents. Lines with intermediate leaf and plant death ratings exhibited intermediate dhurrin levels. Finally, dhurrin levels determined before �耀owering from sorghum grown under irrigated or dryland conditions were related to the degree of pre- or post�耀owering drought tolerance.

Objective: Seedling cold tolerance is one of the important traits for sweet sorghum production. This study is to evaluate the method for identification of sweet sorghum accessions with seedling cold tolerance using both lab cold germination and field early-spring cold planting. Methodology: Sorghum seeds were germinated in growth chamber (under lab cold condition) at a constant 12°C for five weeks. After two weeks, germinated seeds and germination rates were counted and calculated at weekly intervals for four times. The same seed lots for lab cold test were also planted 45 days earlier than the normal planting time in the field (early-spring cold planting). In addition to seedling dry weight, germinated seeds and seed germination rates were also counted and calculated at weekly intervals for three times. Results: In this study, a high correlation coefficient between lab germination rate and field germination rate (R2=0.503, p

Background Climate variability due to fluctuation in temperature is a worldwide concern that imperils crop production. The need to understand how the germplasm variation in major crops can be utilized to aid in discovering and developing breeding lines that can withstand and adapt to temperature fluctuations is more necessary than ever. Here, we analyzed the genetic variation associated with responses to thermal stresses in a sorghum association panel (SAP) representing major races and working groups to identify single nucleotide polymorphisms (SNPs) that are associated with resilience to temperature stress in a major cereal crop. Results The SAP exhibited extensive variation for seedling traits under cold and heat stress. Genome-wide analyses identified 30 SNPs that were strongly associated with traits measured at seedling stage under cold stress and tagged genes that act as regulators of anthocyanin expression and soluble carbohydrate metabolism. Meanwhile, 12 SNPs were significantly associated with seedling traits under heat stress and these SNPs tagged genes that function in sugar metabolism, and ion transport pathways. Evaluation of co-expression networks for genes near the significantly associated SNPs indicated complex gene interactions for cold and heat stresses in sorghum. We focused and validated the expression of four genes in the network of Sb06g025040, a basic-helix-loop-helix (bHLH) transcription factor that was proposed to be involved in purple color pigmentation of leaf, and observed that genes in this network were upregulated during cold stress in a moderately tolerant line as compared to the more sensitive line. Conclusion This study facilitated the tagging of genome regions associated with variation in seedling traits of sorghum under cold and heat stress. These findings show the potential of genotype information for development of temperature resilient sorghum cultivars and further characterization of genes and their networks responsible for adaptation to thermal stresses. Knowledge on the gene networks from this research can be extended to the other cereal crops to better understand the genetic basis of resilience to temperature fluctuations during plant developmental stages. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0966-2) contains supplementary material, which is available to authorized users.

Sorghum (Sorghum bicolor) is a versatile C4 crop and a model for research in family Poaceae. High-quality genome sequence is available for the elite inbred line BTx623, but functional validation of genes remains challenging due to the limited genomic and germplasm resources available for comprehensive analysis of induced mutations. In this study, we generated 6400 pedigreed M4 mutant pools from EMS-mutagenized BTx623 seeds through single-seed descent. Whole-genome sequencing of 256 phenotyped mutant lines revealed >1.8 million canonical EMS-induced mutations, affecting >95% of genes in the sorghum genome. The vast majority (97.5%) of the induced mutations were distinct from natural variations. To demonstrate the utility of the sequenced sorghum mutant resource, we performed reverse genetics to identify eight genes potentially affecting drought tolerance, three of which had allelic mutations and two of which exhibited exact cosegregation with the phenotype of interest. Our results establish that a large-scale resource of sequenced pedigreed mutants provides an efficient platform for functional validation of genes in sorghum, thereby accelerating sorghum breeding. Moreover, findings made in sorghum could be readily translated to other members of the Poaceae via integrated genomics approaches.

This study presents results from a 2-year evaluation of biomass and cellulosic ethanol (EtOH) production potential of forage sorghum (Sorghum bicolor L. Moench) cultivars differing in brown midrib trait (i.e., bmr12) under dryland (no irrigation) and limited irrigation (2.88 mm day−1; subsurface drip) in the semiarid Southern High Plains of the USA. Commercial cultivar Sorghum Partners 1990 (SP 1990, conventional non-bmr) produced significantly more biomass (29–62 %) than a bmr12 cultivar PaceSetter bmr (PS bmr) under irrigated and dryland conditions during both years of this study. However, PS bmr biomass had higher cellulosic EtOH conversion efficiency than SP 1990 in both years according to simultaneous saccharification and fermentation analysis. Irrigation resulted in 26–49 % more biomass and 28–72 % more cellulosic EtOH production during both growing seasons, indicating that limited irrigation had favorable effects on both biomass and biofuel production. In the first year, when precipitation was below average, both cultivars produced similar amounts of cellulosic EtOH. During the second year, when precipitation was above average, higher biomass production of SP 1990 resulted in 28 % higher cellulosic EtOH production than PS bmr when averaged across both irrigated and dryland conditions. The large range of cellulosic EtOH production (1,600 to 3,380 L ha−1) during the 2 years of this study was primarily driven by differences in water availability that resulted from precipitation and irrigation. Our findings indicates that chemical composition and biomass yield potential of sorghum cultivars are critical factors that affect biomass and biofuel production under limited water conditions.

All rights reserved. No part of this periodical may be reproduced or trans mitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. ABSTRACT Genetic stock collections have played an important role in shaping breeding strategies in many crops. With the advance in genomics, they are now essential tools for identifying genes underlying important agronomic traits. The objective of this work is to formally register and document the release of historical sorghum [Sorghum bicolor (L.) Moench] genetic stocks originally collected and curated by the late USDA-ARS sorghum geneticist Keith Schertz. The seeds from the original collection were revitalized at the USDA-ARS Experiment Station at Lubbock, TX in 2004 and 2006, and evaluated for phenotypic variations in 2009. The collection consists of 507 lines and accessions (Reg. No. GS-144, PI 663217 through Reg. No. GS-650, PI 663723). This collection displays signifi cant variation in growth, developmental, and agronomic traits, such as seedling vigor, leaf/vegetative organs, reproduction, maturity, height, and grain quality. Combined with advanced genomic tools, these genetic stocks are vital resources for elucidating the fundamental pathways that make sorghum and other related grasses unusually tolerant to drought and heat stress, have moderate to low requirements for fertilizer, and highly productive under diverse environmental conditions.

Evaluation of biofuel production cropping systems should address not only energy yields but also the impacts on soil attributes. In this study, forage sorghum (Sorghum bicolor L. Moench) cropping systems were initiated on a low organic matter soil (

Genetic diversity was characterized in a collection of 159 sorghum (Sorghum bicolor L. Moench) landraces gathered from the colder region (primarily the northeastern region) of China. A set of 41 microsatellites or simple sequence repeat (SSR) markers distributed throughout the 10 chromosomes of sorghum was utilized in the analysis. Majority (40 out of 41 SSRs, 98%) of the SSR markers were polymorphic and highly informative with polymorphism information content (PIC) value ranging from 0.05 to 0.92. An estimation of genetic similarity coefficients (GSC, with values ranging from 0.42 to 0.96) revealed a range of variability in this collection of sorghum landraces. Eight clusters of accessions were identified at a cut off at 0.50 GSC and these groupings were supported by the analysis of subpopulations using Structure. This study provides evidence that Chinese landraces of sorghum exhibit moderate genetic diversity and results from cluster analysis may well aid in identification of diverse accessions that can serve as parental lines for efficient utilization and application of these germplasm into sorghum breeding programs.

Heat can be one of the major abiotic stresses that adversely affect crop production worldwide at different stages of development. As field screening for heat tolerance can be inconsistent and seasonally-limited, it is important to develop a reliable protocol under controlled conditions that allows simultaneous screening of multiple genotypes. The objective of this research was to develop a straightforward laboratory protocol using acquired thermotolerance (ATT) in peanut seedlings as a measure of one mechanism of heat stress tolerance. Sixteen genotypes, including selected accessions of the US peanut minicore collection along with standard checks, were evaluated for acquired themotolerance in two independent experiments. A change in the temperature sensitivity of chlorophyll accumulation was used as an indicator of acquired thermotolerance. Pre-incubation at 38°C for 4 h before the 30-min 50°C challenge triggered the acquired thermotolerance system of the leaf disks, resulting in chlorophyll accumulation upon exposure to light. There was considerable variation among genotypes for ATT in both experiments. Genotypic ranking for mean ATT values were highly correlated (0.949) in both experiments. The effect of seed weight on ATT was not significant. This method is relatively simple and inexpensive and can be used to screen a large number of genotypes.

The BTx623/IS3620C sorghum [Sorghum bicolor (L.) Moench.] mapping population (Reg. No. MP-1, NSL 469802 MAP) is a set of 430 F 7 and F 9 recombinant inbred lines (RILs) (F 7 lines: PI 658758 through PI 659060; F 9 lines: PI 659144 through PI 659271) from a cross between the inbred lines BTx623 (PI 659985 MAP) and IS3620C (PI 659986 MAP). This sorghum mapping population was released jointly by the Plant Stress and the Germplasm Development Unit, Cropping Systems Research Laboratory, USDA-ARS, Lubbock, TX and the Crop Germplasm Research Unit, USDA-ARS, College Station, TX in 2009. The original 137 RILs of this mapping population served as the basis for the construction of landmark high-density genetic and physical maps, with the integrated genome map subsequently used to aid in assembly of the sorghum genome sequence. This population has been genotyped by a number of investigators with a total of 3348 markers—including amplifi ed fragment length polymorphisms, DArT (Diversity Arrays Technology, Yarralumla, Australia), restriction fragment length polymorphisms, simple sequence repeats, and insertion-deletions—mapped to ten linkage groups corresponding to the ten sorghum chromosomes. The population has been utilized to map quantitative trait loci that underlie various agronomic traits, including tillering, fl oral architecture, plant height, fl owering date, grain size, and abiotic stress tolerance. The registration of this recombinant inbred mapping population will allow public access to the genomic and germplasm resources that can facilitate mapping and gene discovery for complex traits in sorghum and related cereal species.

Soil temperatures at 15°C or below limit germination and seedling establishment for warm season cereal crops such as sorghum (Sorghum bicolor (L.) Moench) during early-season planting. To better understand the genetics of early-season cold tolerance in sorghum, mapping of quantitative trait loci (QTL) associated with germination, emergence and vigor using a recombinant inbred mapping population was carried out. A mapping population consisting of 171 F7–F8 recombinant inbred lines (RILs) derived from the cross between RTx430 (cold-sensitive) and PI610727 (cold-tolerant) was developed and a genetic map was constructed using 141 microsatellites or simple sequence repeat (SSR) markers. The RILs were evaluated for cold and optimal temperature germinability in the laboratory, field emergence, and seedling vigor in two locations during early-season planting. Two or more QTL were detected for all traits, except for seedling vigor, with only one QTL was detected in the population. A QTL for cold germinability (Germ 12-2.1) showed the highest LOD value and was also associated with optimal germinability. One of the QTL for field emergence, Fearlygerm-9.3, a contribution from PI610727, was found significant in both locations used for the study. This study showed alignment of QTL in SBi1 (Fearlygerm-1.2 and FGerm30-1.2) with previously reported QTL associated with late field emergence identified from a different mapping population. This indicates that PI617027 shares some common loci with other known early-season cold-tolerant sorghum germplasm but also harbors novel QTL that could be useful in introgression of enhanced laboratory germination and early-season field emergence.

Cotton (Gossypium hirsutum L.) monoculture under conventional tillage has been the predominant cropping system in the Southern High Plains region of the United States since the 1940s. This study evaluated other cropping systems and land uses for their potential to increase soil quality and enhance soil functioning compared with continuous cotton (Ct-Ct), including a mixture of grasses in the Conservation Reserve Program (CRP), a pasture monoculture [Bothriochloa bladhii (Retz) S.T. Blake] and a cotton-winter wheat (Triticum aestivum L.)-corn (Zea mays L.) rotation (Ct-W-Cr). Soil microbial communities were evaluated according to microbial biomass C (MBC) and N (MBN), fatty acid methyl ester (FAME) profiling, and molecular cloning techniques. Soil MBC was higher under the alternative systems at 0 to 5 cm (CRP > pasture = Ct-W-Cr > Ct-Ct), 5 to 10 cm (CRP = Ct-W-Cr > pasture > Ct-Ct), and 10 to 20 cm (CRP = pasture = Ct-W-Cr > Ct-Ct). Soil DNA concentration was correlated with key soil quality parameters such as microbial biomass (r > 0.52, P < 0.05), total C (r = 0.372, P < 0.1), and total N (r = 0.449, P < 0.05). The 16S rRNA gene banding patterns (0-5 cm) of undisturbed systems (CRP and pasture) were more similar to each other than to Ct-Ct and Ct-W-Cr. Fungal/bacterial FAME ratios were higher under CRP and pasture than under Ct-Ct at 0 to 5 and 5 to 10 cm. This study found increases in sensitive soil quality parameters under alternative management compared with cotton monoculture.

Post-fl owering drought tolerance is an essential trait for increasing the production of sorghum [Sorghum bicolor (L.) Moench] and other cereals in Mediterranean and semiarid tropical climates. Current methodologies for identifying the nonsenescent (stay-green) trait require the right intensity of drought stress at the right developmental stage to visually evaluate lines in the fi eld. Field-based evaluations of drought tolerance are notoriously diffi cult to manage, and oft en require growing lines in multiple locations across several years to acquire a meaningful assessment of the stay-green trait. By means of a 30-min high-temperature challenge to leaf tissue during fl owering of well-watered sorghum and a 30-min room temperature recovery, we show that stay-green lines can be readily identifi ed. Using chlorophyll fl uorescence to monitor tissue injury, we found that tissue with higher intercellular sucrose concentrations exhibited higher chlorophyll fl uorescence yield following the temperature challenge. Stay-green lines evaluated in this study maintained higher dawn leaf sucrose levels than the senescent lines among the fi ve youngest leaf positions. Evaluation of 10 known stay-green and senescent sorghum lines, previously reported in the literature, with this bioassay allowed us to separate the two classes of sorghum from well-watered fl owering plants. Th e stay-green lines can also be separated from senescent lines under well-watered greenhouse conditions from the boot stage onward. Th is technology will greatly reduce the selection time needed to identify drought tolerant sorghum.

Peanut genotypes from the US mini-core collection were analysed for changes in leaf proteins during reproductive stage growth under water-deficit stress. One- and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected tolerant and susceptible genotypes. A total of 102 protein bands/spots were analysed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine non-redundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1l-myo-inositol-1-phosphate synthase, which aid in inter- and intracellular stress signalling, were more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis, increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. Additionally, there was a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of water-deficit stress tolerance in peanut.

The sorghum [Sorghum bicolor (L.) Moench] inbred line BTx623 has served as a parent for development of several mapping populations, also providing a source for the generation of DNA libraries for physical mapping, and as the inbred line selected for sorghum genome sequencing. Since genetic mapping, physical mapping and genome sequencing are all based on the same inbred line, these genetic resources have made the genome study of sorghum very efficient. However, in comparison with other model species, there is one important genetic resource still missing in the sorghum research community, a mutant population. A systematically annotated mutant population will facilitate many avenues of research, especially those focusing on functional genomics and bioenergy research. Here we report the generation of a sorghum mutant population derived from the inbred line BTx623 by treatment with the chemical agent ethyl methanesulfonate (EMS). The mutant population consists of 1,600 pedigreed M3 families; each of them was derived from an independent M1 seed. Many lines displayed traits such as brown midrib (bmr), erect leaves (erl), multiple tillers (mtl), and late flowering (lfl), characteristics useful for bioenergy research. Results from our phenotyping and genotyping studies indicate that this mutant population will be a valuable and useful genetic resource for both sorghum functional genomics and bioenergy research.

Sorghum is distinct from other cereal crops due to its ability to produce profuse amount of epicuticular wax (EW or bloom) on its culm and leaves along with less permeable cuticle which are considered to be important traits contributing to abiotic stress tolerance. Here, we report the molecular mapping and characterization of BL OO M-C UTICLE (BLMC), a locus associated with production of profuse wax, using a mutant mapping population developed from a cross between BTx623 (wild type with profuse wax) and KFS2021 (a mutant with greatly reduced wax). The F2 progenies were genotyped using known and newly developed microsatellite markers to establish a molecular map of BLMC. The locus mapped to a 3.6-centimorgans (cM) interval in the terminal end of sorghum chromosome 10 with flanking markers Xsbarslbk10.47 and Xcup42. Targeted mapping delimited BLMC to as small as 0.7 cM region and facilitated identification of three cosegregating markers with the trait. The BLMC region corresponds to approximately 153,000 bp and candidate genes identified include among others an acyl CoA oxidase (a gene involved in lipid and wax biosynthesis) and seven other putative transcripts. Phenotypic characterization showed that in addition to disrupting the EW production, BLMC mutation reduced culm and leaf cuticle, increased plant death rating in the field at anthesis and significantly reduced the C:28 to C:30 free fatty acid fractions of culm and leaf EW. These results clearly support the important role of BLMC in the expression of profuse wax and enhanced cuticular features of sorghum. Genetic mapping of BLMC opened avenues for identification of genes involved in the cuticle/wax pathway of sorghum and their application for improvement of abiotic stress tolerance.

In peanut hybridization, distinguishing inadvertent selfs from the true hybrids may be difficult. In this study, to differentiate between selfs and hybrids, DNA was extracted from leaf tissue of F1 or F2 plants, and SSR markers were amplified and bands separated by a novel submarine horizontal polyacrylamide gel electrophoresis (H-PAGE). By comparing the resulting banding patterns to those of the parents, 70% of the putative hybrids were shown to be true hybrids on the basis of possessing a marker allele from the male parent. The H-PAGE gels gave better band separation and differentiation of selfed progenies than agarose gels, and were compatible with the common horizontal agarose gel units. This method provides a quick assay to distinguish hybrids from inadvertent selfs, and should result in greater efficiency and more effective use of resources in peanut breeding programs.

An irradiation-induced bloomless mutant of sorghum [Sorghum bicolor (L.) Moench.], KFS2021, which visually exhibits an absence of white fl uffy epicuticular wax in leaf and sheath, was characterized using a combination of genetic and physiological approaches. Study of the phenotypic segregation for the bloomless trait in F 2 and F 2:3 populations from a cross between KFS2021 and BTx623 (a cultivar with bloom showing profuse deposition of white epicuticular wax) suggests that bloomless is controlled by a single nuclear recessive gene. The bloomless parent (KFS2021) and F 2 individuals had lower frequency of guttation, leakier epidermal layer (based on percentage of chlorophyll leaching), and higher rate of seedling water loss than the BTx623 and F 2 bloom individuals. Bloomless F 2 individuals showed 3- to 6-fold higher nighttime transpiration rates relative to F 2 bloom individuals based on nighttime conductance. Correlation analysis showed signifi cant negative associations between leaf epicuticular wax load with epidermal permeability and nighttime conductance, which indicate the important role of epicuticular wax in these traits. These results suggest that epicuticular wax may enhance water use effi ciency of sorghum by regulating nighttime water loss.

Sorghum is a versatile crop used for food, feed, fodder, and biofuel. Because of its resilience to environmental stresses and low soil fertility, sorghum is becoming increasingly important in meeting the growing need for food and energy in the face of declining arable land and fresh water resources. Low grain yield and poor nutritional quality are two major limiting factors for sorghum production. New resources are needed to overcome these limits. We have developed a pedigreed mutant library in BTx623, the inbred line for generating the reference genome sequence. The library consists of 6400 M4 seed pools, each of which was derived from an independent M1 seed through singleseed descent. The mutant library displays a wide range of visible phenotypes, and many are beneficial traits that have potential to be used as breeding materials to improve grain yield and quality. A selection of 256 lines was sequenced to 16x coverage of the whole genome. More than 1.8 million canonical ethyl methanesulfonate (EMS)-induced guanine/cytosine to adenosine/thymine single nucleotide polymorphisms (SNPs) were discovered, covering 94% of the genes in the sorghum genome. Over 57% of the genes carry large effect mutations that may alter the function of the gene product. In comparison with natural variations, 97.5% of the EMS-induced mutations are novel. Thus, the pedigreed mutant library can serve as a unique resource for selection of novel agronomic traits and identifying their causal mutations for sorghum improvement and for elucidating gene function through isolation of mutant series for the traits of interest.