Sorghum Chromosomes Research Articles

Genetic loci associated with sorghum drought tolerance in multiple environments and their sensitivity to environmental covariables.

Climate change can limit yields of naturally resilient crops, like sorghum, challenging global food security. Agriculture under an erratic climate requires tapping into a reservoir of flexible adaptive loci that can lead to lasting yield stability under multiple abiotic stress conditions. Domesticated in the hot and dry regions of Africa, sorghum is considered a harsh crop, which is adapted to important stress factors closely related to climate change. To investigate the genetic basis of drought stress adaptation in sorghum, we used a multi-environment multi-locus genome-wide association study (MEML-GWAS) in a subset of a diverse sorghum association panel (SAP) phenotyped for performance both under well-watered and water stress conditions. We selected environments in Brazil that foreshadow agriculture where both drought and temperature stresses coincide as in many tropical agricultural frontiers. Drought reduced average grain yield (Gy) by up to 50% and also affected flowering time (Ft) and plant height (Ph). We found 15 markers associated with Gy on all sorghum chromosomes except for chromosomes 7 and 9, in addition to loci associated with phenology traits. Loci associated with Gy strongly interacted with the environment in a complex way, while loci associated with phenology traits were less affected by G × E. Studying environmental covariables potentially underpinning G × E, increases in relative humidity and evapotranspiration favored and disfavored grain yield, respectively. High temperatures influenced G × E and reduced sorghum yields, with a ~ 100kgha-1 average decrease in grain yield for each unit increase in maximum temperature between 29 and 38°C. Extreme G × E for sorghum stress resilience poses an additional challenge to breed crops for moving, erratic weather conditions.

Genomic Identification of Callose Synthase (CalS) Gene Family in Sorghum (Sorghum bicolor) and Comparative In Silico Expression Analysis under Aphid (Melanaphis sacchari) Infestation

Callose is widely present in higher plants and plays a significant role in plant growth, development, and response to various stresses. Although numerous studies have highlighted the importance of the callose synthase (CalS) genes, their role in the resistance of sorghum (Sorghum bicolor) to aphids (Melanaphis sacchari) remains limitedly understood. This study identified 11 sorghum callose synthase genes (SbCalS), unevenly distributed across four chromosomes of sorghum. All SbCalS proteins contain glucan synthase and Fks1 domains, with segmental duplication playing a major role in gene diversification. Cis-element prediction revealed the presence of numerous stress-responsive elements, indicating that this gene family is primarily involved in stress resistance. Using published RNA-seq data, we discovered the differential expression of the SbCalS5 gene between resistant and susceptible sorghum varieties. Real-time quantitative PCR (qPCR) analysis confirmed the relative expression levels of all SbCalS members under aphid stress. To further verify the role of callose in sorghum, we measured the callose content in both resistant and susceptible sorghum varieties. The results indicated that callose plays a critical role in aphid resistance in sorghum, particularly the SbCalS5 gene. This study provides a reference for further investigation into the role of callose synthase genes in sorghum aphid resistance.

Improving digestibility of sorghum proteins by CRISPR/Cas9‐based genome editing

AbstractKafirins are the primary storage proteins in sorghum kernels that provide amino acids for seed germination. The highly proteolytic resistant γ‐ and β‐kafirins form the cross‐linked outer layers that encapsulate α‐kafirins to generate protein bodies, resulting in poor digestibility of sorghum grains. The sorghum kafirins thus contribute to the poor quality of the kernels. The nutritional quality and digestibility of sorghum grains can be improved by reducing the contents of kafirin. γ‐Kafirin is encoded by the K2G gene, which is located on sorghum chromosome 2. In this study, we used CRISPR/Cas9 gene editing to target the K2G gene to create new sorghum lines with reduced levels of kafirin. A guide RNA (sgRNA) was designed to introduce mutations in the CDS region that encodes the endoplasmic reticulum signal peptide of γ‐kafirin. The pK2GsgRNA/Cas9 vector was transformed into sorghum using the pollen‐mediated transformation method. Sequencing of the transformants showed that three out of 24 transgenic plants contain genetic mutations in the targeted region. Compared with the wildtype, the γ‐kafirin contents of the mutant plants decreased by 12.75%–19.22%, and the protein digestibility of the mutant kernels increased by 26.91%–74.31% in raw flour. Although the grain weights remained comparable to those of the wildtype, the growth of the mutant plants was more vigorous as the mutant shoots grew taller and thicker compared with those of the wildtype. Our work advances the ability to improve the digestibility of an important crop. The resulting quality improvements can also be rapidly deployed for breeding and generation of transgene‐free, improved cultivars of sorghum, a major crop worldwide.

Genome-wide identification and expression analysis of the U-box E3 ubiquitin ligase gene family related to salt tolerance in sorghum (Sorghum bicolor L.).

Plant U-box (PUB) E3 ubiquitin ligases play essential roles in many biological processes and stress responses, but little is known about their functions in sorghum (Sorghum bicolor L.). In the present study, 59 SbPUB genes were identified in the sorghum genome. Based on the phylogenetic analysis, the 59 SbPUB genes were clustered into five groups, which were also supported by the conserved motifs and structures of these genes. SbPUB genes were found to be unevenly distributed on the 10 chromosomes of sorghum. Most PUB genes (16) were found on chromosome 4, but there were no PUB genes on chromosome 5. Analysis of cis-acting elements showed that SbPUB genes were involved in many important biological processes, particularly in response to salt stress. From proteomic and transcriptomic data, we found that several SbPUB genes had diverse expressions under different salt treatments. To verify the expression of SbPUBs, qRT-PCR analyses also were conducted under salt stress, and the result was consistent with the expression analysis. Furthermore, 12 SbPUB genes were found to contain MYB-related elements, which are important regulators of flavonoid biosynthesis. These results, which were consistent with our previous multi-omics analysis of sorghum salt stress, laid a solid foundation for further mechanistic study of salt tolerance in sorghum. Our study showed that PUB genes play a crucial role in regulating salt stress, and might serve as promising targets for the breeding of salt-tolerant sorghum in the future.

Construction of an ultra-high-density consensus genetic map and analysis of recombination rate variation in Sorghum bicolor

Abstract. Satrio RD, Nikmah IA, Fendiyanto MH, Pratami MP, Awwanah M, Sari NIP, Farah N, Nurhadiyanta. 2022. Construction of an ultra-high-density consensus genetic map and analysis of recombination rate variation in Sorghum bicolor. Asian J Agric 6: 47-54. Sorghum is one of the most widely grown cereal crops on a global scale. A consensus map is a method for combining genetic information from multiple populations, and it is an effective way to increase genome coverage and marker density. This study constructed a consensus map by combining publicly available marker data from four mapping populations. A total of 3449 non-redundant polymorphic markers at the nucleotide level were used to construct a single consensus map on 10 sorghum chromosomes. This study generated an ultra-high-density sorghum consensus map consisting of a large number of markers spanning 1571.68 cM and averaging one marker per 0.46 cM. Due to the high density of the markers, it is only 0.06% of the markers had an interval greater than 5 cM. The rates of local recombination were estimated using a set of all markers genetic and physical positions along each of the 10 chromosomes. The analysis of the recombination rate on 10 sorghum chromosomes revealed that it decreased as the centromere position was getting closer. The consensus map generated in this study can be used to integrate information related to sorghum genetic resources and QTLs to the genome sequence, thereby accelerating the discovery of novel potential genes in sorghum.

Fine mapping of <i>Rf5</i> region for a sorghum fertility restorer gene and microsynteny analysis across grass species

Cytoplasmic male sterility (CMS) is widely used to control pollination in the production of commercial F1 hybrid seed in sorghum. So far, 6 major fertility restorer genes, Rf1 to Rf6, have been reported in sorghum. Here, we fine-mapped the Rf5 locus on sorghum chromosome 5 using descendant populations of a ‘Nakei MS-3A’ × ‘JN43’ cross. The Rf5 locus was narrowed to a 140-kb region in BTx623 genome (161-kb in JN43) with 16 predicted genes, including 6 homologous to the rice fertility restorer Rf1 (PPR.1 to PPR.6). These 6 homologs have tandem pentatricopeptide repeat (PPR) motifs. Many Rf genes encode PPR proteins, which bind RNA transcripts and modulate gene expression at the RNA level. No PPR genes were detected at the Rf5 locus on the corresponding homologous chromosome of rice, foxtail millet, or maize, so this gene cluster may have originated by chromosome translocation and duplication after the divergence of sorghum from these species. Comparison of the sequences of these genes between fertile and CMS lines identified PPR.4 as the most plausible candidate gene for Rf5.

QTL mapping in an interspecific sorghum population uncovers candidate regulators of salinity tolerance

Salt stress impedes plant growth and disrupts normal metabolic processes, resulting in decreased biomass and increased leaf senescence. Therefore, the ability of a plant to maintain biomass when exposed to salinity stress is critical for the production of salt tolerant crops. To identify the genetic basis of salt tolerance in an agronomically important grain crop, we used a recombinant inbred line (RIL) population derived from an interspecific cross between domesticated Sorghum bicolor (inbred Tx7000) and a wild relative, Sorghum propinquum. A high-density genetic map was generated from 177 F3:5 RILs and coveres the 10 Sorghum chromosomes with 1991 markers. The genetic map was used to identify 19 total QTL related to plant growth and overall health in optimal and saline conditions. Of these 19 QTL detected, 10 were specific to the salt stress response. The salt-responsive QTL contained numerous genes that have been previously shown to play a role in ionic tolerance, tissue tolerance, and osmotic tolerance, including many aquaporins.

BADH1 is associated with fragrance in sorghum (Sorghum bicolor (L.) Moench) cultivar ‘Ambemohor’

Aroma is an important trait that can enhance the product value in several crops. Pandan-like fragrance resulting from accumulation of 2-acetyl-1-pyrroline (2AP) is one of the pleasant aromas in food crops which is caused by null or missense mutations in betaine aldehyde dehydrogenase 2 (BADH2) gene. In addition, betaine aldehyde aehydrogenase 1 (BADH1) has shown to be associated with aroma in rice. In this study, we investigated the genetics controlling coconut juice-like fragrance in inflorescence of sorghum cultivar 'Ambemohor'. 2AP analysis in seeds revealed that Ambemohor possessed no 2AP. An F2 population developed from the cross between Ambemohor x KU630 (nonfragrant) segregated into a ratio of 3 (fragrant) : 1 (nonfragrant), suggesting that the coconut juice-like fragrance in Ambemohor is controlled by a single dominant gene, designated 'Aro'. Bulked segregant analysis suggested that the gene controlling fragrance in Ambemohor is located on sorghum chromosome 6. Quantitative trait locus (QTL) analysis identified a major QTL, (qAro6.1, for the fragrance located on chromosome 6 between markers SB3567 and SB3570. Bioinformatics analysis revealed that SB3567 and SB3570 were 217.8 kb apart and there were 29 annotated genes in this region including BADH1. Sequence analysis revealed that BADH1 sequences in Ambemohor and KU630 differed in size, but their coding sequences (CDS) were of same size. CDS alignment revealed four single-nucleotide polymorphisms (SNPs) between Ambemohor and KU630 in which two SNPs caused amino change in BADH1 of Ambemohor. These results suggested that BADH1 is a candidate gene for the coconut juice-like fragrance in Ambemohor.

Genome-Wide Association Study for Biomass Related Traits in a Panel of Sorghum bicolor and S. bicolor × S. halepense Populations.

The efficient use of sorghum as a renewable energy source requires high biomass yields and reduced agricultural inputs. Hybridization of Sorghum bicolor with wild Sorghum halepense can help meet both requirements, generating high-yielding and environment friendly perennial sorghum cultivars. Selection efficiency, however, needs to be improved to exploit the genetic potential of the derived recombinant lines and remove weedy and other wild traits. In this work, we present the results from a Genome-Wide Association Study conducted on a diversity panel made up of S. bicolor and an advanced population derived from S. bicolor × S. halepense multi-parent crosses. The objective was to identify genetic loci controlling biomass yield and biomass-relevant traits for breeding purposes. Plants were phenotyped during four consecutive years for dry biomass yield, dry mass fraction of fresh material, plant height and plant maturity. A genotyping-by-sequencing approach was implemented to obtain 92,383 high quality SNP markers used in this work. Significant marker-trait associations were uncovered across eight of the ten sorghum chromosomes, with two main hotspots near the end of chromosomes 7 and 9, in proximity of dwarfing genes Dw1 and Dw3. No significant marker was found on chromosomes 2 and 4. A large number of significant marker loci associated with biomass yield and biomass-relevant traits showed minor effects on respective plant characteristics, with the exception of seven loci on chromosomes 3, 8, and 9 that explained 5.2–7.8% of phenotypic variability in dry mass yield, dry mass fraction of fresh material, and maturity, and a major effect (R2 = 16.2%) locus on chromosome 1 for dry mass fraction of fresh material which co-localized with a zinc-finger homeodomain protein possibly involved in the expression of the D (Dry stalk) locus. These markers and marker haplotypes identified in this work are expected to boost marker-assisted selection in sorghum breeding.

Genome-wide Identification of WRKY transcription factor family members in sorghum (Sorghum bicolor (L.) moench).

WRKY transcription factors regulate diverse biological processes in plants, including abiotic and biotic stress responses, and constitute one of the largest transcription factor families in higher plants. Although the past decade has seen significant progress towards identifying and functionally characterizing WRKY genes in diverse species, little is known about the WRKY family in sorghum (Sorghum bicolor (L.) moench). Here we report the comprehensive identification of 94 putative WRKY transcription factors (SbWRKYs). The SbWRKYs were divided into three groups (I, II, and III), with those in group II further classified into five subgroups (IIa-IIe), based on their conserved domains and zinc finger motif types. WRKYs from the model plant Arabidopsis (Arabidopsis thaliana) were used for the phylogenetic analysis of all SbWRKY genes. Motif analysis showed that all SbWRKYs contained either one or two WRKY domains and that SbWRKYs within the same group had similar motif compositions. SbWRKY genes were located on all 10 sorghum chromosomes, and some gene clusters and two tandem duplications were detected. SbWRKY gene structure analysis showed that they contained 0-7 introns, with most SbWRKY genes consisting of two introns and three exons. Gene ontology (GO) annotation functionally categorized SbWRKYs under cellular components, molecular functions and biological processes. A cis-element analysis showed that all SbWRKYs contain at least one stress response-related cis-element. We exploited publicly available microarray datasets to analyze the expression profiles of 78 SbWRKY genes at different growth stages and in different tissues. The induction of SbWRKYs by different abiotic stresses hinted at their potential involvement in stress responses. qRT-PCR analysis revealed different expression patterns for SbWRKYs during drought stress. Functionally characterized WRKY genes in Arabidopsis and other species will provide clues for the functional characterization of putative orthologs in sorghum. Thus, the present study delivers a solid foundation for future functional studies of SbWRKY genes and their roles in the response to critical stresses such as drought.

Transmission Genetics of a Sorghum bicolor × S. halepense Backcross Populations.

Despite a “ploidy barrier,” interspecific crosses to wild and/or cultivated sorghum (Sorghum bicolor, 2n = 2x = 20) may have aided the spread across six continents of Sorghum halepense, also exemplifying risks of “transgene escape” from crops that could make weeds more difficult to control. Genetic maps of two BC1F1 populations derived from crosses of S. bicolor (sorghum) and S. halepense with totals of 722 and 795 single nucleotide polymorphism (SNP) markers span 37 and 35 linkage groups, with 2–6 for each of the 10 basic sorghum chromosomes due to fragments covering different chromosomal portions or independent segregation from different S. halepense homologs. Segregation distortion favored S. halepense alleles on chromosomes 2 (1.06–4.68 Mb, near a fertility restoration gene), 7 (1.20–6.16 Mb), 8 (1.81–5.33 Mb, associated with gene conversion), and 9 (47.5–50.1 Mb); and S. bicolor alleles on chromosome 6 (0–40 Mb), which contains both a large heterochromatin block and the Ma1 gene. Regions of the S. halepense genome that are recalcitrant to gene flow from sorghum might be exploited as part a multi-component system to reduce the likelihood of spread of transgenes or other modified genes. Its SNP profile suggests that chromosome segments from its respective progenitors S. bicolor and Sorghum propinquum have extensively recombined in S. halepense. This study reveals genomic regions that might discourage crop-to-weed gene escape, and provides a foundation for marker-trait association analysis to determine the genetic control of traits contributing to weediness, invasiveness, and perenniality of S. halepense.

Tissue-specific gene expression and protein abundance patterns are associated with fractionation bias in maize

BackgroundMaize experienced a whole-genome duplication event approximately 5 to 12 million years ago. Because this event occurred after speciation from sorghum, the pre-duplication subgenomes can be partially reconstructed by mapping syntenic regions to the sorghum chromosomes. During evolution, maize has had uneven gene loss between each ancient subgenome. Fractionation and divergence between these genomes continue today, constantly changing genetic make-up and phenotypes and influencing agronomic traits.ResultsHere we regenerate the subgenome reconstructions for the most recent maize reference genome assembly. Based on both expression and abundance data for homeologous gene pairs across multiple tissues, we observed functional divergence of genes across subgenomes. Although the genes in the larger maize subgenome are often expressing more highly than their homeologs in the smaller subgenome, we observed cases where homeolog expression dominance switches in different tissues. We demonstrate for the first time that protein abundances are higher in the larger subgenome, but they also show tissue-specific dominance, a pattern similar to RNA expression dominance. We also find that pollen expression is uniquely decoupled from protein abundance.ConclusionOur study shows that the larger subgenome has a greater range of functional assignments and that there is a relative lack of overlap between the subgenomes in terms of gene functions than would be suggested by similar patterns of gene expression and protein abundance. Our study also revealed that some reactions are catalyzed uniquely by the larger and smaller subgenomes. The tissue-specific, nonequivalent expression-level dominance pattern observed here implies a change in regulatory control which favors differentiated selective pressure on the retained duplicates leading to eventual change in gene functions.

High Density Genetic Maps of Seashore Paspalum Using Genotyping-By-Sequencing and Their Relationship to The Sorghum Bicolor Genome

As a step towards trait mapping in the halophyte seashore paspalum (Paspalum vaginatum Sw.), we developed an F1 mapping population from a cross between two genetically diverse and heterozygous accessions, 509022 and HI33. Progeny were genotyped using a genotyping-by-sequencing (GBS) approach and sequence reads were analyzed for single nucleotide polymorphisms (SNPs) using the UGbS-Flex pipeline. More markers were identified that segregated in the maternal parent (HA maps) compared to the paternal parent (AH maps), suggesting that 509022 had overall higher levels of heterozygosity than HI33. We also generated maps that consisted of markers that were heterozygous in both parents (HH maps). The AH, HA and HH maps each comprised more than 1000 markers. Markers formed 10 linkage groups, corresponding to the ten seashore paspalum chromosomes. Comparative analyses showed that each seashore paspalum chromosome was syntenic to and highly colinear with a single sorghum chromosome. Four inversions were identified, two of which were sorghum-specific while the other two were likely specific to seashore paspalum. These high-density maps are the first available genetic maps for seashore paspalum. The maps will provide a valuable tool for plant breeders and others in the Paspalum community to identify traits of interest, including salt tolerance.

A Novel QTL for Root-Knot Nematode Resistance is Identified from a South African Sweet Sorghum Line.

Southern root-knot nematodes, Meloidogyne incognita, feed on the underground portions of hundreds of plant species and affect nutrient partitioning and water uptake of the host plants. Sorghum (Sorghum bicolor) is often not significantly damaged by southern root-knot nematodes (RKN) but some sorghum genotypes support greater population densities of RKN than other genotypes. These higher nematode populations increase the risk of damage to subsequently planted susceptible crops. A previous study identified a major quantitative trait locus (QTL) for RKN resistance on sorghum chromosome (chr.) 3. To maintain long-term resistance, multiple resistance genes should be pyramided in a cultivar. In this study, we identified a new source of RKN resistance, created a mapping population, and identified single-nucleotide polymorphism markers using genotyping-by-sequencing of the segregating population. Use of single-marker analysis and composite interval mapping identified a single QTL on chr. 5 that was associated with egg number and egg number per gram of root from the resistant sweet sorghum line PI 144134. This region on chr. 5 and the prior QTL on chr. 3 can be potentially moved from PI 144134 and Honey Drip, respectively, into elite sorghum germplasm via marker-assisted selection for more durable resistance.

Colinearity of putative flowering gene in both sugarcane and sorghum

Sugarcane (Saccharum spp.) and sorghum (Sorghum spp.) have become increasingly important crops for biofuels production. Sugarcane has an autopolyploid complex genome, whereas sorghum has a diploid simple genome. Flowering is one of the sugar-related agronomic traits in both species. Here, we obtained cDNA of inflorescences at 0–15 cm from S. spontaneum using cDNA-amplified restriction fragment length polymorphisms to develop flower transcriptome profiling with 26 primer combinations. A total of 183 transcript-derived fragments (TDFs) were screened and 96 TDFs were sequenced. Out of 96 TDFs, 26 were selected as putative flowering genes to study collinearity with the sorghum genome. For gene collinearity, a genetic linkage map with 169 SSR co-dominant markers and 12 TDF marker loci were mapped to 14 linkage groups collectively spanning 1077.8 cM and corresponding to the 10 sorghum chromosomes. Interestingly, 9 TDF marker loci could be mapped to 5 linkage groups. In this study, we successfully identified the homologous location of sugarcane flowering TDFs in the sorghum genome and found that 4DS_1X and 2DS_3E TDFs may serve as candidate specific markers linked to flowering in both sorghum and sugarcane.

Recent polyploidization events in three Saccharum founding species.

SummaryThe complexity of polyploid Saccharum genomes hindered progress of genome research and crop improvement in sugarcane. To understand their genome structure, transcriptomes of 59 F1 individuals derived from S. officinarum LA Purple and S. robustum Molokai 5829 (2n = 80, x = 10 for both) were sequenced, yielding 11 157 and 8998 SNPs and 83 and 105 linkage groups, respectively. Most markers in each linkage group aligned to single sorghum chromosome. However, 71 interchromosomal rearrangements were detected between sorghum and S. officinarum or S. robustum, and 24 (33.8%) of them were shared between S. officinarum and S. robustum, indicating their occurrence before the speciation event that separated these two species. More than 2000 gene pairs from S. spontaneum, S. officinarum and S. robustum were analysed to estimate their divergence time. Saccharum officinarum and S. robustum diverged about 385 thousand years ago, and the whole‐genome duplication events occurred after the speciation event because of shared interchromosomal rearrangements. The ancestor of these two species diverged from S. spontaneum about 769 thousand years ago, and the reduction in basic chromosome number from 10 to 8 in S. spontaneum occurred after the speciation event but before the two rounds of whole‐genome duplication. Our results proved that S. officinarum is a legitimate species in its own right and not a selection from S. robustum during the domestication process in the past 10 000 years. Our findings rejected a long‐standing hypothesis and clarified the timing of speciation and whole‐genome duplication events in Saccharum.

Development and Applications of Chromosome-Specific Cytogenetic BAC-FISH Probes in S. spontaneum.

Saccharum spontaneum is a major Saccharum species that contributed to the origin of modern sugarcane cultivars, and due to a high degree of polyploidy is considered to be a plant species with one of the most complex genetics. Fluorescence in situ hybridization (FISH) is a powerful and widely used tool in genome studies. Here, we demonstrated that FISH based on bacterial artificial chromosome (BAC) clones can be used as a specific cytological marker to identify S. spontaneum individual chromosomes and study the relationship between S. spontaneum and other related species. We screened low-copy BACs as probes from the sequences of a high coverage of S. spontaneum BAC library based on BLAST search of the sorghum genome. In total, we isolated 49 positive BAC clones, and identified 27 BAC clones that can give specific signals on the S. spontaneum chromosomes. Of the 27 BAC probes, 18 were confirmed to be able to discriminate the eight basic chromosomes of S. spontaneum. Moreover, BAC-24, BAC-66, BAC-78, BAC-69, BAC-71, BAC-73, and BAC-77 probes were used to construct physical maps of chromosome 1 and chromosome 2 of S. spontaneum, which indicated synteny in Sb01 between S. spontaneum and sorghum. Furthermore, we found that BAC-14 and BAC-19 probes, corresponding to the sorghum chromosomes 2 and 8, respectively, localized to different arms of the same S. spontaneum chromosome, suggesting that there was an inter-chromosomal rearrangement event between S. spontaneum and sorghum. Our study provides the first set of chromosome-specific cytogenetic markers in Saccharum and is critical for future advances in cytogenetics and genome sequencing studies in Saccharum.

Genome-Wide Identification and Expression Profiling of Ascorbate Peroxidase (APX) and Glutathione Peroxidase (GPX) Genes Under Drought Stress in Sorghum (Sorghum bicolor L.)

APX and GPX are two crucial plant antioxidant enzymes. By protein homology search, nine APX and seven GPX members were identified in Sorghum bicolor genome. They were annotated based on chromosomal localizations as SbAPX1–9 and SbGPX1–7. APXs were distributed on six Sorghum chromosomes and encoded polypeptides of 250–474 residues with characteristic “peroxidase” domain, whereas GPXs were on five chromosomes and encoded proteins of 136–232 residues characterized by a “GSHPx” domain. The first about 1–90 amino acid residues in SbAPXs and about 60–70 amino acid residues in SbGPXs from N-terminus corresponded to transit peptides, and formed the main source of sequence variations. On the other hand, APXs/GPXs appeared to be significantly conserved at the amino acid sequence level. Residues in active and/or metal binding sites of these enzymes were also revealed with inference to their Arabidopsis counterparts. The combined Sorghum–Arabidopsis APX and GPX phylogenies allowed inferring functional roles to putative Sorghum sequences at cross-species level. In digital RNA-seq data from Sorghum, APXs within sensitive genotypes were relatively more responsive to drought compared to GPXs. Differentially upregulated APX4 and downregulated GPX2 suggested that their performance was synergistic. SbAPXs/GPXs expression in drought-exposed sorghum roots and leaves were quantified by Real-Time quantitative PCR (RT-qPCR). Drought-exposed plants morphologically demonstrated reductions in stem/root elongation and size, retardation in plant growth, and erected leaves. Expressions of APXs/GPXs were mostly upregulated in aboveground parts of drought-exposed plants, e.g., leaves while they were downregulated in roots. Furthermore, APX1 and APX5 in leaves, and APX8, APX9, GPX5, and GPX6 in roots showed significant changes in expression levels; therefore, their synergetic regulation during drought should be considered.

Development of Molecular Markers Linked to Powdery Mildew Resistance Gene Pm4b by Combining SNP Discovery from Transcriptome Sequencing Data with Bulked Segregant Analysis (BSR-Seq) in Wheat.

Powdery mildew resistance gene Pm4b, originating from Triticum persicum, is effective against the prevalent Blumeria graminis f. sp. tritici (Bgt) isolates from certain regions of wheat production in China. The lack of tightly linked molecular markers with the target gene prevents the precise identification of Pm4b during the application of molecular marker-assisted selection (MAS). The strategy that combines the RNA-Seq technique and the bulked segregant analysis (BSR-Seq) was applied in an F2:3 mapping population (237 families) derived from a pair of isogenic lines VPM1/7∗Bainong 3217 F4 (carrying Pm4b) and Bainong 3217 to develop more closely linked molecular markers. RNA-Seq analysis of the two phenotypically contrasting RNA bulks prepared from the representative F2:3 families generated 20,745,939 and 25,867,480 high-quality read pairs, and 82.8 and 80.2% of them were uniquely mapped to the wheat whole genome draft assembly for the resistant and susceptible RNA bulks, respectively. Variant calling identified 283,866 raw single nucleotide polymorphisms (SNPs) and InDels between the two bulks. The SNPs that were closely associated with the powdery mildew resistance were concentrated on chromosome 2AL. Among the 84 variants that were potentially associated with the disease resistance trait, 46 variants were enriched in an about 25 Mb region at the distal end of chromosome arm 2AL. Four Pm4b-linked SNP markers were developed from these variants. Based on the sequences of Chinese Spring where these polymorphic SNPs were located, 98 SSR primer pairs were designed to develop distal markers flanking the Pm4b gene. Three SSR markers, Xics13, Xics43, and Xics76, were incorporated in the new genetic linkage map, which located Pm4b in a 3.0 cM genetic interval spanning a 6.7 Mb physical genomic region. This region had a collinear relationship with Brachypodium distachyon chromosome 5, rice chromosome 4, and sorghum chromosome 6. Seven genes associated with disease resistance were predicted in this collinear genomic region, which included C2 domain protein, peroxidase activity protein, protein kinases of PKc_like super family, Mlo family protein, and catalytic domain of the serine/threonine kinases (STKc_IRAK like super family). The markers developed in the present study facilitate identification of Pm4b during its MAS practice.

Genetic dissection of early-season cold tolerance in sorghum: genome-wide association studies for seedling emergence and survival under field and controlled environment conditions.

A QTL on sorghum chromosome SBI-06 putatively improves field emergence under low-temperature conditions. Low temperatures decisively limit seedling emergence and vigor during early growth of sorghum and, thus, strongly impair geographical expansion. To broaden sorghum cultivation to temperate regions, the establishment of cold-tolerant genotypes is a prioritized breeding goal. The present study aims at the quantification of seedling emergence and survival under chilling temperatures and the detection of marker-trait associations controlling temperature-related seedling establishment. A diversity set consisting of 194 biomass sorghum lines was subjected to extensive phenotyping comprising field trials and controlled environment experiments. The final emergence percentage (FEP) under field conditions was significantly reduced under cold stress. Broad-sense heritability was h 2=0.87 for FEP in the field and h 2=0.93 for seedling survival rate (SR) under controlled conditions. Correlations between FEP in the field and under controlled conditions were low; higher correlations were observed between field FEP and SR in controlled environments. Genome-wide association studies (GWAS) were conducted using 44,515 single nucleotide polymorphisms (SNPs) and revealed eight regions with suggestive marker-trait associations for FEP and SR on chromosomes SBI-01, -02, -03, -06, -09, and -10 (p<5.7×10-5) and a significant association on SBI-06 for field FEP (p<2.9×10-6). Although not significant under controlled conditions, SR of genotypes carrying the minor allele on the field FEP quantitative trait loci (QTL) on SBI-06 was on average 13.1% higher, while FEP under controlled conditions was on average 9.7% higher with a linearly decreasing effect with increasing temperatures (R 2=0.82). Promising candidate genes putatively conferring seedling cold tolerance were identified.