Characterization of a new greenbug resistance gene Gb9 in a synthetic hexaploid wheat.

Greenbug [Schizaphis graminum (Rondani)] is a serious insect pest that not only damages cereal crops, but also transmits several destructive viruses. The emergence of new greenbug biotypes in the field makes it urgent to identify novel greenbug resistance genes in wheat. CWI 76364 (PI 703397), a synthetic hexaploid wheat (SHW) line, exhibits greenbug resistance. Evaluation of an F2:3 population from cross OK 14319 × CWI 76364 indicated that a dominant gene, designated Gb9, conditions greenbug resistance in CWI 76364. Selective genotyping of a subset of F2 plants with contrasting phenotypes by genotyping-by-sequencing identified 25 SNPs closely linked to Gb9 on chromosome arm 7DL. Ten of these SNPs were converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for genotyping the entire F2 population. Genetic analysis delimited Gb9 to a 0.6-Mb interval flanked by KASP markers located at 599,835,668 bp (Stars-KASP872) and 600,471,081 bp (Stars-KASP881) on 7DL. Gb9 was 0.5 cM distal to Stars-KASP872 and 0.5 cM proximal to Stars-KASP881. Allelism tests indicated that Gb9 is a new greenbug resistance gene which confers resistance to greenbug biotypes C, E, H, I, and TX1. TX1 is one of the most widely virulent biotypes and has overcome most known wheat greenbug resistance genes. The introgression of Gb9 into locally adapted wheat cultivars is of economic importance, and the KASP markers developed in this study can be used to tag Gb9 in cultivar development.

Sorghum SbGhd7 is a major regulator of floral transition and directly represses genes crucial for flowering activation.

Molecular genetic understanding of flowering time regulation is crucial for sorghum development. GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (SbGhd7) is one of the six classical loci conferring photoperiod sensitivity of sorghum flowering. However, its functions remain poorly studied. The molecular functions of SbGhd7 were characterized. The gene regulatory network controlled by SbGhd7 was constructed and validated. The biological roles of SbGhd7 and its major targets were studied. SbGhd7 overexpression (OE) completely prevented sorghum flowering. Additionally, we show that SbGhd7 is a major negative regulator of flowering, binding to the promoter motif TGAATG(A/T)(A/T/C) and repressing transcription of the major florigen FLOWERING LOCUS T 10 (SbFT10) and floral activators EARLY HEADING DATE (SbEhd1), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (SbFKF1) and EARLY FLOWERING 3 (SbELF3). Reinforcing the direct effect of SbGhd7, SbEhd1 OE activated the promoters of three functional florigens (SbFT1, SbFT8 and SbFT10), dramatically accelerating flowering. Our studies demonstrate that SbGhd7 is a major repressor of sorghum flowering by directly and indirectly targeting genes for flowering activation. The mechanism appears ancient. Our study extends the current model of floral transition regulation in sorghum and provides a framework for a comprehensive understanding of sorghum photoperiod response.

Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality.

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance in the Uzbekistani Wheat Landrace Teremai Bugdai.

Leaf rust, caused by Puccinia triticina, is a major cause of wheat yield losses globally, and novel leaf rust resistance genes are needed to enhance wheat leaf rust resistance. Teremai Bugdai is a landrace from Uzebekistan that is highly resistant to many races of P. triticina in the United States. To unravel leaf rust resistance loci in Teremai Bugdai, a recombinant inbred line (RIL) population of Teremai Bugdai × TAM 110 was evaluated for response to P. triticina race Pt54-1 (TNBGJ) and genotyped using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). Quantitative trait loci (QTL) analysis using 5,130 high-quality GBS-SNPs revealed three QTLs, QLr-Stars-2DS, QLr-Stars-6BL, and QLr.Stars-7BL, for leaf rust resistance in two experiments. QLr-Stars-2DS, which is either a new Lr2 allele or a new resistance locus, was delimited to an ∼19.47-Mb interval between 46.4 and 65.9 Mb on 2DS and explained 31.3 and 33.2% of the phenotypic variance in the two experiments. QLr-Stars-6BL was mapped in an ∼84.0-kb interval between 719.48 and 719.56 Mb on 6BL, accounting for 33 to 36.8% of the phenotypic variance in two experiments. QLr.Stars-7BL was placed in a 350-kb interval between 762.41 and 762.76 Mb on 7BL and explained 4.4 to 5.3% of the phenotypic variance. Nine GBS-SNPs flanking these QTLs were converted to kompetitive allele specific PCR (KASP) markers, and these markers can be used to facilitate their introgression into locally adapted wheat lines.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion in Medicago truncatula and Nicotiana sylvestris.

WOX family transcription factors regulate multiple developmental programs. The intermediate clade transcriptional activator WOX9 functions together with the modern clade transcriptional repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. STF and LAM1 are WOX1 orthologs required for leaf blade outgrowth in Medicago truncatula and Nicotiana sylvestris, respectively. Using biochemical methods and genome editing technology, here we show that WOX9 is an abaxial factor and functions antagonistically to STF and LAM1 to regulate leaf blade development. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression and knockout of NsWOX9 in N. sylvestris resulted in a range of severe leaf blade distortions, indicating important role in blade development. Our results indicate that direct repression of WOX9 by WUS clade repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that controlling transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in flowering plants.

Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa).

Alfalfa (Medicago sativa) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green (MsSGR) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula.

MAIN CONCLUSION: An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.

Control of leaf blade outgrowth and floral organ development by LEUNIG, ANGUSTIFOLIA3 and WOX transcriptional regulators.

Plant lateral organ development is a complex process involving both transcriptional activation and repression mechanisms. The WOX transcriptional repressor WOX1/STF, the LEUNIG (LUG) transcriptional corepressor and the ANGUSTIFOLIA3 (AN3) transcriptional coactivator play important roles in leaf blade outgrowth and flower development, but how these factors coordinate their activities remains unclear. Here we report physical and genetic interactions among these key regulators of leaf and flower development. We developed a novel in planta transcriptional activation/repression assay and suggest that LUG could function as a transcriptional coactivator during leaf blade development. MtLUG physically interacts with MtAN3, and this interaction appears to be required for leaf and flower development. A single amino acid substitution at position 61 in the SNH domain of MtAN3 protein abolishes its interaction with MtLUG, and its transactivation activity and biological function. Mutations in lug and an3 enhanced each other's mutant phenotypes. Both the lug and the an3 mutations enhanced the wox1 prs leaf and flower phenotypes in Arabidopsis. Our findings together suggest that transcriptional repression and activation mediated by the WOX, LUG and AN3 regulators function in concert to promote leaf and flower development, providing novel mechanistic insights into the complex regulation of plant lateral organ development.

Photoperiod response and floral transition in sorghum.

Sorghum is a short day plant with strong photoperiod response and its cultivation for grain in temperate regions necessitated the development of photoperiod insensitive mutants that can flower rapidly in the long days of summer. Wild type genotypes grow vegetatively in summer accumulating significant biomass before floral transition ensues during the shorter days of fall. Thus, photoperiod insensitive mutants are grown for grain production while photoperiod sensitive wild type genotypes are grown for forage and biomass feedstock production in the United States. However, the molecular mechanism of photoperiod response and floral transition is poorly understood in sorghum. We have previously reported 3 FLOWERING LOCUS T homologues (SbFT1, SbFT8 and SbFT10) that serve as the ultimate mediators of photoperiod response and floral transition, but more work remains to be done to clearly define the molecular function of the upstream regulatory factors. One of the major QTL that accounts for 85% of the flowering time variation, which was reported to be encoding the PRR37 protein is now debated to be encoding the SbFT12 protein, raising further questions as to how SbFT12 may regulate sorghum florigens. Further molecular analyses will uncover the true nature of the day length sensors in sorghum and the mechanisms of their interactions with florigens to modulate photoperiod dependent vegetative growth and floral transition.

Control of floral transition in the bioenergy crop switchgrass.

Switchgrass (Panicum virgatum L.), a perennial warm season bunchgrass native to North America, has been a target in the U.S. as a renewable bioenergy crop because of its ability to produce moderate to high biomass yield on marginal soils. Delaying flowering can increase vegetative biomass production by allowing prolonged growth before switching to the reproductive phase. Despite the identification of flowering time as a biomass trait in switchgrass, the molecular regulatory factors involved in controlling floral transition are poorly understood. Here we identified PvFT1, PvAPL1-3 and PvSL1, 2 as key flowering regulators required from floral transition initiation to development of floral organs. PvFT1 expression in leaves is developmentally regulated peaking at the time of floral transition, and diurnally regulated with peak at approximately 2 h into the dark period. Ectopic expression of PvFT1 in Arabidopsis, Brachypodium and switchgrass led to extremely early flowering, and activation of FT downstream target genes, confirming that it is a strong activator of flowering in switchgrass. Ectopic expression of PvAPL1-3 and PvSL1, 2 in Arabidopsis also activated early flowering with distinct floral organ phenotypes. Our results suggest that switchgrass has conserved flowering pathway regulators similar to Arabidopsis and rice.

Three FLOWERING LOCUS T-like genes function as potential florigens and mediate photoperiod response in sorghum.

Sorghum is a typical short-day (SD) plant and its use in grain or biomass production in temperate regions depends on its flowering time control, but the underlying molecular mechanism of floral transition in sorghum is poorly understood. Here we characterized sorghum FLOWERING LOCUS T (SbFT) genes to establish a molecular road map for mechanistic understanding. Out of 19 PEBP genes, SbFT1, SbFT8 and SbFT10 were identified as potential candidates for encoding florigens using multiple approaches. Phylogenetic analysis revealed that SbFT1 clusters with the rice Hd3a subclade, while SbFT8 and SbFT10 cluster with the maize ZCN8 subclade. These three genes are expressed in the leaf at the floral transition initiation stage, expressed early in grain sorghum genotypes but late in sweet and forage sorghum genotypes, induced by SD treatment in photoperiod-sensitive genotypes, cooperatively repressed by the classical sorghum maturity loci, interact with sorghum 14-3-3 proteins and activate flowering in transgenic Arabidopsis plants, suggesting florigenic potential in sorghum. SD induction of these three genes in sensitive genotypes is fully reversed by 1 wk of long-day treatment, and yet, some aspects of the SD treatment may still make a small contribution to flowering in long days, indicating a complex photoperiod response mediated by SbFT genes.