CRISPR-induced miRNA156-recognition element mutations in TaSPL13 improve multiple agronomic traits in wheat.

Increase in grain yield is always a major objective of wheat genetic improvement. The SQUAMOSA promoter-binding protein-like (SPL) genes, coding for a small family of diverse plant-specific transcription factors, represent important targets for improving grain yield and other major agronomic traits in rice. The function of the SPL genes in wheat remains to be investigated in this respect. In this study, we identified 56 wheat orthologues of rice SPL genes belonging to 19 homoeologous groups. Like in rice, nine orthologous TaSPL genes harbour the microRNA156 recognition elements (MRE) in their last exons except for TaSPL13, which harbour the MRE in its 3'-untranslated region (3'UTR). We modified the MRE of TaSPL13 using CRISPR-Cas9 and generated 12 mutations in the three homoeologous genes. As expected, the MRE mutations led to an approximately two-fold increase in the TaSPL13 mutant transcripts. The phenotypic evaluation showed that the MRE mutations in TaSPL13 resulted in a decrease in flowering time, tiller number, and plant height, and a concomitantly increase in grain size and number. The results show that the TaSPL13 mutants exhibit a combination of different phenotypes observed in Arabidopsis AtSPL3/4/5 mutants and rice OsSPL13/14/16 mutants and hold great potential in improving wheat yield by simultaneously increasing grain size and number and by refining plant architecture. The novel TaSPL13 mutations generated can be utilized in wheat breeding programmes to improve these agronomic traits.

Adaptive laboratory evolution of Rhodococcus rhodochrous DSM6263 for chlorophenol degradation under hypersaline condition.

BACKGROUND: Normally, a salt amount greater than 3.5% (w/v) is defined as hypersaline. Large amounts of hypersaline wastewater containing organic pollutants need to be treated before it can be discharged into the environment. The most critical aspect of the biological treatment of saline wastewater is the inhibitory/toxic effect exerted on bacterial metabolism by high salt concentrations. Although efforts have been dedicated to improving the performance through the use of salt-tolerant or halophilic bacteria, the diversities of the strains and the range of substrate spectrum remain limited, especially in chlorophenol wastewater treatment. RESULTS: In this study, a salt-tolerant chlorophenol-degrading strain was generated from Rhodococcus rhodochrous DSM6263, an original aniline degrader, by adaptive laboratory evolution. The evolved strain R. rhodochrous CP-8 could tolerant 8% NaCl with 4-chlorophenol degradation capacity. The synonymous mutation in phosphodiesterase of strain CP-8 may retard the hydrolysis of cyclic adenosine monophosphate (cAMP), which is a key factor reported in the osmoregulation. The experimentally verified up-regulation of intracellular cAMP level in the evolved strain CP-8 contributes to the improvement of growth phenotype under high osmotic condition. Additionally, a point mutant of the catechol 1,2-dioxygenase, CatAN211S, was revealed to show the 1.9-fold increment on activity, which the mechanism was well explained by molecular docking analysis. CONCLUSIONS: This study developed one chlorophenol-degrading strain with extraordinary capacity of salt tolerance, which showed great application potential in hypersaline chlorophenol wastewater treatment. The synonymous mutation in phosphodiesterase resulted in the change of intracellular cAMP concentration and then increase the osmotic tolerance in the evolved strain. The catechol 1,2-dioxygenase mutant with improved activity also facilitated chlorophenol removal since it is the key enzyme in the degradation pathway.

Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co-transferred plasmid backbone and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium-delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain-regulatory genes, TaCKX2-1, TaGLW7, TaGW2, and TaGW8, in T0 , T1 , and T2 generation plants at an average edit rate of 10% without detecting off-target mutations in the most Cas9-active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160-bp deletion in TaCKX2-D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium-delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding.

Identification and differential regulation of microRNAs in response to methyl jasmonate treatment in Lycoris aurea by deep sequencing.

BACKGROUND: Lycoris aurea is a medicine-valuable and ornamental herb widely distributed in China. Former studied have showed that methyl jasmonate (MJ) treatment could increase the content of glanthamine-a worldwide medicine for symptomatic treatment of Alzheimer's disease in genus Lycoris plants. To explore the possible role of miRNAs in the regulation of jasmonic acid signaling pathway and uncover their potential correlations, we investigated the expression profiles of small RNAs (sRNAs) and their targets in Lycoris aurea, with MJ treatment by using next-generation deep sequencing. RESULTS: A total of 365 miRNAs were identified, comprising 342 known miRNAs (representing 60 miRNA families) and 23 novel miRNAs. Among them, 143 known and 11 novel miRNAs were expressed differently under MJ treatment. Quantitative real-time PCR of eight selected miRNAs validated the expression pattern of these loci in response to MJ treatment. In addition, degradome sequencing analysis showed that 32 target genes were validated to be targeted by the 49 miRNAs, respectively. Gene function and pathway analyses showed that these targets such as auxin response factors (ARFs), squamosa promoter-binding like (SPL) proteins, basic helix-loop-helix (bHLH) proteins, and ubiquitin-conjugating enzyme E2 are involved in different plant processes, indicating miRNAs mediated regulation might play important roles in L. aurea response to MJ treatment. Furthermore, several L. aurea miRNAs associated with their target genes that might be involved in Amaryllidaceae alkloids biosynthehsis were also analyzed. CONCLUSIONS: A number of miRNAs with diverse expression patterns, and complex relationships between expression of miRNAs and targets were identified. This study represents the first transcriptome-based analysis of miRNAs in Lycoris and will contribute to understanding the potential roles of miRNAs involved in regulation of MJ response.

Evaluation of high yielding soybean germplasm under water limitation.

Limited information is available for soybean root traits and their plasticity under drought stress. To date, no studies have focused on examining diverse soybean germplasm for regulation of shoot and root response under water limited conditions across varying soil types. In this study, 17 genetically diverse soybean germplasm lines were selected to study root response to water limited conditions in clay (trial 1) and sandy soil (trial 2) in two target environments. Physiological data on shoot traits was measured at multiple crop stages ranging from early vegetative to pod filling. The phenotypic root traits, and biomass accumulation data are collected at pod filling stage. In trial 1, the number of lateral roots and forks were positively correlated with plot yield under water limitation and in trial 2, lateral root thickness was positively correlated with the hill plot yield. Plant Introduction (PI) 578477A and 088444 were found to have higher later root number and forks in clay soil with higher yield under water limitation. In sandy soil, PI458020 was found to have a thicker lateral root system and higher yield under water limitation. The genotypes identified in this study could be used to enhance drought tolerance of elite soybean cultivars through improved root traits specific to target environments.

Fine mapping of shattering locus Br2 reveals a putative chromosomal inversion polymorphism between the two lineages of Aegilops tauschii.

KEY MESSAGE: This work laid the foundation for cloning of shattering gene Br2 and provided first line of evidence that two major Aegilops tauschii lineages are differentiated by an inversion polymorphism. Chromosome inversions often accompany population differentiation and capture local adaptation during speciation. Aegilops tauschii, the D-genome donor species of hexaploid wheat, consists of two genetically isolated lineages, L1 and L2, but little is known about the genetic mechanisms underlying the population differentiation in this diploid species. During fine mapping of the shattering gene Br2 using a large F2 population derived from a cross between TA1604 (an L1 accession) and AL8/78 (an L2 accession), we found contrasting patterns of crossover distribution in the Br2 interval and neighboring regions despite the high local gene synteny with Brachypodium distachyon and rice. Br2 was localized in a 0.08-cM interval, and 13 marker loci formed a block, where single-crossovers were completely suppressed, but double-crossovers were enriched with a recombination rate of ~11 cM/Mb. In contrast, in a neighboring region no double-crossover was recovered, but single-crossover rate reached 24 cM/Mb, which is much higher than the genome-wide average. This result suggests a putative inversion polymorphism between the parental lines in the Br2 region. Genotyping using the markers from the Br2 region divided a collection of 55 randomly sampled A. tauschii accessions into two major groups, and they are largely genetically isolated. The two groups correspond to the L1 and L2 lineages based on their geographic distribution patterns. This provides first evidence that inversions may underlie the evolution of A. tauschii lineages. The presence of inter-lineage inversions may complicate map-based cloning in A. tauschii and transfer of useful traits to wheat.

The chloroplast view of the evolution of polyploid wheat.

Polyploid wheats comprise four species: Triticum turgidum (AABB genomes) and T. aestivum (AABBDD) in the Emmer lineage, and T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m) A(m) ) in the Timopheevi lineage. Genetic relationships between chloroplast genomes were studied to trace the evolutionary history of the species. Twenty-five chloroplast genomes were sequenced, and 1127 plant accessions were genotyped, representing 13 Triticum and Aegilops species. The A. speltoides (SS genome) diverged before the divergence of T. urartu (AA), A. tauschii (DD) and the Aegilops species of the Sitopsis section. Aegilops speltoides forms a monophyletic clade with the polyploid Emmer and Timopheevi wheats, which originated within the last 0.7 and 0.4 Myr, respectively. The geographic distribution of chloroplast haplotypes of the wild tetraploid wheats and A. speltoides illustrates the possible geographic origin of the Emmer lineage in the southern Levant and the Timopheevi lineage in northern Iraq. Aegilops speltoides is the closest relative of the diploid donor of the chloroplast (cytoplasm), as well as the B and G genomes to Timopheevi and Emmer lineages. Chloroplast haplotypes were often shared by species or subspecies within major lineages and between the lineages, indicating the contribution of introgression to the evolution and domestication of polyploid wheats.

Cell-specific polymerization-driven biomolecular condensate formation fine-tunes root tissue morphogenesis.

Formation of biomolecular condensates can be driven by weak multivalent interactions and emergent polymerization. However, the mechanism of polymerization-mediated condensate formation is less studied. We found lateral root cap cell (LRC)-specific SUPPRESSOR OF RPS4-RLD1 (SRFR1) condensates fine-tune primary root development. Polymerization of the SRFR1 N-terminal domain is required for both LRC condensate formation and optimal root growth. Surprisingly, the first intrinsically disordered region (IDR1) of SRFR1 can be functionally substituted by a specific group of intrinsically disordered proteins known as dehydrins. This finding facilitated the identification of functional segments in the IDR1 of SRFR1, a generalizable strategy to decode unknown IDRs. With this functional information we further improved root growth by modifying the SRFR1 condensation module, providing a strategy to improve plant growth and resilience.

A non-additive interaction in a single locus causes a very short root phenotype in wheat.

Non-additive allelic interactions underlie over dominant and under dominant inheritance, which explain positive and negative heterosis. These heteroses are often observed in the aboveground traits, but rarely reported in root. We identified a very short root (VSR) phenotype in the F1 hybrid between the common wheat (Triticum aestivum L.) landrace Chinese Spring and synthetic wheat accession TA4152-71. When germinated in tap water, primary roots of the parental lines reached ~15 cm 10 days after germination, but those of the F1 hybrid were ~3 cm long. Selfing populations segregated at a 1 (long-root) to 1 (short-root) ratio, indicating that VSR is controlled by a non-additive interaction between two alleles in a single gene locus, designated as Vsr1. Genome mapping localized the Vsr1 locus in a 3.8-cM interval delimited by markers XWL954 and XWL2506 on chromosome arm 5DL. When planted in vermiculite with supplemental fertilizer, the F1 hybrid had normal root growth, virtually identical to the parental lines, but the advanced backcrossing populations segregated for VSR, indicating that the F1 VSR expression was suppressed by interactions between other genes in the parental background and the vermiculite conditions. Preliminary physiological analyses showed that the VSR suppression is independent of light status but related to potassium homeostasis. Phenotyping additional hybrids between common wheat and synthetics revealed a high VSR frequency and their segregation data suggested more Vsr loci involved. Because the VSR plants can be regularly maintained and readily phenotyped at the early developmental stage, it provides a model for studies of non-additive interactions in wheat.

Genetic dissection of yield-related traits in a recombinant inbred line population created using a key breeding parent in China's wheat breeding.

Understanding the genetics underlying yield formation of wheat is important for increasing wheat yield potential in breeding programs. Nanda2419 was a widely used cultivar for wheat production and breeding in China. In this study, we evaluated yield components and a few yield-related traits of a recombinant inbred line (RIL) population created by crossing Nanda2419 with the indigenous cultivar Wangshuibai in three to four trials at different geographical locations. Negative and positive correlations were found among some of these evaluated traits. Five traits had over 50 % trial-wide broad sense heritability. Using a framework marker map of the genome constructed with this population, quantitative trait loci (QTL) were identified for all traits, and epistatic loci were identified for seven of them. Our results confirmed some of the previously reported QTLs in wheat and identified several new ones, including QSn.nau-6D for effective tillers, QGn.nau-4B.2 for kernel number, QGw.nau-4D for kernel weight, QPh.nau-4B.2 and QPh.nau-4A for plant height, and QFlw.nau-5A.1 for flag leaf width. In the investigated population, Nanda2419 contributed all QTLs associated with higher kernel weight, higher leaf chlorophyll content, and a major QTL associated with wider flag leaf. Seven chromosome regions were related to more than one trait. Four QTL clusters contributed positively to breeding goal-based trait improvement through the Nanda2419 alleles and were detected in trials set in different ecological regions. The findings of this study are relevant to the molecular improvement of wheat yield and to the goal of screening cultivars for better breeding parents.

Genome sequence of Pseudomonas aeruginosa DQ8, an efficient degrader of n-alkanes and polycyclic aromatic hydrocarbons.

Pseudomonas aeruginosa DQ8, which was isolated from the crude oil polluted soil in the Daqing oilfield of China, can efficiently degrade diesel, crude oil, n-alkanes, and polycyclic aromatic hydrocarbons (PAHs). Here, we present a 6.8-Mb assembly of its genome sequence. We have annotated 23 coding sequences (CDSs) responsible for catabolism of n-alkanes and PAHs.

[Biomechanical study of flexor tendon and finger motor function].

In order to provide data for clinical approach to Hand-functional rehabilitation, we conducted this study on the relationships among flexor tendon load, tendon excursion and finger joint angle. Using the dynamic biomechanical test, three-dimension motion image analysis and computer analysis, we investigated eight intact normal male cadavers, hand mechanics of flexor tendon load, tendon excursion and joint angle. The results showed that, at the time when the top of finger touched the palm, the mean tendon load of flexor digitorum profundus(FDP) tendon was 7.9 N, the mean tendon excursion 43.4 mm, the mean total range of motion 237.0 degree. When the top of finger touched the palm, the mean tendon load of flexor digitorum superficial(FDS) tendon was 8.9 N, the mean tendon excursion 38.5 mm, and the mean total range of motion 206.3 degree. These findings demonstrated that there are some curvilinear relationships between flexor tendon load, tendon excursion and finger joint motion. When we flex our fists, the proximal interphalangeal(PIP) joint plays an important role in both FDP and FDS tendon.

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm.

Alterations of mitochondrial-encoded subunits of the F(o)F(1)-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF(A)d that encodes the putative F(A)d subunit of the F(o)F(1)-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer '2114' for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF(A)d is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF(A)d-GFP fusion in onion epidermal cells demonstrated TaF(A)d's mitochondrial location. TaF(A)d was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F(o) was down-regulated in a manner similar to TaF(A)d in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F(o) and the gamma subunit of F(1) was unaffected. These observations implied that TaF(A)d is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy.

Tragopogon (Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9-based genome editing platform in Tragopogon will facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 in Tragopogon. We have established a feasible tissue culture and transformation protocol for Tragopogon. Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted for Tragopogon) was capable of introducing site-specific mutations in Tragopogon protoplasts. Agrobacterium-mediated transformation with Cas9-sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles of TraPDS in albino shoots from Tragopogon porrifolius (2x) and Tragopogon mirus (4x), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9-based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed in Tragopogon, application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene-balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems.

Mapping QTLs for tissue culture response of mature wheat embryos.

The mature wheat embryo is arguably one of the best explants for genetic transformation because of its unlimited availability and lack of growth season restriction. However, an efficient regeneration system using mature wheat embryos (Triticum aestivum L.) is still not available. To identify genes related to the tissue culture response (TCR) of wheat, QTLs for callus induction from mature embryos and callus regeneration were mapped using an RIL population derived from the cross of "Wangshuibai" with "Nanda2419" which has a good TCR. By whole genome scanning we identified five, four and four chromosome regions conditioning, respectively, percent embryos forming a callus (PEFC), percent calli regenerating plantlets (PCRP), and number of plantlets per regenerating callus (NPRC). The major QTLs QPefc.nau-2A and QPcrp.nau-2A were mapped to the long arm of chromosome 2A, explaining up to 22.8% and 17.6% of the respective phenotypic variance. Moreover, two major QTLs for NPRC were detected on chromosomes 2D and 5D; these together explained 51.6% of the phenotypic variance. We found that chromosomes 2A, 2D, 5A, 5B and 5D were associated via different intervals with at least two of the three TCR indexes used. Based on this study and other reports, the TCRs of different explant types of wheat may be under the control of shared or tightly linked genes, while different genes or gene combinations may govern the stages from callus induction to plantlet regeneration. The importance of group 2 and 5 chromosomes in controlling the TCRs of Triticeae crops and the likely conservation of the corresponding genes in cereals are discussed.

Silver(I) N-heterocyclic carbene-bridged calix[4]arene analogues as efficient [60]fullerene receptors.

Two silver(I) N-heterocyclic carbene-bridged calix[4]arene analogues 4 and 5 were synthesized by a fragment-coupling approach; the preliminary inclusion properties of 5 with [60]fullerene shows that it is a novel efficient [60]fullerene fluorescent sensor.

Biodesulfurization of DBT in tetradecane and crude oil by a facultative thermophilic bacterium Mycobacterium goodii X7B.

Mycobacterium goodii X7B, a facultative thermophilic bacterium, cleaving the C-S bond of dibenzothiophene via a sulfur-specific pathway, was investigated for DBT in tetradecane and crude oil desulfurization. The extent of growth was improved by fed-batch culture controlled at a constant pH. The total sulfur level of dibenzothiophene in tetradecane, was reduced by 99%, from 200 to 2 ppm within 24h at 40 degrees C. After 72 h treatment, 59% of the total sulfur content in Liaoning crude oil was removed, from 3600 to 1478 ppm.

Cloning and characterization of TOM7-like gene in wheat.

The cDNA encoding TOM7 (translocase of outer mitochondrial membrane subunit 7) like protein in wheat was cloned through RT-PCR, and its genomic DNA fragment was subsequently cloned. This gene was tentatively designated as TaTOM7. It has no intron in the coding region and its product possesses one hydrophobic trans-membrane domain in the middle, and one hydrophilic domain in the N-terminal and C-terminal domain, respectively. The amino acid composition in the trans-membrane domain of the TaTOM7 subunit is highly conserved among plants, animals and fungi. According to the phylogenetic tree, TOM7-like proteins from different species can be classified into three groups, representing plants, animals and fungi respectively. TaTOM7 is a single- or oligo-copy gene in the wheat genome, displaying different expression levels between Ms2 near-isogenic lines in some tissues, suggesting a role of Ms2 in its expression. TaTOM7 was mapped to chromosome group three of wheat.