F2 Mutant Research Articles

Gys1 regulates maternal glycogen reserve essential for embryonic development in zebrafish

The reserve of glycogen is essential for embryonic development. In oviparous fish, egg is an isolated system after egg laying with all the required energy deposits by their mothers. However, the key regulated factor mediates the storage of maternal glycogen reserve which support for embryogenesis in the offspring is largely unknown. Glycogen synthase (GYS) is a central enzyme for glycogen synthesis. In our previous study, we generated a gys1 knockout zebrafish line, showed an embryonic developmental defect in F3 generation. In this study, firstly we determined that the gys1 was maternal origin by backcrossing the F2 mutant with wildtype lines. PAS staining and glycogen content measurement showed that glycogen reserve was reduced both in ovaries and embryos in the mutant group compared to wildtypes. Free glucose measurement analysis showed a 50 % of reduction in gys1 mutant embryos compared to wildtype embryos at 24 hpf; showed an approximal 50 % of reduction in gys1 mutant adults compared to wildtypes. Microinjection of 2-NBDG in embryos and comparison of fluorescent signal demonstrated that glucose uptake ability was decreased in the mutant embryos, indicating an impaired glucose metabolism. Untargeted metabolomics analysis then was employed and revealed that key modified metabolites enriched into vitamin B pathway, carbohydrate and unsaturated fatty acid pathways. These results demonstrated that gys1 played a role on glycogen metabolism, involved into the maternal glycogen reserve which essentially contribute to embryonic development.

Radiation Biology of Structurally Different <i>Drosophila melanogaster</i> Genes. Message 9. General Regularities and Locus-Specific Features of Radiomutability of Sex-Linked and Autosomal Genes

The results of large-scale radiation, genetic, cytogenetic and molecular genetic studies on the nature and frequency of heritable mutations of two sex-linked and three autosomal genes of Drosophila melanogaster after exposure to 60Co γ-rays and 0.85 MeV neutrons are presented. Regularities were established for studied genes and radiations in the induction of five different types of recessively inherited mutations, which can be combined into two main classes - the class of chromosomal mutations in one way or another affecting the gene under study (changes leading to sterility of F1 mutants, multilocus deletions, inversion or translocation breakpoint chromosome in the region of gene localization) and a class of “point” gene mutations with a complex spectrum of DNA changes detected by PCR and sequencing. In this report, the results of classical genetic and cytogenetic analysis of these classes of mutations are considered in detail and the frequency of their induction by γ-rays or neutrons is estimated per locus per dose unit (1 Gy). An important and unexpected result of the assessment was the fact that the frequency of “point” mutations turned out to be invariant (on average 1.15 E-06/locus/Gy) for the studied genes and radiations, in contrast to chromosomal mutations, where a pronounced locus-specificity is observed for individual types of mutations. At the same time, neutrons are two or more times more effective than γ-rays in inducing this class of mutations. It is significant that the ave-rage frequency of “point” mutations induction correlates with the average cDNA length of these genes (1.62 kb), but not with their average size (6.07 kb), indicating that the target for " point mutations is, obviously, not the entire DNA of the gene, but only the informational part of its exons. The dependence of the frequency of chromosomal mutations of one type or another on the position of the gene on the chromosome and in the three-dimensional space of the genome is discussed.

GROWTH AND GENETIC CHARACTERISTICS OF CUCUMBER (CUCUMIS SATIVUS L.) CULTIVAR MERCY F1 HYBRID AND MUTANT POPULATIONS

Phenotypic and genotypic diversity is essential and needs more enhancement in different ways to achieve higher productivity and better quality vegetables. The presented research aimed to study the phenotypic characteristics and determine the genetic diversity of F1 populations compared with the colchicine mutant variants in the Mercy cultivar of cucumber (Cucumis sativus L.), held from March to May 2019 at the experimental field of the Department of Agrotechnology, Islamic University of Riau, Pekanbaru, Indonesia. A total number of 144 studied plants comprised 72 from each of the two plant groups. The data recorded on various parameters attained statistical analysis using paired T-test at a significant P < 0.05. Comparisons between the cucumber plants of both groups included karyotypes and chromosome shapes, idiograms, and sequencing. The results revealed that, on average, the taller plants came from F1 populations (93.49 cm), followed by mutants of the same cultivar (67.83 cm). The F1 hybrids showed early flowering (29.00 days) compared with colchicine-treated mutants (33.31 days). However, the number of fruits and fruit weight were higher in the mutant variants (9.39 fruits and 1055.39 g), followed by F1 populations. The karyotypes of cucumber cultivar Mercy F1 hybrids and mutants have different chromosomes, especially with the arm size. An idiogram also exhibited differences in chromosome length between the variants of both plant groups, while the primers of trnL-F and trnL-R target sequences were the same with the DNA sequence length. Using mutant cucumber seeds demonstrates a change in phenotypic character to increase fruit production. However, polyploidy did not occur, with genetic alterations measured by the length of chromosome arms. As a result, a future study with increased concentrations of colchicine is imperative to obtain significant chromosomal mutations.

GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system

Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.

Gene Editing of the Follicle-Stimulating Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Transcription Activator-like Effector Nuclease Technology with Electroporation

Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P1fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3-74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P1 mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P1 mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F1 embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P1 channel catfish genome. Neither the P1 nor the F1 mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F1 families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish.

Large-scale forward genetic screening of zebrafish affecting thyroid development

The thyroid follicular cells originate from the foregut endoderm and elucidating which genes and signaling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to carry an ENU-based forward mutagenesis screen aiming at identifying genes involved in the development and function of the thyroid follicular cells. ENU is an excellent chemical mutagen due to its high mutation efficiency and an indiscriminate selection of genes. A total of 1606 F2 families from 36 ENU treated founders was raised and embryos from F3 generation were collected at 5dpf to perform the whole embryo in situ hybridization with a cocktail probe of thyroid marker thyroglobulin(tg), pituitary marker thyroid stimulating hormone (tshba) to determine the mutagenic phenotype. Among the 1606 F2 families, 112 F2 mutant families with normal development stages except for thyroid dysfunction were identified and divided into three different groups according to their phenotypic characteristics. Further studies of the mutants are likely to shed more insights into the molecular basis of both the thyroid development and function in the zebrafish and vertebrate.

Effect of Ethylene Sletr1-2 Receptor Allele on Flowering, Fruit Phenotype, Yield, and Shelf-Life of Four F1 Generations of Tropical Tomatoes (Solanum lycopersicum L.)

A longer shelf-life for tomatoes without pleiotropic effects is one of the main goals of breeding programs in tropical countries. Therefore, this study aimed to evaluate the effect of the Sletr1-2 mutant allele on flowering, fruit phenotype, shelf life, and yield-related traits in four F1 hybrids from four tropical tomato genetic backgrounds. The study consisted of four tropical strains, namely ‘Intan’, ‘Mirah’, ‘Ratna’, and ‘Mutiara’, as females crossed with wild type ‘Micro-Tom’ (WT-MT) and mutant Sletr1-2 as males. Each was given three treatments and analyzed separately using a randomized block design with four replications of five samples each. The next test used was the Tukey Alpha 0.05 test. The genetic background of tropical tomatoes affects the phenotype and shelf-life. F1 mutants ‘Intan’ and ‘Ratna’ showed significant results, with a longer shelf-life than F1 WT (10.2 and 14.6 days, respectively). In addition, there were no side effects of the Sletr1-2 mutant allele in the heterozygous form on flowering, fruit phenotype, and yield. In conclusion, the Sletr1-2 allele has the potential to be used in tomato breeding programs in tropical countries.

The Drosophila melanogaster enzyme glycerol-3-phosphate dehydrogenase 1 is required for oogenesis, embryonic development, and amino acid homeostasis

As the fruit fly, Drosophila melanogaster, progresses from one life stage to the next, many of the enzymes that compose intermediary metabolism undergo substantial changes in both expression and activity. These predictable shifts in metabolic flux allow the fly meet stage-specific requirements for energy production and biosynthesis. In this regard, the enzyme glycerol-3-phosphate dehydrogenase 1 (GPDH1) has been the focus of biochemical genetics studies for several decades and, as a result, is one of the most well-characterized Drosophila enzymes. Among the findings of these earlier studies is that GPDH1 acts throughout the fly lifecycle to promote mitochondrial energy production and triglyceride accumulation while also serving a key role in maintaining redox balance. Here, we expand upon the known roles of GPDH1 during fly development by examining how depletion of both the maternal and zygotic pools of this enzyme influences development, metabolism, and viability. Our findings not only confirm previous observations that Gpdh1 mutants exhibit defects in larval development, lifespan, and fat storage but also reveal that GPDH1 serves essential roles in oogenesis and embryogenesis. Moreover, metabolomics analysis reveals that a Gpdh1 mutant stock maintained in a homozygous state exhibits larval metabolic defects that significantly differ from those observed in the F1 mutant generation. Overall, our findings highlight unappreciated roles for GPDH1 in early development and uncover previously undescribed metabolic adaptations that could allow flies to survive the loss of this key enzyme.

CRISPR/Cas-9 induced knockout of myostatin gene improves growth and disease resistance in channel catfish (Ictalurus punctatus)

Channel catfish, Ictalurus punctatus, is the most produced aquaculture species in the United States, although production levels have decreased since their peak in 2003. Myostatin (mstn) regulates skeletal muscle growth and has been identified as an important gene for increasing body weight in aquaculture species. In this study, the effects of CRISPR/Cas9 knockout of the mstn gene in channel catfish were investigated. Three sgRNAs targeting exon 1 of the channel catfish myostatin gene were microinjected with Cas9 protein in embryos. A total of 209 fish survived to 6 mph after microinjection of Cas9 and sgRNA targeting exon 1 of the mstn gene over the 3 yr experiment (2017–2019) with an average mutation rate of 58%. Successful generation of myostatin knockout (MSTN KO) F1 heterozygotes was achieved in 2019 by individually mating two pairs of control females with MSTN homozygous KO males. The offspring of both families inherited the mutation at a mean rate of 88%. Growth was generally higher in MSTN mutants than controls at all life stages and environments. P1 MSTN mutants were 88% larger than controls at the stocker stage (100 to 200 g) and 27% larger than controls at market size. Heterozygous F1 mutants were 218% larger than controls at the stocker stage. MSTN mutants had reduced overall expression levels of mstn compared to controls. When challenged with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, MSTN mutants performed equally or better than controls. With a high mutation rate and inheritance as well as improved growth and disease resistance, using MSTN gene-edited channel catfish could greatly benefit commercial farms.

Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice.

Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F2 lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the beIIb and isa1 single mutant lines. These results suggest that co-occurrence of beIIb and isa1 mutant alleles in a single genetic background mitigates the negative effect of the isa1 allele on grain filling, and contributes to recovery of the amyloplast formation defect in the isa1 single mutant.

The genome-wide rate and spectrum of EMS-induced heritable mutations in the microcrustacean Daphnia: on the prospect of forward genetics.

Forward genetic screening using the alkylating mutagen ethyl methanesulfonate (EMS) is an effective method for identifying phenotypic mutants of interest, which can be further genetically dissected to pinpoint the causal genetic mutations. An accurate estimate of the rate of EMS-induced heritable mutations is fundamental for determining the mutant sample size of a screening experiment that aims to saturate all the genes in a genome with mutations. This study examines the genome-wide EMS-induced heritable base-substitutions in three species of the freshwater microcrustacean Daphnia to help guide screening experiments. Our results show that the 10 mM EMS treatment induces base substitutions at an average rate of 1.17 × 10-6/site/generation across the three species, whereas a significantly higher average mutation rate of 1.75 × 10-6 occurs at 25 mM. The mutation spectrum of EMS-induced base substitutions at both concentration is dominated by G:C to A:T transitions. Furthermore, we find that female Daphnia exposed to EMS (F0 individuals) can asexually produce unique mutant offspring (F1) for at least 3 consecutive broods, suggestive of multiple broods as F1 mutants. Lastly, we estimate that about 750 F1s are needed for all genes in the Daphnia genome to be mutated at least once with a 95% probability. We also recommend 4-5 F2s should be collected from each F1 mutant through sibling crossing so that all induced mutations could appear in the homozygous state in the F2 population at 70-80% probability.

A new generation needle- and adjuvant-free trivalent plague vaccine utilizing adenovirus-5 nanoparticle platform

A plague vaccine with a fusion cassette of YscF, F1, and LcrV encoding genes in an adenovirus-5 vector (rAd5-YFV) is evaluated for efficacy and immune responses in mice. Two doses of the vaccine provides 100% protection when administered intranasally against challenge with Yersinia pestis CO92 or its isogenic F1 mutant in short- or long- term immunization in pneumonic/bubonic plague models. The corresponding protection rates drop in rAd5-LcrV monovalent vaccinated mice in plague models. The rAd5-YFV vaccine induces superior humoral, mucosal and cell-mediated immunity, with clearance of the pathogen. Immunization of mice with rAd5-YFV followed by CO92 infection dampens proinflammatory cytokines and neutrophil chemoattractant production, while increasing Th1- and Th2-cytokine responses as well as macrophage/monocyte chemo-attractants when compared to the challenge control animals. This is a first study showing complete protection of mice from pneumonic/bubonic plague with a viral vector-based vaccine without the use of needles and the adjuvant.

Identification of modifiers of the plant height in wheat using an induced dwarf mutant controlled by RhtB4c allele.

In wheat, 25Rht genes for dwarfness are known, which include both, GA-insensitive and GA-responsive genes. The GA-insensitive Rht genes have been widely used, although, their suitability under abiotic stress conditions has been questioned. This necessitated a search for alternative GA-responsive, spontaneous and induced dwarfing genes. We earlier reported an induced dwarf mutant ('dwarf mutant-3'; 44cm), and the mutant allele was named Rht4c allele (2BL). This dwarf mutant was not suitable for cultivation due to its extra dwarf nature. Therefore, we searched for naturally occurring QTLs, which would modify the phenotype of 'dwarf-mutant-3' using 'mutant-assisted gene identification and characterization' (MAGIC) approach. For this purpose, the 'dwarf mutant-3' was crossed with a tall wheat cv. NP114 and homozygous mutant F2 plants (~ 25% of the progeny) were selected, which were phenotyped for plant height and genotyped using SSR markers. The data were utilized for QTL analysis andplant height. Six modifier QTLs were identified on chromosomes 2A, 2B and 4A. Two QTLs each on 2A and 2B were responsible for increase in plant height (described as 'enhancer modifiers'), whereas the remaining two QTLs located on 4A were responsible for reducing the plant height (described as 'suppressor modifiers'). It was hypothesized that the enhancer QTLs could be exploited for the development of semi-dwarf high yielding genotypes containing Rht4c allele. This is the first study of its kind in wheat demontsrating that the MAGIC approach could be used for identification of modifiers of the mutant phenotypes of other traits for wheat improvement.

A dicamba resistance‐endowing IAA16 mutation leads to significant vegetative growth defects and impaired competitiveness in kochia (Bassia scoparia)†

BACKGROUNDPrecise quantification of the fitness cost of synthetic auxin resistance has been impeded by lack of knowledge about the genetic basis of resistance in weeds. Recent elucidation of a resistance‐endowing IAA16 mutation (G73N) in the key weed species kochia (Bassia scoparia), allows detailed characterization of the contribution of resistance alleles to weed fitness, both in the presence and absence of herbicides. Different G73N genotypes from a segregating resistant parental line (9425) were characterized for cross‐resistance to dicamba, 2,4‐d and fluroxypyr, and changes on stem/leaf morphology and plant architecture. Plant competitiveness and dominance of the fitness effects was quantified through measuring biomass and seed production of three F2 lines in two runs of glasshouse replacement series studies.RESULTS G73N confers robust resistance to dicamba but only moderate to weak resistance to 2,4‐d and fluroxypyr. G73N mutant plants displayed significant vegetative growth defects: (i) they were 30–50% shorter, with a more tumbling style plant architecture, and (ii) they had thicker and more ovate (versus lanceolate and linear) leaf blades with lower photosynthesis efficiency, and 40–60% smaller stems with less‐developed vascular bundle systems. F2 mutant plants had impaired plant competitiveness, which can lead to 80‐90% less biomass and seed production in the replacement series study. The pleiotropic effects of G73N were mostly semidominant (0.5) and fluctuated with the environments and traits measured.CONCLUSION G73N is associated with significant vegetative growth defects and reduced competitiveness in synthetic auxin‐resistant kochia. Management practices should target resistant kochia's high vulnerability to competition in order to effectively contain the spread of resistance.

Phytoene synthase 2 can compensate for the absence of PSY1 in the control of color in Capsicum fruit.

Phytoene synthase 1 (PSY1) and capsanthin-capsorubin synthase (CCS) are two major genes responsible for fruit color variation in pepper (Capsicum spp.). However, the role of PSY2 remains unknown. We used a systemic approach to examine the genetic factors responsible for the yellow fruit color of C. annuum 'MicroPep Yellow' (MY) and to determine the role of PSY2 in fruit color. We detected complete deletion of PSY1 and a retrotransposon insertion in CCS. Despite the loss of PSY1 and CCS function, both MY and mutant F2 plants from a cross between MY and the 'MicroPep Red' (MR) accumulated basal levels of carotenoids, indicating that other PSY genes may complement the loss of PSY1. qRT-PCR analysis indicated that PSY2 was constitutively expressed in both MR and MY fruits, and a color complementation assay using Escherichia coli revealed that PSY2 was capable of biosynthesizing a carotenoid. Virus-induced gene silencing of PSY2 in MY resulted in white fruits. These findings indicate that PSY2 can compensate for the absence of PSY1 in pepper fruit, resulting in the yellow color of MY fruits.

Gene mapping and candidate gene analysis of multi-floret spikelet 3 (mfs3) in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is one of the most important food crops worldwide and a model monocot plant for gene function analysis, so it is an ideal system for studying flower development. This study reports a mutant, named multi-floret spikelet 3 (mfs3), which is related to the spikelet development in rice and derived from the ethylmethane sulfonate (EMS)-treated rice cultivar XIDA 1B. In mfs3, the main body of palea (bop) was degenerated severely and only glume-like marginal regions of palea (mrp) remained, while other floral organs developed normally, indicating that the palea identity was seriously influenced by the mutation. It was also observed that the number of floral organs was increased in some spikelets, including 2 lemmas, 4 mrp, 4 lodicules, 8–10 stamens, and 2 pistils, which meant that the spikelet determinacy was lost to some degree in mfs3. Furthermore, genetic analysis demonstrated that the mfs3 trait was controlled by a single recessive gene. Using 426 F2 mutants derived from the cross between sterile line 56S and mfs3, the MULTI-FLORET SPIKELET 3 (MFS3) gene was mapped between the molecular markers RM19347 and RM19352 on Chr.6, with a physical distance of 106.3 kb. Sequencing of candidate genes revealed that an 83-bp fragment loss and a base substitution occurred in the LOC_Os06g04540 gene in the mutant, confirming preliminarily that the LOC_Os06g04540 gene was the MFS3 candidate gene. Subsequent qPCR analysis showed that the mutation caused the down-regulation of OsMADS1 and FON1 genes, and the up-regulation of OsIDS1 and SNB genes, which are all involved in the regulation of spikelet development. The MFS3 mutation also significantly reduced the transcription of the REP gene, which is involved in palea development. These results indicated that the MFS3 gene might be involved in the spikelet meristem determinacy and palea identity by regulating the expression of these related genes.

Cytological, genetic, and proteomic analysis of a sesame (Sesamum indicum L.) mutant Siyl-1 with yellow\u2013green leaf color

BackgroundBoth photosynthetic pigments and chloroplasts in plant leaf cells play an important role in deciding on the photosynthetic capacity and efficiency in plants. Systematical investigating the regulatory mechanism of chloroplast development and chlorophyll (Chl) content variation is necessary for clarifying the photosynthesis mechanism for crops.ObjectiveThis study aims to explore the critical regulatory mechanism of leaf color mutation in a yellow–green leaf sesame mutant Siyl-1.MethodsWe performed the genetic analysis of the yellow-green leaf color mutation using the F2 population of the mutant Siyl-1. We compared the morphological structure of the chloroplasts, chlorophyll content of the three genotypes of the mutant F2 progeny. We performed the two-dimensional gel electrophoresis (2-DE) and compared the protein expression variation between the mutant progeny and the wild type.ResultsGenetic analysis indicated that there were 3 phenotypes of the F2 population of the mutant Siyl-1, i.e., YY type with light-yellow leaf color (lethal); Yy type with yellow-green leaf color, and yy type with normal green leaf color. The yellow-green mutation was controlled by an incompletely dominant nuclear gene, Siyl-1. Compared with the wild genotype, the chloroplast number and the morphological structure in YY and Yy mutant lines varied evidently. The chlorophyll content also significantly decreased (P < 0.05). The 2-DE comparison showed that there were 98 differentially expressed proteins (DEPs) among YY, Yy, and yy lines. All the 98 DEPs were classified into 5 functional groups. Of which 82.7% DEPs proteins belonged to the photosynthesis and energy metabolism group.ConclusionThe results revealed the genetic character of yellow-green leaf color mutant Siyl-1. 98 DEPs were found in YY and Yy mutant compared with the wild genotype. The regulation pathway related with the yellow leaf trait mutation in sesame was analyzed for the first time. The findings supplied the basic theoretical and gene basis for leaf color and chloroplast development mechanism in sesame.

Development of mlo-based resistance in tetraploid wheat against wheat powdery mildew.

Powdery mildew is a severe disease in wheat. In barley, durable resistance exists, based on non-functionality of the Mlo gene. As a model to analyse the effects of mutagenesis in the homoeologous Mlo genes of wheat, we developed mlo-based powdery mildew resistance in tetraploid durum wheat. To obtain Mlo mutations, we screened a TILLING population developed in tetraploid wheat "Kronos" for which the captured exome sequence of > 1500 lines is available. This resulted in 23 mutants for Mlo-A1 and 26 non-redundant mutants for Mlo-B1. Two Mlo-A1 and four Mlo-B1 mutants were crossed to obtain eight F2 mutant lines that showed a range of phenotypes from susceptibility to full resistance. Pot experiments under semi-field conditions confirmed the resistance levels for six of the mutants without any signs of adverse pleiotropic effects. Resistance ranking was similar across six powdery mildew isolates, indicating no isolate specificity of the mlo-based resistance. The effect of mutations in the Mlo-B1 gene was stronger than in the Mlo-A1 gene, probably reflecting differences in wild-type Mlo gene expression levels. Strong partial resistance effects were observed with single mlo-B1 mutations hence, revealing a dosage effect of mlo mutant alleles. Two of the four mlo-B1 mutations (W163* and P335L) were very strong; however, the highest combined effect was observed with the MloA-P335S/MloB-P335L combination, suggesting that non-functional, but full-length Mlo proteins might have the strongest effect compared with nonsense mutations. Our results show that mlo-based resistance might offer possibilities to introduce durable protection in tetraploid wheat against powdery mildew.

Proteomic Analyses of Thioredoxins f and m Arabidopsis thaliana Mutants Indicate Specific Functions for These Proteins in Plants.

A large number of plastidial thioredoxins (TRX) are present in chloroplast and the specificity versus the redundancy of their functions is currently under discussion. Several results have highlighted the fact that each TRX has a specific target protein and thus a specific function. In this study we have found that in vitro activation of the fructose-1,6-bisphosphatase (FBPase) enzyme is more efficient when f1 and f2 type thioredoxins (TRXs) are used, whilst the m3 type TRX did not have any effect. In addition, we have carried out a two-dimensional electrophoresis-gel to obtain the protein profiling analyses of the trxf1, f2, m1, m2, m3 and m4 Arabidopsis mutants. The results revealed quantitative alteration of 86 proteins and demonstrated that the lack of both the f and m type thioredoxins have diverse effects on the proteome. Interestingly, 68% of the differentially expressed proteins in trxf1 and trxf2 mutants were downregulated, whilst 75% were upregulated in trxm1, trxm2, trxm3 and trxm4 lines. The lack of TRX f1 provoked a higher number of down regulated proteins. The contrary occurred when TRX m4 was absent. Most of the differentially expressed proteins fell into the categories of metabolic processes, the Calvin–Benson cycle, photosynthesis, response to stress, hormone signalling and protein turnover. Photosynthesis, the Calvin–Benson cycle and carbon metabolism are the most affected processes. Notably, a significant set of proteins related to the answer to stress situations and hormone signalling were affected. Despite some studies being necessary to find specific target proteins, these results show signs that are suggest that the f and m type plastidial TRXs most likely have some additional specific functions.

Generation of Acsl4 Gene Knockout Mouse Model by CRISPR/Cas9-Mediated Genome Engineering.

Acyl-CoA synthetase 4 (Acsl4) is involved in lipid synthesis and fatty acid degradation, and disruption of its function causes lipid metabolism disorder in various species. Herein, we report the generation of Acsl4 knockout (KO) mice using the CRISPR/Cas9 gene editing system to study its effects on lipid deposition. In this report, a large 12kb deletion in the Acsl4 gene was performed by coinjection of Cas9 mRNA and two guide RNAs (sgRNAs) into mouse fertilized oocytes. Six mutant mice carrying target mutations were examined by PCR analysis and direct sequencing. The gene modified mice remained healthy and displayed normal behavior. All the mutant F0 mice were mated with wild mice to produce the F1 generation, and only 1 F1 mutant mouse was obtained.