Deletions within intronic T-DNA lead to reversion of T-DNA mutant phenotypes.

Agrobacterium-mediated transformation enables random transfer-DNA (T-DNA) insertion into plant genomes. T-DNA insertion into a gene's exons, introns or untranscribed regions close to the start or stop codon can disrupt gene function. Such T-DNA mutants have been useful for reverse genetics analysis, especially in Arabidopsis thaliana. As T-DNAs are inserted into genomic DNA, they are generally believed to be stably inherited. Here, we report a phenomenon of reversion of intronic T-DNA mutant phenotypes. From a suppressor screen using intronic T-DNA pi4kß1,2 double mutant, we recovered intragenic mutants of pi4kß1, which suppressed the autoimmunity of the double mutant. These mutants carried deletions in the intronic T-DNAs, resulting in elevated transcription of normal PI4Kß1. Such reversion of T-DNA insertional mutant phenotype stresses the need for caution when using intronic T-DNA mutants and reiterates the importance of using irreversible null mutant alleles in genetic analyses.

Balancing roles between phosphatidylinositols and sphingolipids in regulating immunity and ER stress responses in pi4kß1,2.

Plant immune regulation is complex. In addition to proteins, lipid molecules play critical roles in modulating immune responses. The mutant pi4kß1,2 is mutated in two phosphatidylinositol 4-kinases PI4Kß1 and ß2 involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P). The mutant displays autoimmunity, short roots, aberrant root hairs, and a heightened sensitivity to ER stress. In a forward genetic screen designed to dissect pi4kß1,2 autoimmunity, we found that Orosomucoid-like 1 (ORM1) is required for the phenotypes of pi4kß1,2, including short root and ER stress sensitivity. The orm1 mutations lead to increased long-chain base and ceramide levels in the suppressors. We also found that the basic region/leucine Zipper motif (bZIP) 28 and 60 transcription factors, central regulators of ER stress response, are required for its autoimmunity and root defect. In comparison, the defense-related phytohormones salicylic acid (SA) and N-hydroxypipecolic acid (NHP) are required for its autoimmunity but plays a minor role in its root phenotypes. Further, we found that wild-type plants overexpressing ORM1 are autoimmune, displaying short roots and increased ceramide levels. The autoimmunity of the ORM1 overexpression lines is dependent on SA, NHP, and bZIP60. As ORM1 is a known negative regulator of sphingolipid biosynthesis, our study uncovers a balancing role between PIs and sphingolipids in regulating immunity and ER stress responses in pi4kß1,2.

A sucrose-binding protein and ß-conglycinins regulate soybean seed protein content and control multiple seed traits.

Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max (L.) Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins. We isolated two high-seed protein (HP) soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes ß-conglycinin (GmCG-1) and HP2 encodes Sucrose Binding Protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits.

Direct access to millions of mutations by whole genome sequencing of an oilseed rape mutant population.

Induced mutations are an essential source of genetic variation in plant breeding. Ethyl methanesulfonate (EMS) mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In the present study, a rapeseed M2 population was derived from M1 parent cultivar 'Express' treated with EMS. Whole genomes were sequenced from fourfold (4×) pools of 1988 M2 plants representing 497 M2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284 442 single nucleotide polymorphisms (SNPs) per M2 DNA pool were found resulting from EMS mutagenesis. 55% of the SNPs were C → T and G → A transitions, characteristic for EMS induced ('canonical') mutations, whereas the remaining SNPs were 'non-canonical' transitions (15%) or transversions (30%). Additionally, we detected 88 725 high confidence insertions and deletions per pool. On average, each M2 plant carried 39 120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Approximately 82% of such mutations were located either 5 kb upstream or downstream (56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool 'EMSBrassica' (http://www.emsbrassica.plantbreeding.uni-kiel.de), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

Enhancing cold resistance in Banana (Musa spp.) through EMS-induced mutagenesis, L-Hyp pressure selection: phenotypic alterations, biomass composition, and transcriptomic insights.

BACKGROUND: The cultivation of bananas encounters substantial obstacles, particularly due to the detrimental effects of cold stress on their growth and productivity. A potential remedy that has gained attention is the utilization of ethyl mesylate (EMS)-induced mutagenesis technology, which enables the creation of a genetically varied group of banana mutants. This complex procedure entails subjecting the mutants to further stress screening utilizing L-Hyp in order to identify those exhibiting improved resistance to cold. This study conducted a comprehensive optimization of the screening conditions for EMS mutagenesis and L-Hyp, resulting in the identification of the mutant cm784, which exhibited remarkable cold resistance. Subsequent investigations further elucidated the physiological and transcriptomic responses of cm784 to low-temperature stress. RESULTS: EMS mutagenesis had a substantial effect on banana seedlings, resulting in modifications in shoot and root traits, wherein a majority of seedlings exhibited delayed differentiation and limited elongation. Notably, mutant leaves displayed altered biomass composition, with starch content exhibiting the most pronounced variation. The application of L-Hyp pressure selection aided in the identification of cold-resistant mutants among seedling-lethal phenotypes. The mutant cm784 demonstrated enhanced cold resistance, as evidenced by improved survival rates and reduced symptoms of chilling injury. Physiological analyses demonstrated heightened activities of antioxidant enzymes and increased proline production in cm784 when subjected to cold stress. Transcriptome analysis unveiled 946 genes that were differentially expressed in cm784, with a notable enrichment in categories related to 'Carbohydrate transport and metabolism' and 'Secondary metabolites biosynthesis, transport, and catabolism'. CONCLUSION: The present findings provide insights into the molecular mechanisms that contribute to the heightened cold resistance observed in banana mutants. These mechanisms encompass enhanced carbohydrate metabolism and secondary metabolite biosynthesis, thereby emphasizing the adaptive strategies employed to mitigate the detrimental effects induced by cold stress.

EMS-induced mutagenesis in Choy sum (Brassica chinensis var. parachinensis) and selection for low light tolerance using abiotic stress indices.

BACKGROUND: Choy Sum (Brassica rapa ssp. chinensis var. parachinensis), grown in a controlled environment, is vulnerable to changes in indoor light quality and displays distinct photo-morphogenesis responses. The scarcity of Choy Sum germplasm for indoor cultivation necessitates the development of new cultivars. Hence, this study attempted to develop mutants through chemical mutagenesis and select low-light-tolerant mutants by using abiotic stress tolerance indices. RESULTS: A mutant population of Choy Sum created using 1.5% ethyl methane sulfonate (EMS) at 4 h was manually pollinated to obtain the M2 generation. 154 mutants with reduced hypocotyl length were initially isolated from 3600 M2 seedlings screened under low light (R: FR = 0.5). Five mutants that showed reduced plant height at mature stages were selected and screened directly for shade tolerance in the M3 generation. Principal component analysis based on phenotypic data distinguished the M3 mutants from the wild type. Abiotic stress tolerance indices such as relative stress index (RSI), stress tolerance index (STI), geometric mean productivity (GMP), yield stability index (YSI), and stress resistance index (SRI) showed significant (P < 0.05), and positive associations with leaf yield under shade. M3-12-2 was selected as a shade-tolerant mutant based on high values of STI, YSI, and SRI with low values for tolerance (TOL) and stress susceptibility index (SSI). CONCLUSIONS: The results demonstrate that mutation breeding can be used to create dominant mutants in Choy Sum. Furthermore, we show that screening for low light and selection based on abiotic tolerance indices allowed the identification of mutants with high resilience under shade. This method should apply to developing new cultivars in other crop plants that can be suitable for controlled environments with stable yield performance.

Novel evolved Yarrowia lipolytica strains for enhanced growth and lipid content under high concentrations of crude glycerol.

BACKGROUND: Yarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance. RESULTS: Y. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9-20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation. CONCLUSION: ALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable "chassis" for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.

Pheno- and genotyping in vitro dauer juvenile recovery in the nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H2O2; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.

A CCaMK/Cyclops response element in the promoter of Lotus japonicus calcium-binding protein 1 (CBP1) mediates transcriptional activation in root symbioses.

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein 1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter : reporter fusions to transactivation and spatio-temporal expression analysis and screened ethyl methanesulphonate (EMS)-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the calcium and calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

A single amino acid residue substitution in BraA04g017190.3C, a histone methyltransferase, results in premature bolting in Chinese cabbage (Brassica rapa L. ssp. Pekinensis).

BACKGROUND: Flowering is an important inflection point in the transformation from vegetative to reproductive growth, and premature bolting severely decreases crop yield and quality. RESULTS: In this study, a stable early-bolting mutant, ebm3, was identified in an ethyl methanesulfonate (EMS)-mutagenized population of a Chinese cabbage doubled haploid (DH) line 'FT'. Compared with 'FT', ebm3 showed early bolting under natural cultivation in autumn, and curled leaves. Genetic analysis showed that the early-bolting phenotype was controlled by a single recessive nuclear gene. Modified MutMap sequencing, genotyping analyses and allelism test provide strong evidence that BrEBM3 (BraA04g017190.3 C), encoding the histone methyltransferase CURLY LEAF (CLF), was the strongly candidate gene of the emb3. A C to T base substitution in the 14th exon of BrEBM3 resulted in an amino acid change (S to F) and the early-bolting phenotype of emb3. The mutation occurred in the SET domain (Suppressor of protein-effect variegation 3-9, Enhancer-of-zeste, Trithorax), which catalyzes site- and state-specific lysine methylation in histones. Tissue-specific expression analysis showed that BrEBM3 was highly expressed in the flower and bud. Promoter activity assay confirmed that BrEBM3 promoter was active in inflorescences. Subcellular localization analysis revealed that BrEBM3 localized in the nucleus. Transcriptomic studies supported that BrEBM3 mutation might repress H3K27me3 deposition and activate expression of the AGAMOUS (AG) and AGAMOUS-like (AGL) loci, resulting in early flowering. CONCLUSIONS: Our study revealed that an EMS-induced early-bolting mutant ebm3 in Chinese cabbage was caused by a nonsynonymous mutation in BraA04g017190.3 C, encoding the histone methyltransferase CLF. These results improve our knowledge of the genetic and genomic resources of bolting and flowering, and may be beneficial to the genetic improvement of Chinese cabbage.

A highly differentiated region of wheat chromosome 7AL encodes a Pm1a immune receptor that recognizes its corresponding AvrPm1a effector from Blumeria graminis.

Pm1a, the first powdery mildew resistance gene described in wheat, is part of a complex resistance (R) gene cluster located in a distal region of chromosome 7AL that has suppressed genetic recombination. A nucleotide-binding, leucine-rich repeat (NLR) immune receptor gene was isolated using mutagenesis and R gene enrichment sequencing (MutRenSeq). Stable transformation confirmed Pm1a identity which induced a strong resistance phenotype in transgenic plants upon challenge with avirulent Blumeria graminis (wheat powdery mildew) pathogens. A high-density genetic map of a B. graminis family segregating for Pm1a avirulence combined with pathogen genome resequencing and RNA sequencing (RNAseq) identified AvrPm1a effector gene candidates. In planta expression identified an effector, with an N terminal Y/FxC motif, that induced a strong hypersensitive response when co-expressed with Pm1a in Nicotiana benthamiana. Single chromosome enrichment sequencing (ChromSeq) and assembly of chromosome 7A suggested that suppressed recombination around the Pm1a region was due to a rearrangement involving chromosomes 7A, 7B and 7D. The cloning of Pm1a and its identification in a highly rearranged region of chromosome 7A provides insight into the role of chromosomal rearrangements in the evolution of this complex resistance cluster.

Genome-wide identification and analysis of soybean acyl-ACP thioesterase gene family reveals the role of GmFAT to improve fatty acid composition in soybean seed.

KEY MESSAGE: Soybean acyl-ACP thioesterase gene family have been characterized; GmFATA1A mutants were discovered to confer high oleic acid, while GmFATB mutants presented low palmitic and high oleic acid seed content. Soybean oil stability and quality are primarily determined by the relative proportions of saturated versus unsaturated fatty acids. Commodity soybean typically contains 11% palmitic acid, as the primary saturated fatty acids. Reducing palmitic acid content is the principal approach to minimize the levels of saturated fatty acids in soybean. Though high palmitic acid enhances oxidative stability of soybean oil, it is negatively correlated with oil and oleic acid content and can cause coronary heart diseases for humans. For plants, acyl-acyl carrier protein (ACP) thioesterases (TEs) are a group of enzymes to hydrolyze acyl group and release free fatty acid from plastid. Among them, GmFATB1A has become the main target to genetically reduce the palmitic acid content in soybean. However, the role of members in soybean acyl-ACP thioesterase gene family is largely unknown. In this study, we characterized two classes of TEs, GmFATA, and GmFATB in soybean. We also denominated two GmFATA members and discovered six additional members that belong to GmFATB gene family through phylogenetic, syntenic, and in silico analysis. Using TILLING-by-Sequencing+, we identified an allelic series of mutations in five soybean acyl-ACP thioesterase genes, including GmFATA1A, GmFATB1A, GmFATB1B, GmFATB2A, and GmFATB2B. Additionally, we discovered mutations at GmFATA1A to confer high oleic acid (up to 34.5%) content, while mutations at GmFATB presented low palmitic acid (as low as 5.6%) and high oleic acid (up to 36.5%) phenotypes. The obtained soybean mutants with altered fatty acid content can be used in soybean breeding program for improving soybean oil composition traits.

Improving isobutanol tolerance and titers through EMS mutagenesis in Saccharomyces cerevisiae.

Improving yeast tolerance toward isobutanol is a critical issue enabling high-titer industrial production. Here, we used EMS mutagenesis to screen Saccharomyces cerevisiae with greater tolerance toward isobutanol. By this method, we obtained EMS39 with high-viability in medium containing 16 g/L isobutanol. Then, we metabolically engineered isobutanol synthesis in EMS39. About 2µ plasmids carrying PGK1p-ILV2, PGK1p-ILV3 and TDH3p-cox4-ARO10 were used to over-express ILV2, ILV3 and ARO10 genes, respectively, in EMS39 and wild type W303-1A. And the resulting strains were designated as EMS39-20 and W303-1A-20. Our results showed that EMS39-20 increased isobutanol titers by 49.9% compared to W303-1A-20. Whole genome resequencing analysis of EMS39 showed that more than 59 genes had mutations in their open reading frames or regulatory regions. These 59 genes are enriched mainly into cell growth, basal transcription factors, cell integrity signaling, translation initiation and elongation, ribosome assembly and function, oxidative stress response, etc. Additionally, transcriptomic analysis of EMS39-20 was carried out. Finally, reverse engineering tests showed that overexpression of CWP2 and SRP4039 could improve tolerance of S.cerevisiae toward isobutanol. In conclusion, EMS mutagenesis could be used to increase yeast tolerance toward isobutanol. Our study supplied new insights into mechanisms of tolerance toward isobutanol and enhancing isobutanol production in S. cerevisiae.

Combining next-generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals.

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M2 ) family derived from a heterozygous (M1 ) parent were identified using whole-genome shotgun (WGS) sequencing of a small number of (M2 ) individuals with mutant and wild-type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self-pollinated (M3 ) offspring. Finally, we filtered for segregating EMS-induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M2 ) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non-synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.

Reporter-based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana.

The evolution of C4 photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre-condition for the introduction of a functional C4 cycle is the photosynthetic activation of the C3 bundle sheath by increasing its volume and organelle number. Therefore, to engineer C4 photosynthesis into existing C3 crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C4 -like bundle sheath. To this end, an ethylmethanesulfonate (EMS)-based forward genetic screen was established in the Brassicaceae C3  species Arabidopsis thaliana. To ensure a high-throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast-targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping-by-sequencing approach the genomic segments that contained mutated candidate genes were identified.

The SAP function in pistil development was proved by two allelic mutations in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

BACKGROUND: Pistil development is a complicated process in plants, and female sterile mutants are ideal material for screening and cloning pistil development-related genes. Using the female sterile mutant (fsm1), BraA04g009730.3C was previously predicted as a candidate mutant gene encoding the STERILE APETALA (SAP) transcriptional regulator. In the current study, a parallel female sterile mutant (fsm2) was derived from EMS mutagenesis of a Chinese cabbage DH line 'FT' seeds. RESULTS: Both fsm2 and fsm1 mutant phenotypes exhibited pistil abortion and smaller floral organs. Genetic analysis indicated that the phenotype of mutant fsm2 was also controlled by a single recessive nuclear gene. Allelism testing showed that the mutated fsm1 and fsm2 genes were allelic. A single-nucleotide mutation (G-to-A) in the first exon of BraA04g009730.3C caused a missense mutation from GAA (glutamic acid) to GGA (glycine) in mutant fsm2 plants. Both allelic mutations of BraA04g009730.3C in fsm1 and fsm2 conferred the similar pistil abortion phenotype, which verified the SAP function in pistil development. To probe the mechanism of SAP-induced pistil abortion, we compared the mutant fsm1 and wild-type 'FT' pistil transcriptomes. Among the 3855 differentially expressed genes obtained, 29 were related to ovule development and 16 were related to organ size. CONCLUSION: Our study clarified the function of BraA04g009730.3C and revealed that it was responsible for ovule development and organ size. These results lay a foundation to elucidate the molecular mechanism of pistil development in Chinese cabbage.

Characterization of a high-growth-rate mutant strain of Pyropia yezoensis using physiology measurement and transcriptome analysis.

A mutant strain of Pyropia yezoensis, strain E, was isolated from the free-living conchocelis of a pure strain (NA) treated with ethyl methane sulfonate. The incremental quantities of young strain E blades were higher than those of NA after 14 d of cultivation, indicating that young blades of mutant strain E released more archeospores. The mean length and weight of large E blades were both over three times greater than those of NA after 4 weeks of cultivation. The photosynthetic parameters (Fv/Fm, Y[I], Y[II], and O2 evolution rate) and pigment contents (including phycoerythrin and phycocyanin) of strain E blades were higher than those of NA (P < 0.05). The cellular respiratory rate of strain E blades was lower than that of NA (P < 0.05). In order to investigate the causes of changes in strain E blades, total RNA in strain E and NA blades were sequenced using the Illumina Hiseq platform. Compared with NA, 1,549 unigenes were selected in strain E including 657 up-regulated and 892 down-regulated genes. According to the physiology measurement and differentially expressed genes analysis, cell respiration in strain E might decrease, whereas anabolic-like photosynthesis and protein biosynthesis might increase compared with NA. This means substance accumulation might be greater than decomposition in strain E. This might explain why strain E blades showed improved growth compared with NA. In addition, several genes related to stress resistance were up-regulated in strain E indicating that strain E might have a higher stress resistance. The sequencing dataset may be conducive to Pyropia yezoensis molecular breeding research.

Development of a Dunaliella tertiolecta Strain with Increased Zeaxanthin Content Using Random Mutagenesis.

Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10-15% higher cellular zeaxanthin content than the parent strain (zea1), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3, was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65-85 µmol photons·m-2·s-1, and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140-160 µmol photons·m-2·s-1. The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L-1, which was obtained at 0.6 M NaCl and 140-160 µmol photons·m-2·s-1. These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains.

Development and utilization of a new chemically-induced soybean library with a high mutation density .

Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate (EMS)-induced soybean (Glycine max) population, consisting of 21,600 independent M2 lines, was developed. Over 1,000 M4 (5) families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll (Chl) content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to MinnGold, where a different single nucleotide polymorphism variation in the Mg-chelatase subunit gene (ChlI1a) results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker-assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.

Biomass and terpenoids produced by mutant strains of Arthrospira under low temperature and light conditions.

The filamentous Cyanobacterium Arthrospira is commercially produced and is a functional, high-value, health food. We identified 5 low temperature and low light intensity tolerant strains of Arthrospira sp. (GMPA1, GMPA7, GMPB1, GMPC1, and GMPC3) using ethyl methanesulfonate mutagenesis and low temperature screening. The 5 Arthrospira strains grew rapidly below 14 °C, 43.75 µmol photons m-2 s-1 and performed breed conservation at 2.5 °C, 8.75 µmol photons m-2 s-1. We used morphological identification and molecular genetic analysis to identify GMPA1, GMPA7, GMPB1 and GMPC1 as Arthrospira platensis, while GMPC3 was identified as Arthrospira maxima. Growth at different culture temperatures was determined at regular intervals using dry biomass. At 16 °C and 43.75 µmol photons m-2 s-1, the maximum dry biomass production and the mean dry biomass productivity of GMPA1, GMPB1, and GMPC1 were 2057 ± 80 mg l-1, 68.7 ± 2.5 mg l-1 day-1, 1839 ± 44 mg l-1, 60.6 ± 1.8 mg l-1 day-1, and 2113 ± 64 mg l-1, 77.7 ± 2.5 mg l-1 day-1 respectively. GMPB1 was chosen for additional low temperature tolerance studies and growth temperature preference. In winter, GMPB1 grew well at mean temperatures <10 °C, achieving 3258 mg dry biomass from a starting 68 mg. In summer, GMPB1 grew rapidly at mean temperatures more than 28 °C, achieving 1140 mg l-1 dry biomass from a starting 240 mg. Phytonutrient analysis of GMPB1 showed high levels of C-phycocyanin and carotenoids. Arthrospira metabolism relates to terpenoids, and the methyl-D-erythritol 4-phosphate pathway is the only terpenoid biosynthetic pathway in Cyanobacteria. The 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene from GMPB1 was cloned and phylogenetic analysis showed that GMPB1 is closest to the Cyanobacterium Oscillatoria nigro-viridis PCC711. Low temperature tolerant Arthrospira strains could broaden the areas suitable for cultivation, extend the seasonal cultivation time, and lower production costs.