EMS Mutant Research Articles

TaTCP21-A negatively regulates wheat cold tolerance via repressing expression of TaDREB1C

Cold stress is one of the important harmful factors that seriously affect wheat (Triticum aestivum) yield and quality. TCP transcription factor plays important roles in the process of plant cell proliferation and growth. In this study, we identified 60 TaTCP genes expressed in strong cold resistant winter wheat variety Dongnongdongmai1 (Dn1) under cold stress by previous transcriptome data, of which 13 TaTCPs showed significant differences in expression. The evolution of TaTCPs was analyzed, and the results showed that there were 2 homologous pairs in TaTCPs with AtTCPs and 90 homologous pairs in TaTCPs with OsTCPs. Expression patterns of 20 TaTCPs under cold stress were analyzed by qRT-PCR, and TCP21-A with significant expression differences was screened. We obtained tcp21-A mutant from the EMS mutant library of winter wheat Kenong9204. We observed that the mutation of TaTCP21-A significantly improved its cold resistance. Subsequently, transcriptome analysis revealed that TCP21-A inhibited expression of cold responsive gene TaDREB1C. Finally, subcellular localization and yeast one hybrid were used to verify that TCP21-A can act as a transcription factor to bind to the GGTCCC promoter element. Luciferase reporter gene experiment showed that TCP21-A inhibits the transcriptional activity of the TaDREB1C promoter. In summary, we systematically analyzed the expression patterns of TaTCP family members in Dn1 under cold stress and demonstrated that TaTCP21-A negatively regulated wheat cold tolerance by inhibiting expression of TaDREB1C. These results provide new insights into the functional mechanism of TaTCP transcription factors in response to cold stress.

Screening and Validation of Leaf Width-Related Genes in Inbred Maize Lines.

Leaf width is a key determinant of planting density and photosynthetic efficiency. In an effort to determine which genes regulate maize plant leaf width, we performed a genome-wide association study (GWAS) of 1.49 × 106 single nucleotide polymorphisms (SNPs) in 80 sequenced backbone inbred maize lines in Jilin Province, China, based upon phenotypic leaf width data from two years. In total, 14 SNPs were identified as being significantly related to leaf width (p < 0.000001), with these SNPs being located on chromosomes 1, 2, 3, 5, 6, 7, 8, and 9. A total of five candidate genes were identified within a mean linkage disequilibrium (LD) distance of 9.7 kb, with a significant SNP being identified within the Zm00001d044327 candidate gene. RNA was then isolated from 12 different inbred maize lines from this GWAS study cohort and was used to conduct qPCR analyses which revealed significant differences in Zm00001d044327 expression among strains exhibiting significant differences in leaf width. Based on an assessment of EMS mutant lines harboring a conserved amino acid stop mutation and two non-synonymous mutations in Zm00001d044327 that exhibited a narrow leaf width, these data suggested that Zm00001d044327 is a key regulator of maize leaf width.

A fully sequenced collection of homozygous EMS mutants for forward and reverse genetic screens in Arabidopsis thaliana.

Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

Stem lodging Resistance-1 controls stem strength by positively regulating the biosynthesis of cell wall components in Capsicum annuum L.

Lodging presents a significant challenge in cultivating high-yield crops with extensive above-ground biomass, yet the molecular mechanisms underlying this phenomenon in the Solanaceae family remain largely unexplored. In this study, we identified a gene, CaSLR1 (Capsicum annuum Stem Lodging Resistance 1), which encodes a MYELOBLASTOSIS (MYB) family transcription factor, from a lodging-affected C. annuum EMS mutant. The suppression of CaSLR1 expression in pepper led to notable stem lodging, reduced thickness of the secondary cell wall, and decreased stem strength. A similar phenotype was observed in tomato with the knockdown of SlMYB61, the orthologous gene to CaSLR1. Further investigations demonstrated that CaNAC6, a gene involved in secondary cell wall (SCW) formation, is co-expressed with CaSLR1 and acts as a positive regulator of its expression, as confirmed through yeast one-hybrid, dual-luciferase reporter assays, and electrophoretic mobility shift assays. These findings elucidate the CaNAC6-CaSLR1 module that contributes to lodging resistance, emphasizing the critical role of CaSLR1 in the lodging resistance regulatory network.

Impairment in a member of AP2/ERF and F-box family protein enhances complete panicle exsertion in rice.

Complete panicle exsertion (CPE) is an economically important quantitative trait that contributes to grain yield in rice. We deployed an integrated approach for understanding the molecular mechanism of CPE using a stable ethyl methanesulfonate mutant line, CPE-109 of the Samba Mahsuri (SM) variety of rice (Oryza sativa), which exhibits CPE. Two consistent genomic regions were identified for CPE through quantitative trait locus (QTL) mapping [qCPE-4 (28.24-31.22 Mb) and qCPE-12 (2.30-3.18 Mb)] and QTL-sequencing [chr 4 (31.21-33.69 Mb) and chr 12 (0.12-3.15 Mb)]. Two non-synonymous single nucleotide polymorphisms, namely KASP 12-12 (T→C; chr12:1269983) in Os12g0126300, encoding an AP2/ERF transcription factor, and KASP 12-16 (G→A; chr12:1515198) in Os12g0131400, encoding an F-box domain-containing protein, explained 81.05% and 59.61% of the phenotypic variance, respectively, and exhibited strong co-segregation with CPE in F2 mapping populations, advanced generation lines, and CPE-exhibiting SM mutants through KASP assays. Down-regulation of these genes in CPE-109 compared with SM (wild type) was observed in transcriptome sequencing of flag leaves, which was validated through qRT-PCR. We propose that the abrogation of Os12g0126300 and Os12g0131400 in CPE-109 combinatorially influences down-regulation of ethylene biosynthetic genes, Os01g0192900 (ACC synthase) and Os05g0497300 (ethylene-responsive factor-2), and up-regulation of a gibberellic acid synthetic gene, Os06g0569900 (ent-kaurene synthase) and the two cytokinin biosynthetic genes Os07g0486700 (cytokinin-O-glucosyltransferase 2) and Os10g0479500 (similar to carboxy-lyase), which results in complete panicle exsertion.

ZmABF4-ZmVIL2/ZmFIP37 module enhances drought tolerance in maize seedlings.

Drought, as a primary environmental factor, imposes significant constraints on developmental processes and productivity of plants. PHDs were identified as stress-responsive genes in a wide range of eukaryotes. However, the regulatory mechanisms governing PHD genes in maize under abiotic stress conditions are still largely unknown and require further investigation. Here, we identified a mutant, zmvil2, in the EMS mutant library with a C to T mutation in the exon of the Zm00001d053875 (VIN3-like protein 2, ZmVIL2), resulting in premature termination of protein coding. ZmVIL2 belongs to PHD protein family. Compared to WT, zmvil2 mutant exhibited increased sensitivity to drought stress. Consistently, overexpression of ZmVIL2 enhances drought resistance in maize. Y2H, BiFC, and Co-IP experiments revealed that ZmVIL2 directly interacts with ZmFIP37 (FKBP12-interacting protein of 37). zmfip37 knockout mutants also exhibit decreased drought tolerance. Interestingly, we demonstrated that ZmABF4 directly binds to the ZmVIL2 promoter to enhance its activity in yeast one hybrid (Y1H), electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assays. Therefore, we uncovered a novel model ZmABF4-ZmVIL2/ZmFIP37 that promotes drought tolerance in maize. Overall, these findings have enriched the knowledge of the functions of PHD genes in maize and provides genetic resources for breeding stress-tolerant maize varieties.

Rice Brown Planthopper, Nilaparvata lugens (Stål) Feeding Behaviour in Resistant NAGINA22 Rice Mutants

Background: Rice (Oryza sativa L.), is an important staple food and an excellent source of calories in India. Of the different insect pests attacking rice, brown planthopper, BPH, Nilaparvata lugens (Stål) is an important sucking pest causing upto 100% yield losses. Host plant resistance is the most important method in keeping BPH under control and understanding the underlying mechanisms of resistance is necessary to breed the resistant varieties with desirable characters. Methods: Selected EMS mutant lines of Nagina22 (N22) variety, which were previously mass screened under controlled greenhouse conditions were assessed for their antixenosis mechanism to brown planthopper feeding by measuring probing marks and honeydew excretion area by following standard procedures and statistical analysis was done by using Statistix 8.1 software. Result: Both the brown planthopper nymphs and adults probed more in the resistant mutants compared to the susceptible mutants whereas the BPH adults probed more times (24.4) compared to the nymphs (22.2). The recorded honeydew excretion area by the resistant mutants was lower than that of susceptible mutants. In general, the BPH adults fed more and excreted more honeydew (79.5 mm2) compared to the nymphs (59.0 mm2). The damage score has a negative correlation with the probing marks of adults (-0.207) and nymphs (-0.411); and a positive correlation with honeydew excretion of adults (0.547) and nymphs (0.200). In this study, the EMS N22 mutants resistant to BPH, with a greater number of probing marks and less honeydew excretion area can serve as best donors in the breeding programmes to develop brown planthopper resistant varieties.

ERECTA Modulates Seed Germination and Fruit Development via Auxin Signaling in Tomato.

Tomato (Solanum lycopersicum) breeding for improved fruit quality emphasizes selecting for desirable taste and characteristics, as well as enhancing disease resistance and yield. Seed germination is the initial step in the plant life cycle and directly affects crop productivity and yield. ERECTA (ER) is a receptor-like kinase (RLK) family protein known for its involvement in diverse developmental processes. We characterized a Micro-Tom EMS mutant designated as a knock-out mutant of sler. Our research reveals that SlER plays a central role in controlling critical traits such as inflorescence development, seed number, and seed germination. The elevation in auxin levels and alterations in the expression of ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI5 in sler seeds compared to the WT indicate that SlER modulates seed germination via auxin and abscisic acid (ABA) signaling. Additionally, we detected an increase in auxin content in the sler ovary and changes in the expression of auxin synthesis genes YUCCA flavin monooxygenases 1 (YUC1), YUC4, YUC5, and YUC6 as well as auxin response genes AUXIN RESPONSE FACTOR 5 (ARF5) and ARF7, suggesting that SlER regulates fruit development via auxin signaling.

Dynamic transcriptome profiling revealed a key gene ZmJMJ20 and pathways associated with cadmium stress in maize

Cadmium (Cd) pollution in soil poses a global concern due to its serious impacts on human health and ecological security. In plants, tremendous efforts have been made to identify some key genes and pathways in Cd stress responses. However, studies on the roles of epigenetic factors in response to Cd stress were still limited. In the study, we first gain insight into the gene expression dynamics for maize seedlings under 0 h, 12 h, and 72 h Cd stress. As a result, six distinct groups of genes were identified by hierarchical clustering and principal component analysis. The key pathways associated with 12 h Cd stress were protein modifications including protein ubiquitination, signal transduction by protein phosphorylation, and histone modification. Whereas, under 72 h stress, main pathways were involved in biological processes including phenylalanine metabolism, response to oxygen-containing compounds and metal ions. Then to be noted, one of the most highly expressed genes at 12 h under Cd treatment is annotated as histone demethylases (ZmJMJ20). The evolutionary tree analysis and domain analysis showed that ZmJMJ20 belonged to the JmjC-only subfamily of the Jumonji-C (JmjC) family, and ZmJMJ20 was conserved in rice and Arabidopsis. After 72 h of Cd treatment, the zmjmj20 mutant created by EMS treatment manifested less severe chlorosis/leaf yellowing symptoms compared with wild-type plants, and there was no significant difference in Fv/Fm and φPSII value before and after Cd treatment. Moreover, the expression levels of several photosynthesis-related down-regulated genes in EMS mutant plants were dramatically increased compared with those in wild-type plants at 12 h under Cd treatment. Our results suggested that ZmJMJ20 plays an important role in the Cd tolerance response pathway and will facilitate the development of cultivars with improved Cd stress tolerance.

Identification and functional analysis of a chromosome 2D fragment harboring TaFPF1 gene with the potential for yield improvement using a late heading wheat mutant.

A chromosome fragment influencing wheat heading and grain size was identified using mapping of m406 mutant. The study of TaFPF1 in this fragment provides more insights into wheat yield improvement. In recent years, wheat production has faced formidable challenges driven by rapid population growth and climate change, emphasizing the importance of improving specific agronomic traits such as heading date, spike length, and grain size. To identify potential genes for improving these traits, we screened a wheat EMS mutant library and identified a mutant, designated m406, which exhibited a significantly delayed heading date compared to the wild-type. Intriguingly, the mutant also displayed significantly longer spike and larger grain size. Genetic analysis revealed that a single recessive gene was responsible for the delayed heading. Surprisingly, a large 46.58 Mb deletion at the terminal region of chromosome arm 2DS in the mutant was identified through fine mapping and fluorescence in situ hybridization. Thus, the phenotypes of the mutant m406 are controlled by a group of linked genes. This deletion encompassed 917 annotated high-confidence genes, including the previously studied wheat genes Ppd1 and TaDA1, which could affect heading date and grain size. Multiple genes in this region probably contribute to the phenotypes of m406. We further investigated the function of TaFPF1 using gene editing. TaFPF1 knockout mutants showed delayed heading and increased grain size. Moreover, we identified the direct upstream gene of TaFPF1 and investigated its relationship with other important flowering genes. Our study not only identified more genes affecting heading and grain development within this deleted region but also highlighted the potential of combining these genes for improvement of wheat traits.

Genome-wide association study and haplotype analysis reveal novel candidate genes for resistance to powdery mildew in soybean.

Powdery mildew disease (PMD) is caused by the obligate biotrophic fungus Microsphaera diffusa Cooke & Peck (M. diffusa) and results in significant yield losses in soybean (Glycine max (L.) Merr.) crops. By identifying disease-resistant genes and breeding soybean accessions with enhanced resistance, we can effectively mitigate the detrimental impact of PMD on soybeans. We analyzed PMD resistance in a diversity panel of 315 soybean accessions in two locations over 3 years, and candidate genes associated with PMD resistance were identified through genome-wide association studies (GWAS), haplotype analysis, qRT-PCR, and EMS mutant analysis. Based on the GWAS approach, we identified a region on chromosome 16 (Chr16) in which 21 genes form a gene cluster that is highly correlated with PMD resistance. In order to validate and refine these findings, we conducted haplotype analysis of 21 candidate genes and indicated there are single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) variations of Glyma.16G214000, Glyma.16G214200, Glyma.16G215100 and Glyma.16G215300 within the coding and promoter regions that exhibit a strong association with resistance against PMD. Subsequent structural analysis of candidate genes within this cluster revealed that in 315 accessions, the majority of accessions exhibited resistance to PMD when Glyma.16G214300, Glyma.16G214800 and Glyma.16G215000 were complete; however, they demonstrated susceptibility to PMD when these genes were incomplete. Quantitative real-time PCR assays (qRT-PCR) of possible candidate genes showed that 14 candidate genes (Glyma.16G213700, Glyma.16G213800, Glyma.16G213900, Glyma.16G214000, Glyma.16G214200, Glyma.16G214300, Glyma.16G214500, Glyma.16G214585, Glyma.16G214669, Glyma.16G214700, Glyma.16G214800, Glyma.16G215000, Glyma.16G215100 and Glyma.16G215300) were involved in PMD resistance. Finally, we evaluated the PMD resistance of mutant lines from the Williams 82 EMS mutations library, which revealed that mutants of Glyma.16G214000, Glyma.16G214200, Glyma.16G214300, Glyma.16G214800, Glyma.16G215000, Glyma.16G215100 and Glyma.16G215300, exhibited sensitivity to PMD. Combined with the analysis results of GWAS, haplotypes, qRT-PCR and mutants, the genes Glyma.16G214000, Glyma.16G214200, Glyma.16G214300, Glyma.16G214800, Glyma.16G215000, Glyma.16G215100 and Glyma.16G215300 were identified as highly correlated with PMD resistance. The candidate genes identified above are all NLR family genes, and these discoveries deepen our understanding of the molecular basis of PMD resistance in soybeans and will be useful for guiding breeding strategies.

A novel QTL conferring Fusarium crown rot resistance on chromosome 2A in a wheat EMS mutant.

A novel QTL on chromosome 2A for Fusarium crown rot resistance was identified and validated in wheat. Fusarium crown rot (FCR) is a fungal disease that causes significant yield losses in many cereal growing regions in the world. In this study, genetic analysis was conducted for a wheat EMS mutant C549 which showed stable resistance to FCR at seedling stage. A total of 10 QTL were detected on chromosomes 1A, 2A, 3B, 4A, 6B, and 7B using a population of 138 F7 recombinant inbred lines (RILs) derived from a cross between C549 and a Chinese germplasm 3642. A novel locus Qfcr.cau-2A, which accounted for up to 24.42% of the phenotypic variation with a LOD value of 12.78, was consistently detected across all six trials conducted. Furthermore, possible effects of heading date (HD) and plant height on FCR severity were also investigated in the mapping population. While plant height had no effects on FCR resistance, a weak and negative association between FCR resistance and HD was observed. A QTL for HD (Qhd.cau-2A.2) was coincident with Qfcr.cau-2A. Conditional QTL mapping indicated that although Qfcr.cau-2A and Qhd.cau-2A.2 had significant interactions, Qfcr.cau-2A remained significant after the effects of HD was removed. It is unlikely that genes underlying these two loci are same. Nevertheless, the stable expression of Qfcr.cau-2A in the validation population of 148 F7 RILs developed between C549 and its wild parent Chuannong 16 demonstrated the potential value of this locus in FCR resistance breeding programs.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Sorghum bicolor INDETERMINATE1 is a conserved primary regulator of flowering.

A fundamental developmental switch for plants is transition from vegetative to floral growth, which integrates external and internal signals. INDETERMINATE1 (Id1) family proteins are zinc finger transcription factors that activate flowering in grasses regardless of photoperiod. Mutations in maize Id1 and rice Id1 (RID1) cause very late flowering. RID1 promotes expression of the flowering activator genes Early Heading Date1 (Ehd1) and Heading date 1 (Hd1), a rice homolog of CONSTANS (CO). Mapping of two recessive late flowering mutants from a pedigreed sorghum EMS mutant library identified two distinct mutations in the Sorghum bicolor Id1 (SbId1) homolog, mutant alleles named sbid1-1 and sbid1-2. The weaker sbid1-1 allele caused a 35 day delay in reaching boot stage in the field, but its effect was limited to 6 days under greenhouse conditions. The strong sbid1-2 allele delayed boot stage by more than 60 days in the field and under greenhouse conditions. When sbid1-1 and sbid1-2 were combined, the delayed flowering phenotype remained and resembled that of sbid1-2, confirming late flowering was due to loss of SbId1 function. Evaluation of major flowering time regulatory gene expression in sbid1-2 showed that SbId1 is needed for expression of floral activators, like SbCO and SbCN8, and repressors, like SbPRR37 and SbGhd7. These results demonstrate a conserved role for SbId1 in promotion of flowering in sorghum, where it appears to be critical to allow expression of most major flowering regulatory genes.

Identification of candidate genes associated with less-photosensitive anthocyanin phenotype using an EMS mutant (pind) in eggplant (Solanum melongena L.).

Eggplant (Solanum melongena L.) is a highly nutritious and economically important vegetable crop. However, the fruit peel of eggplant often shows poor coloration owing to low-light intensity during cultivation, especially in the winter. The less-photosensitive varieties produce anthocyanin in low light or even dark conditions, making them valuable breeding materials. Nevertheless, genes responsible for anthocyanin biosynthesis in less-photosensitive eggplant varieties are not characterized. In this study, an EMS mutant, named purple in the dark (pind), was used to identify the key genes responsible for less-photosensitive coloration. Under natural conditions, the peel color and anthocyanin content in pind fruits were similar to that of wildtype '14-345'. The bagged pind fruits were light purple, whereas those of '14-345' were white; and the anthocyanin content in the pind fruit peel was significantly higher than that in '14-345'. Genetic analysis revealed that the less-photosensitive trait was controlled by a single dominant gene. The candidate gene was mapped on chromosome 10 in the region 7.72 Mb to 11.71 Mb. Thirty-five differentially expressed genes, including 12 structural genes, such as CHS, CHI, F3H, DFR, ANS, and UFGT, and three transcription factors MYB113, GL3, and TTG2, were identified in pind using RNA-seq. Four candidate genes EGP21875 (myb domain protein 113), EGP21950 (unknown protein), EGP21953 (CAAX amino-terminal protease family protein), and EGP21961 (CAAX amino-terminal protease family protein) were identified as putative genes associated with less-photosensitive anthocyanin biosynthesis in pind. These findings may clarify the molecular mechanisms underlying less-photosensitive anthocyanin biosynthesis in eggplant.

The red/far-red light photoreceptor FvePhyB regulates tissue elongation and anthocyanin accumulation in woodland strawberry.

Light is an important environmental signal that influences plant growth and development. Among the photoreceptors, phytochromes can sense red/far-red light to coordinate various biological processes. However, their functions in strawberry are not yet known. In this study, we identified an EMS mutant, named P8, in woodland strawberry (Fragaria vesca) that showed greatly increased plant height and reduced anthocyanin content. Mapping-by-sequencing revealed that the causal mutation in FvePhyB leads to premature termination of translation. The light treatment assay revealed that FvePhyB is a bona fide red/far-red light photoreceptor, as it specifically inhibits hypocotyl length under red light. Transcriptome analysis showed that the FvePhyB mutation affects the expression levels of genes involved in hormone synthesis and signaling and anthocyanin biosynthesis in petioles and fruits. The srl mutant with a longer internode is caused by a mutation in the DELLA gene FveRGA1 (Repressor of GA1) in the gibberellin pathway. We found that the P8 srl double mutant has much longer internodes than srl, suggesting a synergistic role of FvePhyB and FveRGA1 in this process. Taken together, these results demonstrate the important role of FvePhyB in regulating plant architecture and anthocyanin content in woodland strawberry.

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.

Quantitative trait loci for rolled leaf in a wheat EMS mutant from Jagger.

Two QTLs with major effects on rolled leaf trait were consistently detected on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in the field experiments. Rolled leaf (RL) is a morphological strategy to protect plants from dehydration under stressed field conditions. Identification of quantitative trait loci (QTLs) underlining RL is essential to breed drought-tolerant wheat cultivars. A mapping population of 154 recombinant inbred lines was developed from the cross between JagMut1095, a mutant of Jagger, and Jagger to identify quantitative trait loci (QTLs) for the RL trait. A linkage map of 3106cM was constructed with 1003 unique SNPs from 21 wheat chromosomes. Two consistent QTLs were identified for RL on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in all field experiments. QRl.hwwg-1AS explained 24-56% of the phenotypic variation and QRl.hwwg-5AL explained up to 20% of the phenotypic variation. The combined percent phenotypic variation associated with the two QTLs was up to 61%. Analyses of phenotypic and genotypic data of recombinants generated from heterogeneous inbred families of JagMut1095 × Jagger delimited QRl.hwwg-1AS to a 6.04Mb physical interval. This work lays solid foundation for further fine mapping and map-based cloning of QRl.hwwg-1AS.

Identification and validation of coding and non-coding RNAs involved in high-temperature-mediated seed dormancy in common wheat.

Seed dormancy (SD) significantly decreases under high temperature (HT) environment during seed maturation, resulting in pre-harvest sprouting (PHS) damage under prolonged rainfall and wet weather during wheat harvest. However, the molecular mechanism underlying HT-mediated SD remains elusiveSeed dormancy (SD) significantly decreases under high temperature (HT) environment during seed maturation, resulting in pre-harvest sprouting (PHS) damage under prolonged rainfall and wet weather during wheat harvest. However, the molecular mechanism underlying HT-mediated SD remains elusive. Here, the wheat landrace 'Waitoubai' with strong SD and PHS resistance was treated with HT from 21 to 35 days post anthesis (DPA). Then, the seeds under HT and normal temperature (NT) environments were collected at 21 DPA, 28 DPA, and 35 DPA and subjected to whole-transcriptome sequencing. The phenotypic data showed that the seed germination percentage significantly increased, whereas SD decreased after HT treatment compared with NT, consistent with the results of previous studies. In total, 5128 mRNAs, 136 microRNAs (miRNAs), 273 long non-coding RNAs (lncRNAs), and 21 circularRNAs were found to be responsive to HT, and some of them were further verified through qRT-PCR. In particular, the known gibberellin (GA) biosynthesis gene TaGA20ox1 (TraesCS3D02G393900) was proved to be involved in HT-mediated dormancy by using the EMS-mutagenized wheat cultivar Jimai 22. Similarly, a novel gene TaCDPK21 (TraesCS7A02G267000) involved in the calcium signaling pathway was validated to be associated with HT-mediated dormancy by using the EMS mutant. Moreover, TaCDPK21 overexpression in Arabidopsis and functional complementarity tests supported the negative role of TaCDPK21 in SD. We also constructed a co-expression regulatory network based on differentially expressed mRNAs, miRNAs, and lncRNAs and found that a novel miR27319 was located at a key node of this regulatory network. Subsequently, using Arabidopsis and rice lines overexpressing miR27319 precursor or lacking miR27319 expression, we validated the positive role of miR27319 in SD and further preliminarily dissected the molecular mechanism of miR27319 underlying SD regulation through phytohormone abscisic acid and GA biosynthesis, catabolism, and signaling pathways. These findings not only broaden our understanding of the complex regulatory network of HT-mediated dormancy but also provide new gene resources for improving wheat PHS resistance to minimize PHS damage by using the molecular pyramiding approach.

STEVIA (STEVIA REBAUDIANA B.) GENOTYPES ASSESSMENT FOR LEAF YIELD STABILITY THROUGH GENOTYPE BY ENVIRONMENT INTERACTIONS, AMMI, AND GGE BIPLOT ANALYSES

Multilocation testing plays a vital role in the release of new high-yielding cultivars of stevia (Stevia rebaudiana B.) in Indonesia. In stevia, the leaf yield potential demonstrates an important characteristic for superior genotype selection. The study sought to identify the effects of genotype by environment interactions (GEI) on stevia yield and select the genotypes with stable yield resulting from radiation and hybridization through AMMI and GGE biplot analyses under three growing environments. The experiments took place in three locations, namely, Bandung, Sumedang, and Garut, West Java, Indonesia, consisting of a randomized complete block design (RCBD), with three replications. The combined analysis of variance (ANOVA) attained the genotype by environment interactions measurements. Calculating yield stability used the additive main effects and multiplicative interaction (AMMI), AMMI stability value (ASV), genotype stability index (GSI), and genotype plus genotype by environments (GGE) biplot measurements. The results revealed significant effects of environments (92.38%), followed by GEI (5.20%), and genotype effects (2.43%) of the total variation on stevia yield. The stevia genotypes viz., G11, G27, G2, and G5 gave a higher and more stable yield based on the AMMI-1 biplot. Based on the GGE biplot and the genotypes’ sustainable performances, the stevia genotypes, i.e., G27, G2, G11, G20, and G26, gained selection as stable. The three selected stevia genotypes displayed the highest yields and proved stable in three environments viz., G2 (Tamangwangu EMS mutant number A), G11 (Bogor mutant with gamma ray radiation 5 number C), and G27 (a hybrid from Garut × Bogor-3). These promising genotypes exhibit the potential for further development into new superior stevia genotypes.