A novel natural variation in the promoter of GmCHX1 regulates conditional gene expression to improve salt tolerance in soybean.

Identification and characterization of soybean germplasm and gene(s)/allele(s) for salt tolerance is an effective way to develop improved varieties for saline soils. Previous studies identified GmCHX1 (Glyma03g32900) as a major salt tolerance gene in soybean, and two main functional variations were found in the promoter region (148/150 bp insertion) and the third exon with a retrotransposon insertion (3.78 kb). In the current study, we identified four salt-tolerant soybean lines, including PI 483460B (Glycine soja), carrying the previously identified salt-sensitive variations at GmCHX1, suggesting new gene(s) or new functional allele(s) of GmCHX1 in these soybean lines. Subsequently, we conducted quantitative trait locus (QTL) mapping in a recombinant-inbred line population (Williams 82 (salt-sensitive) × PI 483460B) to identify the new salt tolerance loci/alleles. A new locus, qSalt_Gm18, was mapped on chromosome 18 associated with leaf scorch score. Another major QTL, qSalt_Gm03, was identified to be associated with chlorophyll content ratio and leaf scorch score in the same chromosomal region of GmCHX1 on chromosome 3. Novel variations in a STRE (stress response element) cis-element in the promoter region of GmCHX1 were found to regulate the salt-inducible expression of the gene in these four newly identified salt-tolerant lines including PI 483460B. This new allele of GmCHX1 with salt-inducible expression pattern provides an energy cost efficient (conditional gene expression) strategy to protect soybean yield in saline soils without yield penalty under non-stress conditions. Our results suggest that there might be no other major salt tolerance locus similar to GmCHX1 in soybean germplasm, and further improvement of salt tolerance in soybean may rely on gene-editing techniques instead of looking for natural variations.

Breeding for disease resistance in soybean: a global perspective.

KEY MESSAGE: This review provides a comprehensive atlas of QTLs, genes, and alleles conferring resistance to 28 important diseases in all major soybean production regions in the world. Breeding disease-resistant soybean [Glycine max (L.) Merr.] varieties is a common goal for soybean breeding programs to ensure the sustainability and growth of soybean production worldwide. However, due to global climate change, soybean breeders are facing strong challenges to defeat diseases. Marker-assisted selection and genomic selection have been demonstrated to be successful methods in quickly integrating vertical resistance or horizontal resistance into improved soybean varieties, where vertical resistance refers to R genes and major effect QTLs, and horizontal resistance is a combination of major and minor effect genes or QTLs. This review summarized more than 800 resistant loci/alleles and their tightly linked markers for 28 soybean diseases worldwide, caused by nematodes, oomycetes, fungi, bacteria, and viruses. The major breakthroughs in the discovery of disease resistance gene atlas of soybean were also emphasized which include: (1) identification and characterization of vertical resistance genes reside rhg1 and Rhg4 for soybean cyst nematode, and exploration of the underlying regulation mechanisms through copy number variation and (2) map-based cloning and characterization of Rps11 conferring resistance to 80% isolates of Phytophthora sojae across the USA. In this review, we also highlight the validated QTLs in overlapping genomic regions from at least two studies and applied a consistent naming nomenclature for these QTLs. Our review provides a comprehensive summary of important resistant genes/QTLs and can be used as a toolbox for soybean improvement. Finally, the summarized genetic knowledge sheds light on future directions of accelerated soybean breeding and translational genomics studies.

Genome-wide identification and characterization of NBS-encoding genes in Raphanus sativus L. and their roles related to Fusarium oxysporum resistance.

BACKGROUND: The nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes are important for plant development and disease resistance. Although genome-wide studies of NBS-encoding genes have been performed in several species, the evolution, structure, expression, and function of these genes remain unknown in radish (Raphanus sativus L.). A recently released draft R. sativus L. reference genome has facilitated the genome-wide identification and characterization of NBS-encoding genes in radish. RESULTS: A total of 225 NBS-encoding genes were identified in the radish genome based on the essential NB-ARC domain through HMM search and Pfam database, with 202 mapped onto nine chromosomes and the remaining 23 localized on different scaffolds. According to a gene structure analysis, we identified 99 NBS-LRR-type genes and 126 partial NBS-encoding genes. Additionally, 80 and 19 genes respectively encoded an N-terminal Toll/interleukin-like domain and a coiled-coil domain. Furthermore, 72% of the 202 NBS-encoding genes were grouped in 48 clusters distributed in 24 crucifer blocks on chromosomes. The U block on chromosomes R02, R04, and R08 had the most NBS-encoding genes (48), followed by the R (24), D (23), E (23), and F (17) blocks. These clusters were mostly homogeneous, containing NBS-encoding genes derived from a recent common ancestor. Tandem (15 events) and segmental (20 events) duplications were revealed in the NBS family. Comparative evolutionary analyses of orthologous genes among Arabidopsis thaliana, Brassica rapa, and Brassica oleracea reflected the importance of the NBS-LRR gene family during evolution. Moreover, examinations of cis-elements identified 70 major elements involved in responses to methyl jasmonate, abscisic acid, auxin, and salicylic acid. According to RNA-seq expression analyses, 75 NBS-encoding genes contributed to the resistance of radish to Fusarium wilt. A quantitative real-time PCR analysis revealed that RsTNL03 (Rs093020) and RsTNL09 (Rs042580) expression positively regulates radish resistance to Fusarium oxysporum, in contrast to the negative regulatory role for RsTNL06 (Rs053740). CONCLUSIONS: The NBS-encoding gene structures, tandem and segmental duplications, synteny, and expression profiles in radish were elucidated for the first time and compared with those of other Brassicaceae family members (A. thaliana, B. oleracea, and B. rapa) to clarify the evolution of the NBS gene family. These results may be useful for functionally characterizing NBS-encoding genes in radish.

QTL mapping for Fusarium wilt resistance based on the whole-genome resequencing and their association with functional genes in Raphanus sativus.

KEY MESSAGE: Two major QTL associated with resistance to Fusarium wilt (FW) were identified using whole-genome resequencing. Sequence variations and gene expression level differences suggest that TIR-NBS and LRR-RLK are candidate genes associated with FW-resistance. Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. raphani is an important disease in radish, leading to severe decrease in yield and quality. YR4 as a novel genetic source to resistant to FW was confirmed through screening with five pathogen isolates. We have generated F2 and F2:3 populations segregated with FW resistance using YR4 and YR18 inbred lines. The disease symptom was evaluated in F2:3 population (n = 180) in three independent studies over two years. We identified 4 QTL including the two major QTL (FoRsR7.159A and FoRsR9.359A). FoRsR7.159A and FoRsR9.359A were detected in three replicated experiments. FoRsR7.159A was delimited to the 2.18-Mb physical interval on chromosome R07, with a high LOD value (5.17-12.84) and explained phenotypic variation (9.34%-27.97%). The FoRsR9.359A represented relatively low LOD value (3.38-4.52) and explained phenotypic variation (6.24%-8.82%). On the basis of the re-sequencing data for the parental lines, we identified five putative resistance-related genes and 13 unknown genes with sequence variations at the gene and protein levels. A semi-quantitative RT-PCR analysis revealed that Rs382940 (TIR-NBS) and Rs382200 (RLK) were expressed only in 'YR4' from 0 to 6 days after the inoculation. Moreover, Rs382950 (TIR-NBS-LRR) was more highly expressed in 'YR4' from 3 to 6 days after the inoculation. These three genes might be important for FW-resistance in radish. We identified several markers based on these potential candidate genes. The marker set should be useful for breeding system to introduce the FW resistance loci from 'YR4' to improve tolerance to FW.

Single-base cytosine methylation analysis in fruits of three Capsicum species.

Single-base cytosine methylation analysis across fruits of Capsicum annuum, C. chinense and C. frutescens showed global average methylation ranging from 82.8-89.1%, 77.6-83.9%, and 22.4-25% at CG, CHG and CHH contexts, respectively. High gene-body methylation at CG and CHG was observed across Capsicum species. The C. annuum showed the highest proportion (>80%) of mCs at different genomic regions compared to C. chinense and C. frutescens. Cytosine methylation for transposable-elements were lower in C. frutescens compared to C. annuum and C. chinense. A total of 510,165 CG, 583112 CHG and 277,897 CHH DMRs were identified across three Capsicum species. The differentially methylated regions (DMRs) distribution analysis revealed C. frutescens as more hypo-methylated compared to C. annuum and C. chinense, and also the presence of more intergenic DMRs in Capsicum genome. At CG and CHG context, gene expression and promoter methylation showed inverse correlations. Furthermore, the observed correlation between methylation and expression of genes suggested the potential role of methylation in Capsicum fruit development/ripening.

Development and characterization of non-coding RNA based simple sequence repeat markers in Capsicum species.

Plant growth and development are largely regulated by non-coding RNAs (ncRNA); thus ncRNA based markers would be rewarding in molecular breeding. In the present study, for the first time we developed total 623 ncRNA based SSRs including 119 microRNASSRs (miRNASSRs) and 504 long non-coding RNASSRs (lncRNASSRs) distributed across 12 Capsicum chromosomes. Out of 623 ncRNASSRs, 120 (including 60 each miRNASSRs and lncRNASSRs) were used for genotyping of 96 Capsicum accessions belonging to C. annuum, C. chinense and C. frutescens; and 75% SSRs were polymorphic. Model-based and distance-based cluster analyses identified three species specific clusters, i.e. cluster-I (C. annuum), cluster-II (C. frutescens) and cluster-III (C. chinense); therefore, these SSRs may have a potential role to play in interspecific Capsicum breeding. Tissue specific expression of SSR containing ncRNAs and versatile functions of their targets suggested the usefulness of SSRs for mapping of genes/QTLs and breeding of wide range of traits in Capsicum.

Genome-Wide Identification, Evolution, and Comparative Analysis of B-Box Genes in Brassica rapa, B. oleracea, and B. napus and Their Expression Profiling in B. rapa in Response to Multiple Hormones and Abiotic Stresses.

The B-box zinc-finger transcription factors are important for plant growth, development, and various physiological processes such as photomorphogenesis, light signaling, and flowering, as well as for several biotic and abiotic stress responses. However, there is relatively little information available regarding Brassica B-box genes and their expression. In this study, we identified 51, 52, and 101 non-redundant genes encoding B-box proteins in Brassica rapa (BrBBX genes), B. oleracea (BoBBX genes), and B. napus (BnBBX genes), respectively. A whole-genome identification, characterization, and evolutionary analysis (synteny and orthology) of the B-box gene families in the diploid species B. rapa (A genome) and B. oleracea (C genome) and in the allotetraploid species B. napus (AC genome) revealed segmental duplications were the major contributors to the expansion of the BrassicaBBX gene families. The BrassicaBBX genes were classified into five subgroups according to phylogenetic relationships, gene structures, and conserved domains. Light-responsive cis-regulatory elements were detected in many of the BBX gene promoters. Additionally, BrBBX expression profiles in different tissues and in response to various abiotic stresses (heat, cold, salt, and drought) or hormones (abscisic acid, methyl jasmonate, and gibberellic acid) were analyzed by qRT-PCR. The data indicated that many B-box genes (e.g., BrBBX13, BrBBX15, and BrBBX17) may contribute to plant development and growth as well as abiotic stress tolerance. Overall, the identified BBX genes may be useful as functional genetic markers for multiple stress responses and plant developmental processes.

Integrating Omics and Gene Editing Tools for Rapid Improvement of Traditional Food Plants for Diversified and Sustainable Food Security.

Indigenous communities across the globe, especially in rural areas, consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many TFPs are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. Traditional food plants are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in situations such as COVID-19 and in times of other pandemics. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realizing the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of TFPs for their improvements, cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in several TFPs. This suggests that there is ample scope for improvement of TFPs for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques.

MiR1885 Regulates Disease Tolerance Genes in Brassica rapa during Early Infection with Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a severe disease of cruciferous crops that decreases crop quality and productivity. Several clubroot resistance-related quantitative trait loci and candidate genes have been identified. However, the underlying regulatory mechanism, the interrelationships among genes, and how genes are regulated remain unexplored. MicroRNAs (miRNAs) are attracting attention as regulators of gene expression, including during biotic stress responses. The main objective of this study was to understand how miRNAs regulate clubroot resistance-related genes in P. brassicae-infected Brassica rapa. Two Brassica miRNAs, Bra-miR1885a and Bra-miR1885b, were revealed to target TIR-NBS genes. In non-infected plants, both miRNAs were expressed at low levels to maintain the balance between plant development and basal immunity. However, their expression levels increased in P. brassicae-infected plants. Both miRNAs down-regulated the expression of the TIR-NBS genes Bra019412 and Bra019410, which are located at a clubroot resistance-related quantitative trait locus. The Bra-miR1885-mediated down-regulation of both genes was detected for up to 15 days post-inoculation in the clubroot-resistant line CR Shinki and in the clubroot-susceptible line 94SK. A qRT-PCR analysis revealed Bra019412 expression was negatively regulated by miR1885. Both Bra019412 and Bra019410 were more highly expressed in CR Shinki than in 94SK; the same expression pattern was detected in multiple clubroot-resistant and clubroot-susceptible inbred lines. A 5' rapid amplification of cDNA ends analysis confirmed the cleavage of Bra019412 by Bra-miR1885b. Thus, miR1885s potentially regulate TIR-NBS gene expression during P. brassicae infections of B. rapa.

Fine-mapping of a major QTL (Fwr1) for fusarium wilt resistance in radish.

KEY MESSAGE: A major radish QTL (Fwr1) for fusarium wilt resistance was fine-mapped. Sequence and expression analyses suggest that a gene encoding a serine/arginine-rich protein kinase is a candidate gene for Fwr1. Fusarium wilt resistance locus 1 (Fwr1) is a major quantitative trait locus (QTL) mediating the resistance of radish inbred line 'B2' to Fusarium oxysporum, which is responsible for fusarium wilt. We previously detected Fwr1 on radish linkage group 3 (i.e., chromosome 5). In this study, a high-resolution genetic map of the Fwr1 locus was constructed by analyzing 354 recombinant F2 plants derived from a cross between 'B2' and '835', the latter of which is susceptible to fusarium wilt. The Fwr1 QTL was fine-mapped to a 139.8-kb region between markers FM82 and FM87 in the middle part of chromosome 5. Fifteen candidate genes were predicted in this region based on a sequence comparison with the 'WK10039' radish reference genome. Additionally, we examined the time-course expression patterns of these predicted genes following an infection by the fusarium wilt pathogen. The ORF4 expression level was significantly higher in the resistant 'B2' plants than in the susceptible '835' plants. The ORF4 sequence was predicted to encode a serine/arginine-rich protein kinase and includes SNPs that result in nonsynonymous mutations, which may have important functional consequences. This study reveals a novel gene responsible for fusarium wilt resistance in radish. Further analyses of this gene may elucidate the molecular mechanisms underlying the fusarium wilt resistance of plants.

Analysis of bioactive components in Ghost chili (Capsicum chinense) for antioxidant, genotoxic, and apoptotic effects in mice.

Apart from using traditionally in culinary preparations, chili peppers are also important constituents of herbal medicines. Although the bioactive components are studied mostly in the fruits of Capsicum annuum, no such study reports till date is available for Ghost chili (C. chinense) from North East India. Therefore, the aim of this study was to carry out an analysis of the bioactive constituents in the naturally occurring hottest chili Ghost chili (C. chinense), and evaluate its antioxidant, pro/anti-genotoxic, and apoptotic effects in in vitro and in vivo models. Three different antioxidant assays showed that lower doses of Ghost chili extract showed higher DNA protective and antioxidant activities. Furthermore, the administration for 7 alternate days into 6 week old Swiss albino mice showed that the lower doses (50 and 100 mg/kg bw) reduced DMBA induced genotoxicity beside significantly enhancing the activities of hepatic antioxidant enzymes, while higher dose (200 mg/kg bw) induced genotoxic effect in bone marrow cells. The administration of higher dose (200 mg/kg bw) also induced apoptosis and upregulation of Bax (pro) and downregulation of Bcl-2 (anti) apoptotic genes. Dose dependent increase of apoptosis was also observed in Hep G2 and Hep 3B liver cancer cell lines. Our findings in the present study suggest that low doses of C. chinense can exert cancer chemopreventive effects. The induction of apoptosis in both cancer cell lines and mouse bone marrow cells, and up-regulation of proapoptotic genes suggests that the higher dose of C. chinense can be used for targeted cancer therapy.

Identification of genes involved in fruit development/ripening in Capsicum and development of functional markers.

The molecular mechanism of the underlying genes involved in the process of fruit ripening in Capsicum (family Solanaceae) is not clearly known. In the present study, we identified orthologs of 32 fruit development/ripening genes of tomato in Capsicum, and validated their expression in fruit development stages in C. annuum, C. frutescens, and C. chinense. In silico expression analysis using transcriptome data identified a total of 12 out of 32 genes showing differential expression during different stages of fruit development in Capsicum. Real time expression identified gene LOC107847473 (ortholog of MADS-RIN) had substantially higher expression (>500 folds) in breaker and mature fruits, which suggested the non-climacteric ripening behaviour of Capsicum. However, differential expression of Ehtylene receptor 2-like (LOC107873245) gene during fruit maturity supported the climacteric behaviour of only C. frutescens (hot pepper). Furthermore, development of 49 gene based simple sequence repeat (SSR) markers would help in selection of identified genes in Capsicum breeding.

Quantitative Trait Locus Mapping of Clubroot Resistance and Plasmodiophora brassicae Pathotype Banglim-Specific Marker Development in Brassica rapa.

Clubroot resistance is an economically important trait in Brassicaceae crops. Although many quantitative trait loci (QTLs) for clubroot resistance have been identified in Brassica, disease-related damage continues to occur owing to differences in host variety and constant pathogen variation. Here, we investigated the inheritance of clubroot resistance in a double haploid population developed by crossing clubroot resistant and susceptible lines "09CR500" and "09CR501", respectively. The resistance of "09CR500" to Plasmodiophora brassicae pathotype "Banglim" was controlled as a single dominant gene, with the segregation of resistance and susceptibility being nearly 1:1. PbBrA08Banglim was identified as having a logarithm of odds value of 7.9-74.8, and a phenotypic variance of 26.0-97.1% with flanking marker "09CR.11390652" in A08. After aligning QTL regions to the B. rapa reference genome, 11 genes were selected as candidates. PbBrA08Banglim was located near Crr1, CRs, and Rcr9 loci, but differences were validated by marker analysis, gene structural variations, and gene expression levels, as well as phenotypic responses to the pathotype. Genotyping using the "09CR.11390652" marker accurately distinguished the Banglim-resistance phenotypes in the double haploid population. Thus, the developed marker will be useful in Brassica breeding programs, marker-assisted selection, and gene pyramiding to identify and develop resistant cultivars.

Red Chinese Cabbage Transcriptome Analysis Reveals Structural Genes and Multiple Transcription Factors Regulating Reddish Purple Color.

Reddish purple Chinese cabbage (RPCC) is a popular variety of Brassica rapa (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC. Analyses of transcriptome data revealed 32,395 genes including 3345 differentially expressed genes (DEGs) between 3-week-old RPCC and green Chinese cabbage (GCC). The DEGs included 218 transcription factor (TF) genes and some functionally uncharacterized genes. Sixty DEGs identified from the transcriptome data were analyzed in 3-, 6- and 9-week old seedlings by RT-qPCR, and 35 of them had higher transcript levels in RPCC than in GCC. We detected cis-regulatory motifs of MYB, bHLH, WRKY, bZIP and AP2/ERF TFs in anthocyanin biosynthetic gene promoters. A network analysis revealed that MYB75, MYB90, and MYBL2 strongly interact with anthocyanin biosynthetic genes. Our results show that the late biosynthesis genes BrDFR, BrLDOX, BrUF3GT, BrUGT75c1-1, Br5MAT, BrAT-1, BrAT-2, BrTT19-1, and BrTT19-2 and the regulatory MYB genes BrMYB90, BrMYB75, and BrMYBL2-1 are highly expressed in RPCC, indicative of their important roles in anthocyanin biosynthesis, modification, and accumulation. Finally, we propose a model anthocyanin biosynthesis pathway that includes the unique anthocyanin pigments and genes specific to RPCC.

Genetic and physiological analyses of root cracking in radish (Raphanus sativus L.).

KEY MESSAGE: A major QTL conferring tolerance to radish (Raphanus sativus) root cracking was mapped for the first time and two calcium regulatory genes were identified that positively associated with the cracking phenomenon. Root cracking is a severe physiological disorder that significantly decreases the yield and commercial value of radish. The genetic and physiological mechanisms underlying this root cracking disorder have not been characterized. In this study, quantitative trait loci (QTLs) putatively associated with radish root cracking were mapped. Ten QTLs were distributed in six linkage groups, among these QTLs, 'RCr1' in LG1 was detected over 3 consecutive years and was considered to be a major QTL for root cracking. The QTL 'RCr1' was responsible for 4.47-18.11% of variance in the root cracking phenotype. We subsequently identified two candidate genes, RsANNAT and RsCDPK. Both genes encode proteins involved in calcium binding, ion transport, and Ca2+ signal transduction, which are important for regulating plant development and adaptations to the environment. These genes were co-localized to the major QTL region. Additionally, we analyzed physiological changes (i.e., root firmness, cell wall content, and cell-wall-bound calcium content) in two parental lines during different developmental stages. Moreover, we observed that the RsANNAT and RsCDPK expression levels are positively correlated with Ca2+ contents in the roots of the cracking-tolerant '835' cultivar. Thus, these genes may influence root cracking. The data provided herein may support the useful information to understand root cracking behavior in radish and may enable breeders to develop new cultivars exhibiting increased tolerance to root and fruit cracking.

Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate.

KEY MESSAGE: Highly tolerant herbicide-resistant transgenic rice was developed by expressing codon-modified synthetic CP4--EPSPS. The transformants could tolerate up to 1% commercial glyphosate and has the potential to be used for DSR (direct-seeded rice). Weed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T0, T1 and T2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1% of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.

Integrated analysis of DNA methylation, transcriptome, and global metabolites in interspecific heterotic Capsicum F1 hybrid.

Hybrid breeding is one of the efficacious methods of crop improvement. Here, we report our work towards understanding the molecular basis of F1 hybrid heterosis from Capsicum chinense and C. frutescens cross. Bisulfite sequencing identified a total of 70597 CG, 108797 CHG, and 38418 CHH differentially methylated regions (DMRs) across F1 hybrid and parents, and of these, 4891 DMRs showed higher methylation in F1 compared to the mid-parental methylation values (MPMV). Transcriptome analysis showed higher expression of 46-55% differentially expressed genes (DE-Gs) in the F1 hybrid. The qRT-PCR analysis of 24 DE-Gs with negative promoter methylation revealed 91.66% expression similarity with the transcriptome data. A few metabolites and 65-72% enriched genes in metabolite biosynthetic pathways showed overall increased expression in the F1 hybrid compared to parents. These findings, taken together, provided insights into the integrated role of DNA methylation, and genes and metabolites expression in the manifestation of heterosis in Capsicum.

Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli.

Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and spectacular socioeconomic gains. To attain the enhanced level of protein expression, the cry genes have to be extensively modified for RNA stability and translation efficiency in the plant systems. However, such modifications in nucleotide sequences make it difficult to express the cry genes in Escherichia coli because of the presence of E. coli rare codons. Induction of gene expression through the T7 promoter/lac operator system results in high levels of transcription but limits the availability of activated tRNA corresponding to rare codons that leads to translation stalling at ribosomes. In the present study, an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)/rifampicin combination-based approach was adopted to induce transcription of cry genes through T7 promoter/lac operator while simultaneously inhibiting the transcription of host genes through rifampicin. The results show that the IPTG/rifampicin combination leads to high-level expression of four plant codon-optimized cry genes (cry2Aa, cry1F, cry1Ac, and cry1AcF). Northern blot analysis of the cry gene expressing E. coli samples showed that the RNA expression level in the IPTG-induced samples was higher as compared to that in the IPTG/rifampicin-induced samples. Diet overlay insect bioassay of IPTG/rifampicin-induced Cry toxins with Helicoverpa armigera larvae showed bioactivity (measured as LC50) similar to the previous studies. The experiment has proved that recombinant synthetic gene (plant codon-optimized gene) with the combination of Rifampicin which inhibits DNA-dependent bacterial RNA polymerase and reduces the excessive baggage of translational machinery of the bacterial cell triggers the production of synthetic protein. Purification of protein using high pH buffer increases the solubility of the protein. Further, LC50 analysis shows no reduction of protein activity leads to protein stability. Further, purified cry toxin protein can be used for crop protection against pests and a purified form of the synthetic protein can be used for antibody production and perform the immunoassay for the identification of the transgenic plant. The crystallographic structure of synthetic protein could be used for interaction study with another insect to see insecticidal activity.

Starch content changes and metabolism-related gene regulation of Chinese cabbage synergistically induced by Plasmodiophora brassicae infection.

Clubroot is one of the major diseases adversely affecting Chinese cabbage (Brassica rapa) yield and quality. To precisely characterize the Plasmodiophora brassicae infection on Chinese cabbage, we developed a dual fluorescent staining method for simultaneously examining the pathogen, cell structures, and starch grains. The number of starch (amylopectin) grains increased in B. rapa roots infected by P. brassicae, especially from 14 to 21 days after inoculation. Therefore, the expression levels of 38 core starch metabolism genes were investigated by quantitative real-time PCR. Most genes related to starch synthesis were up-regulated at seven days after the P. brassicae inoculation, whereas the expression levels of the starch degradation-related genes increased at 14 days after the inoculation. Then genes encoding the core enzymes involved in starch metabolism were investigated by assessing their chromosomal distributions, structures, duplication events, and synteny among Brassica species. Genome comparisons indicated that 38 non-redundant genes belonging to six core gene families related to starch metabolism are highly conserved among Arabidopsis thaliana, B. rapa, Brassica nigra, and Brassica oleracea. Genome sequencing projects have revealed that P. brassicae obtained host nutrients by manipulating plant metabolism. Starch may serve as a carbon source for P. brassicae colonization as indicated by the histological observation and transcriptomic analysis. Results of this study may elucidate the evolution and expression of core starch metabolism genes and provide researchers with novel insights into the pathogenesis of clubroot in B. rapa.