Identifying long non-coding RNAs involved in heat stress response during wheat pollen development.

Introduction: Wheat is a staple food crop for over one-third of the global population. However, the stability of wheat productivity is threatened by heat waves associated with climate change. Heat stress at the reproductive stage can result in pollen sterility and failure of grain development. Methods: This study used transcriptome data analysis to explore the specific expression of long non-coding RNAs (lncRNAs) in response to heat stress during pollen development in four wheat cultivars. Results and discussion: We identified 11,054 lncRNA-producing loci, of which 5,482 lncRNAs showed differential expression in response to heat stress. Heat-responsive lncRNAs could target protein-coding genes in cis and trans and in lncRNA-miRNA-mRNA regulatory networks. Gene ontology analysis predicted that target protein-coding genes of lncRNAs regulate various biological processes such as hormonal responses, protein modification and folding, response to stress, and biosynthetic and metabolic processes. We also noted some paired lncRNA/protein-coding gene modules and some lncRNA-miRNA-mRNA regulatory modules shared in two or more wheat cultivars. These modules were related to regulating plant responses to heat stress, such as heat-shock proteins and transcription factors, and protein domains, such as MADS-box, Myc-type, and Alpha crystallin/Hsp20 domain. Conclusion: Our results provide the basic knowledge and molecular resources for future functional studies investigating wheat reproductive development under heat stress.

Overexpression of GmPIF4b affects morpho-physiological traits to reduce heat-induced grain loss in soybean.

Heat waves associated with climate change seriously threaten crop productivity. Crop seed yield depends on the success of reproduction. However, reproductive development is most vulnerable to heat stress conditions. Perception of heat and its conversion into cellular signals is a complex process. The basic helix loop helix (bHLH) transcription factor, Phytochrome Interacting Factor 4 (PIF4), plays a significant role in this process. However, studies on PIF4- mediated impacts on crop grain yield at a higher temperature are lacking. We investigated the overexpression of GmPIF4b in soybean to alleviate heat-induced damage and yield using a transgenic approach. Our results showed that under high-temperature conditions (38°C/28°C), overexpressing soybeans plants had higher chlorophyll a and b, and lower proline accumulation compared to WT. Further, overexpression of GmPIF4b improved pollen viability under heat stress and reduced heat-induced structural abnormalities in the male and female reproductive organs. Consequently, the transgenic plants produced higher pods and seeds per plant at high temperatures. Quantitative RT-PCR analysis showed that the overexpressing GmPIF4b soybeans had higher transcripts of heat shock factor, GmHSF-34, and heat-shock protein, GmHSP90A2. Collectively, our results suggest that GmPIF4b regulates multiple morpho-physiological traits for better yield under warmer climatic conditions.

Pokkali: A Naturally Evolved Salt-Tolerant Rice Shows a Distinguished Set of lncRNAs Possibly Contributing to the Tolerant Phenotype.

Pokkali is a strong representation of how stress-tolerant genotypes have evolved due to natural selection pressure. Numerous omics-based investigations have indicated different categories of stress-related genes and proteins, possibly contributing to salinity tolerance in this wild rice. However, a comprehensive study towards understanding the role of long-noncoding RNAs (lncRNAs) in the salinity response of Pokkali has not been done to date. We have identified salt-responsive lncRNAs from contrasting rice genotypes IR64 and Pokkali. A total of 63 and 81 salinity-responsive lncRNAs were differentially expressed in IR64 and Pokkali, respectively. Molecular characterization of lncRNAs and lncRNA-miRNA-mRNA interaction networks helps to explore the role of lncRNAs in the stress response. Functional annotation revealed that identified lncRNAs modulate various cellular processes, including transcriptional regulation, ion homeostasis, and secondary metabolite production. Additionally, lncRNAs were predicted to bind stress-responsive transcription factors, namely ERF, DOF, and WRKY. In addition to salinity, expression profiling was also performed under other abiotic stresses and phytohormone treatments. A positive modulation in TCONS_00035411, TCONS_00059828, and TCONS_00096512 under both abiotic stress and phytohormone treatments could be considered as being of potential interest for the further functional characterization of IncRNA. Thus, extensive analysis of lncRNAs under various treatments helps to delineate stress tolerance mechanisms and possible cross-talk.

Circular RNAs modulate the floral fate acquisition in soybean shoot apical meristem.

BACKGROUND: Soybean (Glycine max), a major oilseed and protein source, requires a short-day photoperiod for floral induction. Though key transcription factors controlling flowering have been identified, the role of the non-coding genome is limited. Circular RNAs (circRNAs) recently emerged as a novel class of RNAs with critical regulatory functions. However, a study on circRNAs during the floral transition of a crop plant is lacking. We investigated the expression and potential function of circRNAs in floral fate acquisition by soybean shoot apical meristem in response to short-day treatment. RESULTS: Using deep sequencing and in-silico analysis, we denoted 384 circRNAs, with 129 exhibiting short-day treatment-specific expression patterns. We also identified 38 circRNAs with predicted binding sites for miRNAs that could affect the expression of diverse downstream genes through the circRNA-miRNA-mRNA network. Notably, four different circRNAs with potential binding sites for an important microRNA module regulating developmental phase transition in plants, miR156 and miR172, were identified. We also identified circRNAs arising from hormonal signaling pathway genes, especially abscisic acid, and auxin, suggesting an intricate network leading to floral transition. CONCLUSIONS: This study highlights the gene regulatory complexity during the vegetative to reproductive transition and paves the way to unlock floral transition in a crop plant.

Melatonin and Abiotic Stress Tolerance in Crop Plants.

Increasing food demand by the growing human population and declining crop productivity due to climate change affect global food security. To meet the challenges, developing improved crops that can tolerate abiotic stresses is a priority. Melatonin in plants, also known as phytomelatonin, is an active component of the various cellular mechanisms that alleviates oxidative damage in plants, hence supporting the plant to survive abiotic stress conditions. Exogenous melatonin strengthens this defence mechanism by enhancing the detoxification of reactive by-products, promoting physiological activities, and upregulating stress-responsive genes to alleviate damage during abiotic stress. In addition to its well-known antioxidant activity, melatonin protects against abiotic stress by regulating plant hormones, activating ER stress-responsive genes, and increasing protein homoeostasis, heat shock transcription factors and heat shock proteins. Under abiotic stress, melatonin enhances the unfolded protein response, endoplasmic reticulum-associated protein degradation, and autophagy, which ultimately protect cells from programmed cell death and promotes cell repair resulting in increased plant survival.

Functional analysis of soybean miR156 and miR172 in tobacco highlights their role in plant morphology and floral transition.

Soybean (Glycine max), a significant oilseed and protein source for humans and livestock feed, needs short day photoperiod for floral induction. Further, soybean has a paleopolyploid genome with multiple copies of flowering genes adding to the complexity of genetic regulation of flowering, and seed set, especially in investigating the role of the noncoding genome. microRNAs, a class of noncoding RNA, play a regulatory role in plant development. miR156 and miR172 are major components of the essential regulatory hub controlling juvenile and vegetative developments and initiation of reproductive phase change leading to flowering. These microRNAs have been originally isolated and studied from model plant, Arabidopsis. However, a study on soybean microRNAs is lacking. We investigated the temporal expression patterns of gma-miR156a and gma-miR172a and found inversely related - gma-miR156a expression was higher in the vegetative stage, and gma-miR172a expression was elevated under inductive flowering conditions. The functions of gma-miR156a and gma-miR172a were evaluated via heterologous expressions in transgenic tobacco plants (Nicotiana tabacum L.). The analysis of overexpression transgenic lines highlighted that gma-miR156a plays a role in juvenile development via repression of the SPL transcription factor family. In contrast, gma-miR172a plays a pivotal role in the reproductive development phase by down-regulating its target genes, AP2. In addition, ectopic expression of gma-miR156a and gma-miR172a affected plant morphology and physiology during plant growth. Collectively, our results suggest that gma-miR156a and gma-miR172a regulate multiple morpho-physiological traits that could be used to enhance crop yield under changing climate conditions.

Genome-wide analysis reveals the crucial role of lncRNAs in regulating the expression of genes controlling pollen development.

KEY MESSAGE: The genomic location and stage-specific expression pattern of many long non-coding RNAs reveal their critical role in regulating protein-coding genes crucial in pollen developmental progression and male germ line specification. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with no apparent protein-coding potential. Multiple investigations have revealed high expression of lncRNAs in plant reproductive organs in a cell and tissue-specific manner. However, their potential role as essential regulators of molecular processes involved in sexual reproduction remains largely unexplored. We have used developing field mustard (Brassica rapa) pollen as a model system for investigating the potential role of lncRNAs in reproductive development. Reference-based transcriptome assembly performed to update the existing genome annotation identified novel expressed protein-coding genes and long non-coding RNAs (lncRNAs), including 4347 long intergenic non-coding RNAs (lincRNAs, 1058 expressed) and 2,045 lncRNAs overlapping protein-coding genes on the opposite strand (lncNATs, 780 expressed). The analysis of expression profiles reveals that lncRNAs are significant and stage-specific contributors to the gene expression profile of developing pollen. Gene co-expression networks accompanied by genome location analysis identified 38 cis-acting lincRNA, 31 cis-acting lncNAT, 7 trans-acting lincRNA and 14 trans-acting lncNAT to be substantially co-expressed with target protein-coding genes involved in biological processes regulating pollen development and male lineage specification. These findings provide a foundation for future research aiming at developing strategies to employ lncRNAs as regulatory tools for gene expression control during reproductive development.

Role of long non-coding RNAs in rice reproductive development.

Rice is a staple crop, feeding over half of the global population. The future demand of population growth and climate change requires substantial rice improvement. Recent advances in rice genomics have highlighted the vital role of the non-coding part of the genome. The protein-coding regions account for only a tiny portion of the eukaryotic genome, and most of the genomic regions transcribe copious amounts of non-coding RNAs. Of these, the long non-coding RNAs, including linear non-coding RNAs (lncRNAs) and circular non-coding RNAs (circRNAs), have been shown to play critical roles in various developmental processes by regulating the expression of genes and functions of proteins at transcriptional, post-transcriptional and post-translational levels. With the advances in next-generation sequencing technologies, a substantial number of long non-coding RNAs have been found to be expressed in plant reproductive organs in a cell- and tissue-specific manner suggesting their reproductive development-related functions. Accumulating evidence points towards the critical role of these non-coding RNAs in flowering, anther, and pollen development, ovule and seed development and photoperiod and temperature regulation of male fertility. In this mini review, we provide a brief overview of the role of the linear and circular long non-coding RNAs in rice reproductive development and control of fertility and crop yield.

Rapid Transcriptional Reprogramming Associated With Heat Stress-Induced Unfolded Protein Response in Developing Brassica napus Anthers.

Climate change associated increases in the frequency and intensity of extreme temperature events negatively impact agricultural productivity and global food security. During the reproductive phase of a plant's life cycle, such high temperatures hinder pollen development, preventing fertilization, and seed formation. At the molecular level, heat stress-induced accumulation of misfolded proteins activates a signaling pathway called unfolded protein response (UPR) in the endoplasmic reticulum (ER) and the cytoplasm to enhance the protein folding capacity of the cell. Here, we report transcriptional responses of Brassica napus anthers exposed to high temperature for 5, 15, and 30 min to decipher the rapid transcriptional reprogramming associated with the unfolded protein response. Functional classification of the upregulated transcripts highlighted rapid activation of the ER-UPR signaling pathway mediated by ER membrane-anchored transcription factor within 5 min of heat stress exposure. KEGG pathway enrichment analysis also identified "Protein processing in ER" as the most significantly enriched pathway, indicating that the unfolded protein response (UPR) is an immediate heat stress-responsive pathway during B. napus anther development. Five minutes of heat stress also led to robust induction of the cytosolic HSF-HSP heat response network. Our results present a perspective of the rapid and massive transcriptional reprogramming during heat stress in pollen development and highlight the need for investigating the nature and function of very early stress-responsive networks in plant cells. Research focusing on very early molecular responses of plant cells to external stresses has the potential to reveal new stress-responsive gene networks that can be explored further for developing climate change resilient crops.

Biological Parts for Engineering Abiotic Stress Tolerance in Plants.

It is vital to ramp up crop production dramatically by 2050 due to the increasing global population and demand for food. However, with the climate change projections showing that droughts and heatwaves becoming common in much of the globe, there is a severe threat of a sharp decline in crop yields. Thus, developing crop varieties with inbuilt genetic tolerance to environmental stresses is urgently needed. Selective breeding based on genetic diversity is not keeping up with the growing demand for food and feed. However, the emergence of contemporary plant genetic engineering, genome-editing, and synthetic biology offer precise tools for developing crops that can sustain productivity under stress conditions. Here, we summarize the systems biology-level understanding of regulatory pathways involved in perception, signalling, and protective processes activated in response to unfavourable environmental conditions. The potential role of noncoding RNAs in the regulation of abiotic stress responses has also been highlighted. Further, examples of imparting abiotic stress tolerance by genetic engineering are discussed. Additionally, we provide perspectives on the rational design of abiotic stress tolerance through synthetic biology and list various bioparts that can be used to design synthetic gene circuits whose stress-protective functions can be switched on/off in response to environmental cues.

Towards Developing Drought-smart Soybeans.

Drought is one of the significant abiotic stresses threatening crop production worldwide. Soybean is a major legume crop with immense economic significance, but its production is highly dependent on optimum rainfall or abundant irrigation. Also, in dry periods, it may require supplemental irrigation for drought-susceptible soybean varieties. The effects of drought stress on soybean including osmotic adjustments, growth morphology and yield loss have been well studied. In addition, drought-resistant soybean cultivars have been investigated for revealing the mechanisms of tolerance and survival. Advanced high-throughput technologies have yielded remarkable phenotypic and genetic information for producing drought-tolerant soybean cultivars, either through molecular breeding or transgenic approaches. Further, transcriptomics and functional genomics have led to the characterisation of new genes or gene families controlling drought response. Interestingly, genetically modified drought-smart soybeans are just beginning to be released for field applications cultivation. In this review, we focus on breeding and genetic engineering approaches that have successfully led to the development of drought-tolerant soybeans for commercial use.

Circular RNAs Repertoire and Expression Profile during Brassica rapa Pollen Development.

Circular RNAs (circRNAs) are covalently closed RNA molecules generated by the back-splicing of exons from linear precursor mRNAs. Though various linear RNAs have been shown to play important regulatory roles in many biological and developmental processes, little is known about the role of their circular counterparts. In this study, we performed high-throughput RNA sequencing to delineate the expression profile and potential function of circRNAs during the five stages of pollen development in Brassica rapa. A total of 1180 circRNAs were detected in pollen development, of which 367 showed stage-specific expression patterns. Functional enrichment and metabolic pathway analysis showed that the parent genes of circRNAs were mainly involved in pollen-related molecular and biological processes such as mitotic and meiotic cell division, DNA processes, protein synthesis, protein modification, and polysaccharide biosynthesis. Moreover, by predicting the circRNA-miRNA network from our differentially expressed circRNAs, we found 88 circRNAs with potential miRNA binding sites, suggesting their role in post-transcriptional regulation of the genes. Finally, we confirmed the back-splicing sites of nine selected circRNAs using divergent primers and Sanger sequencing. Our study presents the systematic analysis of circular RNAs during pollen development and forms the basis of future studies for unlocking complex gene regulatory networks underpinning reproduction in flowering plants.

The Role of Endoplasmic Reticulum Stress Response in Pollen Development and Heat Stress Tolerance.

Endoplasmic reticulum (ER) stress is defined by a protracted disruption in protein folding and accumulation of unfolded or misfolded proteins in the ER. This accumulation of unfolded proteins can result from excessive demands on the protein folding machinery triggered by environmental and cellular stresses such as nutrient deficiencies, oxidative stress, pathogens, and heat. The cell responds to ER stress by activating a protective pathway termed unfolded protein response (UPR), which comprises cellular mechanisms targeted to maintain cellular homeostasis by increasing the ER's protein folding capacity. The UPR is especially significant for plants as being sessile requires them to adapt to multiple environmental stresses. While multiple stresses trigger the UPR at the vegetative stage, it appears to be active constitutively in the anthers of unstressed plants. Transcriptome analysis reveals significant upregulation of ER stress-related transcripts in diploid meiocytes and haploid microspores. Interestingly, several ER stress-related genes are specifically upregulated in the sperm cells. The analysis of gene knockout mutants in Arabidopsis has revealed that defects in ER stress response lead to the failure of normal pollen development and enhanced susceptibility of male gametophyte to heat stress conditions. In this mini-review, we provide an overview of the role of ER stress and UPR in pollen development and its protective roles in maintaining male fertility under heat stress conditions.

A dynamic intron retention program regulates the expression of several hundred genes during pollen meiosis.

KEY MESSAGE: Intron retention is a stage-specific mechanism of functional attenuation of a subset of co-regulated, functionally related genes during early stages of pollen development. To improve our understanding of the gene regulatory mechanisms that drive developmental processes, we performed a genome-wide study of alternative splicing and isoform switching during five key stages of pollen development in field mustard, Brassica rapa. Surprisingly, for several hundred genes (12.3% of the genes analysed), isoform switching results in stage-specific expression of intron-retaining transcripts at the meiotic stage of pollen development. In such cases, we report temporally regulated switching between expression of a canonical, translatable isoform and an intron-retaining transcript that is predicted to produce a truncated and presumably inactive protein. The results suggest a new pervasive mechanism underlying modulation of protein levels in a plant developmental program. The effect is not based on gene expression induction but on the type of transcript produced. We conclude that intron retention is a stage-specific mechanism of functional attenuation of a subset of co-regulated, functionally related genes during meiosis, especially genes related to ribosome biogenesis, mRNA transport and nuclear envelope architecture. We also propose that stage-specific expression of a non-functional isoform of Brassica rapa BrSDG8, a non-redundant member of histone methyltransferase gene family, linked to alternative splicing regulation, may contribute to the intron retention observed.

Genome-Wide In Silico Identification and Comparative Analysis of Dof Gene Family in Brassica napus.

DNA binding with one finger (DOF) proteins are plant-specific transcription factors that play roles in diverse plant functions. However, little is known about the DOF protein repertoire of the allopolyploid crop, Brassica napus. This in silico study identified 117 Brassica napus Dof genes (BnaDofs) and classified them into nine groups (A, B1, B2, C1, C2.1, C2.2, C3, D1, and D2), based on phylogenetic analysis. Most members belonging to a particular group displayed conserved gene structural organisation and protein motif distribution. Evolutionary analysis exemplified that the divergence of the Brassica genus from Arabidopsis, the whole-genome triplication event, and the hybridisation of Brassica oleracea and Brassica rapa to form B. napus, followed by gene loss and rearrangements, led to the expansion and divergence of the Dof transcription factor (TF) gene family in B. napus. So far, this is the largest number of Dof genes reported in a single eudicot species. Functional annotation of BnaDof proteins, cis-element analysis of their promoters, and transcriptomic analysis suggested potential roles in organ development, the transition from the vegetative to the reproductive stage, light responsiveness, phytohormone responsiveness, as well as potential regulatory roles in abiotic stress. Overall, our results provide a comprehensive understanding of the molecular structure, evolution, and possible functional roles of Dof genes in plant development and abiotic stress response.

Molecular characterization of a soybean FT homologue, GmFT7.

Soybean (Glycine max) is a vital oilseed legume crop that provides protein and oil for humans and feedstock for animals. Flowering is a prerequisite for seed production. Floral transition, from vegetative to reproductive stage, in a plant, is regulated by environmental (light, temperature) and endogenous factors. In Arabidopsis, Flowering Locus T (FT) protein is shown to be a mobile signal that moves from leaf to shoot apical meristem to induce flowering. However, FTs role in soybean is not fully resolved due to the presence of multiple (ten) homologs in the genome. Two of the ten FT homologs (GmFT2a and GmFT5a) have a role in the floral transition while GmFT1a and GmFT4 suppress soybean flowering. Recent deep sequencing data revealed that six FT homologs are expressed in shoot apical meristem and leaves during floral transition. One FT homolog, GmFT7 showed strong expression during soybean floral transition. Though bioinformatic analyses revealed that GmFT7 had high similarity with GmFT2a, ectopic GmFT7 expression in Arabidopsis could not promote flowering or rescue the late-flowering phenotype of Arabidopsis ft-10 mutant.

Global Role of Crop Genomics in the Face of Climate Change.

The development of climate change resilient crops is necessary if we are to meet the challenge of feeding the growing world's population. We must be able to increase food production despite the projected decrease in arable land and unpredictable environmental conditions. This review summarizes the technological and conceptual advances that have the potential to transform plant breeding, help overcome the challenges of climate change, and initiate the next plant breeding revolution. Recent developments in genomics in combination with high-throughput and precision phenotyping facilitate the identification of genes controlling critical agronomic traits. The discovery of these genes can now be paired with genome editing techniques to rapidly develop climate change resilient crops, including plants with better biotic and abiotic stress tolerance and enhanced nutritional value. Utilizing the genetic potential of crop wild relatives (CWRs) enables the domestication of new species and the generation of synthetic polyploids. The high-quality crop plant genome assemblies and annotations provide new, exciting research targets, including long non-coding RNAs (lncRNAs) and cis-regulatory regions. Metagenomic studies give insights into plant-microbiome interactions and guide selection of optimal soils for plant cultivation. Together, all these advances will allow breeders to produce improved, resilient crops in relatively short timeframes meeting the demands of the growing population and changing climate.

Rice 3D chromatin structure correlates with sequence variation and meiotic recombination rate.

Genomes of many eukaryotic species have a defined three-dimensional architecture critical for cellular processes. They are partitioned into topologically associated domains (TADs), defined as regions of high chromatin inter-connectivity. While TADs are not a prominent feature of A. thaliana genome organization, they have been reported for other plants including rice, maize, tomato and cotton and for which TAD formation appears to be linked to transcription and chromatin epigenetic status. Here we show that in the rice genome, sequence variation and meiotic recombination rate correlate with the 3D genome structure. TADs display increased SNP and SV density and higher recombination rate compared to inter-TAD regions. We associate the observed differences with the TAD epigenetic landscape, TE composition and an increased incidence of meiotic crossovers.

Engineering Multiple Abiotic Stress Tolerance in Canola, Brassica napus.

Impacts of climate change like global warming, drought, flooding, and other extreme events are posing severe challenges to global crop production. Contribution of Brassica napus towards the oilseed industry makes it an essential component of international trade and agroeconomics. Consequences from increasing occurrences of multiple abiotic stresses on this crop are leading to agroeconomic losses making it vital to endow B. napus crop with an ability to survive and maintain yield when faced with simultaneous exposure to multiple abiotic stresses. For an improved understanding of the stress sensing machinery, there is a need for analyzing regulatory pathways of multiple stress-responsive genes and other regulatory elements such as non-coding RNAs. However, our understanding of these pathways and their interactions in B. napus is far from complete. This review outlines the current knowledge of stress-responsive genes and their role in imparting multiple stress tolerance in B. napus. Analysis of network cross-talk through omics data mining is now making it possible to unravel the underlying complexity required for stress sensing and signaling in plants. Novel biotechnological approaches such as transgene-free genome editing and utilization of nanoparticles as gene delivery tools are also discussed. These can contribute to providing solutions for developing climate change resilient B. napus varieties with reduced regulatory limitations. The potential ability of synthetic biology to engineer and modify networks through fine-tuning of stress regulatory elements for plant responses to stress adaption is also highlighted.

RNA-Seq Highlights Molecular Events Associated With Impaired Pollen-Pistil Interactions Following Short-Term Heat Stress in Brassica napus.

The global climate change is leading to increased frequency of heatwaves with crops getting exposed to extreme temperature events. Such temperature spikes during the reproductive stage of plant development can harm crop fertility and productivity. Here we report the response of short-term heat stress events on the pollen and pistil tissues in a commercially grown cultivar of Brassica napus. Our data reveals that short-term temperature spikes not only affect pollen fitness but also impair the ability of the pistil to support pollen germination and pollen tube growth and that the heat stress sensitivity of pistil can have severe consequences for seed set and yield. Comparative transcriptome profiling of non-stressed and heat-stressed (40°C for 30 min) pollen and pistil (stigma + style) highlighted the underlying cellular mechanisms involved in heat stress response in these reproductive tissues. In pollen, cell wall organization and cellular transport-related genes possibly regulate pollen fitness under heat stress while the heat stress-induced repression of transcription factor encoding transcripts is a feature of the pistil response. Overall, high temperature altered the expression of genes involved in protein processing, regulation of transcription, pollen-pistil interactions, and misregulation of cellular organization, transport, and metabolism. Our results show that short episodes of high-temperature exposure in B. napus modulate key regulatory pathways disrupted reproductive processes, ultimately translating to yield loss. Further investigations on the genes and networks identified in the present study pave a way toward genetic improvement of the thermotolerance and reproductive performance of B. napus varieties.