Summary
Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid compared with its parents. The underlying molecular basis for heterosis, particularly ...for allopolyploids, remains elusive. In this study we analyzed the transcriptomes of Brassica napus parental lines and their F1 hybrids at three stages of early flower development. Phenotypically, the F1 hybrids show remarkable heterosis in silique number and grain yield. Transcriptome analysis revealed that various phytohormone (auxin and salicylic acid) response genes are significantly altered in the F1 hybrids relative to the parental lines. We also found evidence for decreased expression divergence of the homoeologous gene pairs in the allopolyploid F1 hybrids and suggest that high‐parental expression‐level dominance plays an important role in heterosis. Small RNA and methylation studies aimed at examining the epigenetic effect of the changes in gene expression level in the F1 hybrids showed that the majority of the small interfering RNA (siRNA) clusters had a higher expression level in the F1 hybrids than in the parents, and that there was an increase in genome‐wide DNA methylation in the F1 hybrid. Transposable elements associated with siRNA clusters had a higher level of methylation and a lower expression level in the F1 hybrid, implying that the non‐additively expressed siRNA clusters resulted in lower activity of the transposable elements through DNA methylation in the hybrid. Our data provide insights into the role that changes in gene expression pattern and epigenetic mechanisms contribute to heterosis during early flower development in allopolyploid B. napus.
Significance Statement
We found that the majority of small interfering RNA clusters had a higher expression level in F1 hybrids than in the parents, and there was an increase in the genome‐wide DNA methylation of F1 hybrids. Our data provide insights into the role that changes in gene expression pattern and epigenetic mechanisms play in heterosis during early flower development in allopolyploid B. napus.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Flower color is an important trait for the ornamental value of colored rapeseed (
L.), as the plant is becoming more popular. However, the color fading of red petals of rapeseed is a problem for its ...utilization. Unfortunately, the mechanism for the process of color fading in rapeseed is unknown. In the current study, a red flower line, Zhehuhong, was used as plant material to analyze the alterations in its morphological and physiological characteristics, including pigment and phytohormone content, 2 d before flowering (T1), at flowering (T2), and 2 d after flowering (T3). Further, metabolomics and transcriptomics analyses were also performed to reveal the molecular regulation of petal fading. The results show that epidermal cells changed from spherical and tightly arranged to totally collapsed from T1 to T3, according to both paraffin section and scanning electron microscope observation. The pH value and all pigment content except flavonoids decreased significantly during petal fading. The anthocyanin content was reduced by 60.3% at T3 compared to T1. The content of three phytohormones, 1-aminocyclopropanecarboxylic acid, melatonin, and salicylic acid, increased significantly by 2.2, 1.1, and 30.3 times, respectively, from T1 to T3. However, auxin, abscisic acid, and jasmonic acid content decreased from T1 to T3. The result of metabolomics analysis shows that the content of six detected anthocyanin components (cyanidin, peonidin, pelargonidin, delphinidin, petunidin, and malvidin) and their derivatives mainly exhibited a decreasing trend, which was in accordance with the trend of decreasing anthocyanin. Transcriptomics analysis showed downregulation of genes involved in flavonol, flavonoid, and anthocyanin biosynthesis. Furthermore, genes regulating anthocyanin biosynthesis were preferentially expressed at early stages, indicating that the degradation of anthocyanin is the main issue during color fading. The corresponding gene-encoding phytohormone biosynthesis and signaling, JASMONATE-ZIM-DOMAIN PROTEIN, was deactivated to repress anthocyanin biosynthesis, resulting in fading petal color. The results clearly suggest that anthocyanin degradation and phytohormone regulation play essential roles in petal color fading in rapeseed, which is a useful insight for the breeding of colored rapeseed.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The utilization of heterosis is a successful strategy in increasing yield for many crops. However, it consumes tremendous manpower to test the combining ability of the parents in fields. Here, we ...applied the genomic-selection (GS) strategy and developed models that significantly increase the predictability of heterosis by introducing the concept of a regional parental genetic-similarity index (PGSI) and reducing dimension in the calculation matrix in a machine-learning approach. Overall, PGSI negatively affected grain yield and several other traits but positively influenced the thousand-seed weight of the hybrids. It was found that the C subgenome of rapeseed had a greater impact on heterosis than the A subgenome. We drew maps with overviews of quantitative-trait loci that were responsible for the heterosis (h-QTLs) of various agronomic traits. Identifications and annotations of genes underlying high impacting h-QTLs were provided. Using models that we elaborated, combining abilities between an Ogu-CMS-pool member and a potential restorer can be simulated in silico, sidestepping laborious work, such as testing crosses in fields. The achievements here provide a case of heterosis prediction in polyploid genomes with relatively large genome sizes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Colorful flowers of rapeseed (
Brassica napus
L.) have been a hotspot for researchers, but the underlying mechanisms of pigment formation still need to be clarified. In this study, two stages of ...unopened rapeseed petals with red, white, and yellow colors were selected to identify the metabolites and genes involved in red pigment formation. Metabolomic analysis showed that flavonoids enriched the most co-differentially accumulated metabolites among all categories, and showed higher accumulation in red petal rapeseed than in white and yellow petal ones. RNA-seq analysis showed that among co-differentially expressed genes involved in red pigment formation, genes involved in anthocyanin (belonging to flavonoids) biosynthesis pathway were largely regulated by
ANS, DFR
, and
UF3GT
. The expression of those genes was higher in red petals of rapeseed than in white and yellow petals ones as well. Results of RNA interference of
BnaA03.ANS
in red rapeseed altered petal colors from raspberry red to beige red and zinc yellow under different interference levels, with the contents of pelargonidin, cyanidin, lutein, neoxanthin, β-carotene, and lycopene significantly decreased. However, overexpression of
BnaA03.ANS
in yellow rapeseed petals did not change the color of yellow petals. This study confirmed the important function of flavonoids, especially anthocyanins on red pigment formation, and for the first time, identified the irreplaceable role of
BnaA03.ANS
on red-flowered rapeseed.
Accelerating the differentiation of floral meristem (FM) from shoot apical meristems (SAM) which determines the conversion from vegetative to reproductive growth is of great significance for the ...production of rapeseed (
Brassica napus
L.). In this research, the mechanisms of different nitrogen (N) application rates (low N, N1; normal N, N2; and high N, N3) on different FM development stages triggering the regulation of FM differentiation genes through the auxin biosynthetic and signal transduction were investigated. We found that the stage of FM differentiation, which was identified through a stereomicroscope and scanning electron microscope, came 4 and 7 days earlier under high N rate than under normal and low N levels, with the seed yield increased by 11.1 and 22.6%, respectively. Analysis of the auxin and its derivatives contents showed that the main biosynthesis way of auxin was the indole acetaldehyde oxime (IAOx) pathway, with 3-Indole acetonitrile dramatically accumulated during FM differentiation. At the same time, an obvious decrease of IAA contents at each FM differentiation stage was detected, and then gradually rose. Results of the expression of genes involved in auxin biosynthesis, auxin signaling transduction, and FM identification under five FM differentiation stages and three nitrogen application rates showed that genes involved in auxin biosynthesis were regulated before the FM differentiation stage, while the regulation of FM identity genes appeared mainly at the middle and later periods of the five stages, and the regulation level of genes varied under different N rates. Taken together, a high nitrogen rate could accelerate the initiation of FM differentiation, and auxin involved a lot in this regulation.
Enhancing oil content is one of the major goals in
Brassica napus
breeding; however, genetic regulation of seed oil content in plants is complex and not fully elucidated. In this study, we report ...proteins that were differentially accumulated in immature seeds of 35 days after anthesis between two recombinant inbred lines with contrasting seed oil content, high oil content line (HOCL) and low oil content line (LOCL) using a multiplex isobaric tandem mass tags (TMT)-based quantitative proteomic approach. Over 4,600 proteins were quantified in seeds of the two lines, and 342 proteins showed differential accumulation between seeds of HOCL and LOCL. Gene Ontology enrichment analysis revealed that the differentially accumulated proteins were enriched in proteins involved in lipid biosynthesis and metabolism, photosynthesis, and nutrient reservoir activity. Western blot confirmed the increased abundance of a late embryogenesis abundant protein (BnLEA57) in HOCL seeds compared with LOCL seeds, and overexpression of either
BnLEA57
gene or its homology
BnLEA55
in transgenic
Arabidopsis thaliana
enhanced oil content in
Arabidopsis
seeds. Our work provides new insights into the molecular regulatory mechanism of seed oil content in
B. napus
.
Nitrogen is essential for improving the seed oil yield of rapeseed (Brassica napus L.). However, the molecular mechanism by which increased nitrogen rates impact seed oil content is largely unknown. ...Therefore, a field experiment was conducted to determine how three nitrogen application rates (120, 240, and 360 kg ha−1) regulated seed oil content via transcriptomic analysis. The results showed that the seed yield and the protein and total N contents increased from N1 to N3, with average increases of 57.2%, 16.9%, and 79.5%, respectively. However, the seed oil content significantly decreased from N1 to N3, with an average decrease of 8.6%. These results were repeated over a number of years. The quantity of oil protein bodies observed under a transmission electron microscope was in accordance with the ultimate seed oil and protein contents. As the nitrogen application rate increased, a substantial number of genes involved in the photosynthesis, glycolysis, and phenylpropanoid biosynthesis pathways were up-regulated, as were TF families, such as AP2/ERF, MYB, and NAC. The newly identified genes were mainly involved in carbohydrate, lipid, and amino acid metabolism. Metabolic flux analysis showed that most of the genes involved in glycolysis and fatty acid biosynthesis had higher transcript levels in the early development stages. Our results provide new insights into the molecular regulation of rapeseed seed oil content through increased nitrogen application rates.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Long noncoding RNAs (lncRNAs) are transcripts longer than 200 bp that do not encode proteins but nonetheless have been shown to play important roles in various biological processes in plants. ...Brassica napus is an important seed oil crop worldwide and the target of many genetic improvement activities. To understand better the function of lncRNAs in regulating plant metabolic activities, we carried out a genome-wide lncRNA identification of lncRNAs in Brassica napus with a focus on lncRNAs involved in lipid metabolism. Twenty ribosomal RNA depleted strand specific RNA-seq (ssRNA-seq) datasets were generatred using RNAs isolated from B. napus seeds at four developmental stages. For comparison we also included 30 publically available RNA-seq datasets generated from poly(A) enriched mRNAs isolated from from various Brassica napus tissues in our analysis.
A total of 8905 lncRNA loci were identified, including 7100 long intergenic noncoding RNA (lincRNA) loci and 1805 loci generating long noncoding natural antisense transcript (lncNAT). Many lncRNAs were identified only in the ssRNA-seq and poly(A) RNA-seq dataset, suggesting that B. napus has a large lncRNA repertoire and it is necessary to use libraries prepared from different tissues and developmental stages as well as different library preparation approaches to capture the whole spectrum of lncRNAs. Analysis of coexpression networks revealed that among the regulatory modules are networks containing lncRNAs and protein-coding genes related to oil biosynthesis indicating a possible role of lncRNAs in the control of lipid metabolism. One such example is that several lncRNAs are potential regulators of BnaC08g11970D that encodes oleosin1, a protein found in oil bodies and involved in seed lipid accumulation. We also observed that the expression levels of B. napus lncRNAs is positively correlated with their conservation levels.
We demonstrated that the B. napus genome has a large number of lncRNA and that these lncRNAs are expressed broadly across many developmental times and in different tissue types. We also provide evidence indicating that specific lncRNAs appear to be important regulators of lipid biosynthesis forming regulatory networks with transcripts involved in lipid biosynthesis. We also provide evidence that these lncRNAs are conserved in other species of the Brassicaceae family.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Creating a homologous restorer line for Ogura cytoplasmic male sterility (Ogu-CMS) in Brassica napus is meaningful for the wider application of Ogu-CMS system in rapeseed production. Previously, an ...independent development of a new Ogu-CMS restorer line (CLR650) was reported locally from crossing between Raphanobrassica (AACCRR, 2n = 56) and B. napus and a new version of Ogu CMS lines CLR6430 derived from CLR650 was characterized in this study. The results showed that the fertility restoration gene in CLR6430 presented a distorted segregation in different segregating populations. However, the majority of somatic cells from roots had a regular chromosome number (2n = 38) and no radish signal covered a whole chromosome was detected using GISH. Thirty-two specific markers derived from the introgressed radish fragments were developed based on the re-sequencing results. Unique radish insertions and differences between CLR6430 and R2000 were also identified through both radish-derived markers and PCR product sequences. Further investigations on the genetic behaviors, interactions between the fertility restoration and other traits and specific molecular markers to the introgression in CLR6430 were also conducted in this study. These results should provide the evidence of nucleotide differences between CLR6430 and R2000, and the specific markers will be helpful for breeding new Ogura restore lines in future.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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