Homoeologous exchanges contribute to branch angle variations in rapeseed: Insights from transcriptome, QTL-seq and gene functional analysis.

Branch angle (BA) is a critical morphological trait that significantly influences planting density, light interception and ultimately yield in plants. Despite its importance, the regulatory mechanism governing BA in rapeseed remains poorly understood. In this study, we generated 109 transcriptome data sets for 37 rapeseed accessions with divergent BA phenotypes. Relative to adaxial branch segments, abaxial segments accumulated higher levels of auxin and exhibited lower expression of six TCP1 homologues and one GA20ox3. A co-expression network analysis identified two modules highly correlated with BA. The modules contained homologues to known BA control genes, such as FUL, YUCCA6, TCP1 and SGR3. Notably, a homoeologous exchange (HE), occurring at the telomeres of A09, was prevalent in large BA accessions, while an A02-C02 HE was common in small BA accessions. In their corresponding regions, these HEs explained the formation of hub gene hotspots in the two modules. QTL-seq analysis confirmed that the presence of a large A07-C06 HE (~8.1 Mb) was also associated with a small BA phenotype, and BnaA07.WRKY40.b within it was predicted as candidate gene. Overexpressing BnaA07.WRKY40.b in rapeseed increased BA by up to 20°, while RNAi- and CRISPR-mediated mutants (BnaA07.WRKY40.b and BnaC06.WRKY40.b) exhibited decreased BA by up to 11.4°. BnaA07.WRKY40.b was exclusively localized to the nucleus and exhibited strong expression correlations with many genes related to gravitropism and plant architecture. Taken together, our study highlights the influence of HEs on rapeseed plant architecture and confirms the role of WRKY40 homologues as novel regulators of BA.

Comparative genome-wide analysis of circular RNAs in Brassica napus L.: target-site versus non-target-site resistance to herbicide stress.

Circular RNAs (circRNAs), a class of non-coding RNA molecules, are recognized for their unique functions; however, their responses to herbicide stress in Brassica napus remain unclear. In this study, the role of circRNAs in response to herbicide treatment was investigated in two rapeseed cultivars: MH33, which confers non-target-site resistance (NTSR), and EM28, which exhibits target-site resistance (TSR). The genome-wide circRNA profiles of herbicide-stressed and non-stressed seedlings were analyzed. The findings indicate that NTSR seedlings exhibited a greater abundance of circRNAs, shorter lengths of circRNAs and their parent genes, and more diverse functions of parent genes compared with TSR seedlings. Compared to normal-growth plants, the herbicide-stressed group exhibited similar trends in the number of circRNAs, functions of parent genes, and differentially expressed circRNAs as observed in NTSR seedlings. In addition, a greater number of circRNAs that function as competing microRNA (miRNA) sponges were identified in the herbicide stress and NTSR groups compared to the normal-growth and TSR groups, respectively. The differentially expressed circRNAs were validated by qPCR. The differntially expressed circRNA-miRNA networks were predicted, and the mRNAs targeted by these miRNAs were annotated. Our results suggest that circRNAs play a crucial role in responding to herbicide stress, exhibiting distinct responses between NTSR and TSR in rapeseed. These findings offer valuable insights into the mechanisms underlying herbicide resistance in rapeseed.

Synergistic mutations of two rapeseed AHAS genes confer high resistance to sulfonylurea herbicides for weed control.

KEY MESSAGE: A double mutant 5N of rapeseed was obtained with a synergistic effect of high resistance to sulfonylurea herbicide. Excellent weed control was observed in Ning R201 created by 5N resources. Sulfonylurea herbicides, which inhibit acetohydroxyacid synthase (AHAS), have become the most widely used herbicides worldwide. However, weed control in rapeseed crop production remains challenging in China due to the shortage of available herbicide-resistant cultivars. In this study, we developed a rapeseed line (PN19) with sulfonylurea herbicide resistance through seed mutagenesis. Molecular analysis revealed a Trp-574-Leu mutation in BnAHAS1-2R of PN19 according to the sequence of Arabidopsis thaliana, and an allele-specific cleaved amplified polymorphic sequence marker was developed to target the point mutation. A double mutant (5N) with very high sulfonylurea resistance was then created through pyramiding two mutant genes of PN19 and M342 by molecular marker-assisted selection. Herbicide resistance identification, toxicology testing, and an in vitro enzyme activity assay of AHAS in 5N indicated that each mutant was four and eight times more resistant to sulfonylurea than M342 and PN19, respectively. Protein structure analysis of AHAS1 demonstrated that the leucine of mutant Trp-574-Leu destroyed the original π-plane stacking effect of the local region for tribenuron-methyl binding, leading to herbicide tolerance. Isobole graph analysis showed a significant synergistic effect of the combination of two mutant genes in 5N for improved tolerance to sulfonylurea herbicides. Finally, we bred rapeseed variety Ning R201 using 5N herbicide resistance resources, and observed excellent weed control performance. Together, these results demonstrate the practical value of 5N application for optimizing and simplifying rapeseed cultivation in China.

Identification and application of markers closely linked to the restorer gene (Rfm) in rapeseed (Brassica napus L.).

The Mutsu-Isuzu cytoplasmic male sterility (MI CMS) system is one of the three-line hybrid systems used in China. As we know, the hybrid system is tightly associated with the yield variation in F1 heterosis, while the restorer gene for the MI CMS (Rfm) has not been finely mapped for further application in marker-assisted selection (MAS). In this study, the sets of near-isogenic lines (NILs) of Rfm in two different genetic backgrounds were hybridized with the genome-wide 60 K single-nucleotide polymorphism (SNP) chip of Brassica for screening the possible associated genomic region of Rfm. Through screening genotypes with SNP loci and sequencing the candidate loci, one 2.5 Mb physical region (covering three scaffolds) on chrA09 was identified as the candidate for the Rfm region. Then, the SSR markers for the target scaffolds were used to detect the recombination in an F2 population and narrowed the Rfm gene within the genetic distance of 0.52 cM, equivalent to a 350 kb physical segment. Moreover, the markers were tested to improve new elite restoration lines and to assess the percentage of hybrid seeds. Our results could potentially accelerate the map-based cloning of the Rfm gene to benefit rapeseed breeding.

Unconditional and conditional QTL analyses of seed fatty acid composition in Brassica napus L.

BACKGROUND: The fatty acid composition of B. napus' seeds determines the oil's nutritional and industrial values, and affects seed germination. Many studies have reported correlations among C16:0, C18:0, C18:1, C18:2 and C18:3 based on phenotypic data; however, the genetic basis of the fatty acid composition in B. napus is still not well understood. RESULTS: In this study, unconditional and conditional quantitative trail locus (QTL) mapping analyses were conducted using a recombinant inbred line in six environments. In total, 21 consensus QTLs each for C16:0, C18:0 and C18:2, 16 for C18:1 and 22 for C18:3 were detected by unconditional mapping. The QTLs with overlapping confidence intervals were integrated into 71 pleiotropically unique QTLs by meta-analysis. Two major QTLs, uuqA5-6 and uuqA5-7, simultaneously affected the fatty acids, except C18:0, in most of environments, with the homologous genes fatty acid desaturase 2 (FAD2) and glycerol-3-phosphate sn-2-acyltransferase 5 (GPAT5) occurring in the confidence interval of uuqA5-6, while phosphatidic acid phosphohydrolase 1 (PAH1) was assigned to uuqA5-7. Moreover, 49, 30, 48, 60 and 45 consensus QTLs were detected for C16:0, C18:0, C18:1, C18:2 and C18:3, respectively, by the conditional mapping analysis. In total, 128 unique QTLs were subsequently integrated from the 232 conditional consensus QTLs. A comparative analysis revealed that 63 unique QTLs could be identified by both mapping methodologies, and 65 additional unique QTLs were only identified in conditional mapping. CONCLUSIONS: Thus, conditional QTL mapping for fatty acids may uncover numerous additional QTLs that were inhibited by the effects of other traits. These findings provide useful information for better understanding the genetic relationships among fatty acids at the QTL level.

Neck circumference and waist circumference associated with cardiovascular events in type 2 diabetes (Beijing Community Diabetes Study 23).

Obesity increases the risk of developing cardiovascular disease and other metabolic diseases. We intended to compare three different anthropometric indicators of obesity, in predicting the incidence of cardiovascular events in Chinese type 2 diabetes. Beijing Community Diabetes Study was a prospective multi-center study conducted in Beijing community health centers. Type 2 diabetes patients from fourteen community health centers were enrolled at baseline. The primary endpoint was cardiovascular events. The upper quartile of neck circumference (NC) was set as greater NC. A total of 3299 diabetes patients were enrolled. In which, 941 (28.52%) had cardiovascular disease at baseline. Logistic analysis showed that central obesity (waist circumference (WC) above 90 cm in men and 85 cm in women) and greater NC were all related to baseline cardiovascular disease (adjusted OR = 1.49, and 1.55). After 10-year follow-up, 340 (10.31%) had cardiovascular events. Compared with patients without cardiovascular events, those having cardiovascular events had higher BMI, larger WC and NC. Cox regression analysis showed that greater WC and NC were all associated with the occurrence of cardiovascular events (adjusted HR = 1.41, and 1.38). A higher NC and WC might increase the risk of cardiovascular events by about 40% in type 2 diabetes patients in Beijing communities.

Association between Neck Circumference and the Occurrence of Cardiovascular Events in Type 2 Diabetes: Beijing Community Diabetes Study 20 (BCDS-20).

BACKGROUND: Neck circumference (NC) was found to be related to the risk factors of cardiovascular disease. However, the effects of NC on cardiovascular disease are still controversial. A prospective study of Chinese patients with type 2 diabetes was performed to evaluate the relationship between NC and cardiovascular disease. METHODS: A multicenter prospective study with eight-year follow-up was conducted in Beijing communities. Cardiovascular events were defined as myocardial infarction, unstable angina pectoris, hospitalization for heart failure, coronary revascularization, cardiac death, stroke, transient ischemic attack, and cerebral hemorrhage. RESULTS: A total of 3,009 diabetic patients were recruited. Following an eight-year follow-up, 211 patients with cardiovascular events (105 in men and 106 in women) were identified. All patients were categorized into two groups according to the upper quartile of NC (43 cm in men and 39 cm in women). The prevalence of cardiovascular events in men with an NC >43 cm (16.48%) was higher than that in the group with an NC <43 cm (8.16%, p=0.007). The prevalence of cardiovascular events in women with the NC >39 cm (10.67%) was higher compared to the group with NC <39 cm (5.31%, p=0.004). The longitudinal prevalence of cardiovascular events in groups with different NC increased with the increasing duration of follow-up (p < 0.001). Cox regression analysis showed that higher NC was associated with the occurrence of cardiovascular events after adjusting for confounding variables (adjusted HR = 2.305 (1.535-3.460)). CONCLUSIONS: NC was associated with the occurrence of cardiovascular events in type 2 diabetes in Chinese communities, and greater NC may increase the risk of cardiovascular events by about 2.3-fold.

Identification and Functional Analysis of Two New Mutant BnFAD2 Alleles That Confer Elevated Oleic Acid Content in Rapeseed.

Rapeseed (Brassica napus L.) is a vital oil crop worldwide. High oleic acid content is a desirable quality trait for rapeseed oil, which makes it more beneficial to human health. However, many germplasm resources with high oleic acid content in rapeseed have not been evaluated with regard to their genotypes, making it difficult to select the best strains with this trait for the breeding of high oleic acid rapeseed variety. This work was to explore the gene-regulation mechanism of this trait using a new super-high oleic acid content (∼85%) line N1379T as genetic material. In this study, the sequences of four homologous fatty acid desaturase (BnFAD2) genes were compared between super-high (∼85%, N1379T) and normal (∼63%) oleic acid content lines. Results showed that there were two single-nucleotide polymorphisms (SNPs) in BnFAD2-1 and BnFAD2-2, respectively, which led to the amino acid changes (E106K and G303E) in the corresponding proteins. Functional analysis of both genes in yeast confirmed that these SNPs were loss-of-function mutations, thus limiting the conversion of oleic acid to linoleic acid and resulting in the considerable accumulation of oleic acid. Moreover, two specific cleaved amplified polymorphic sequences (CAPS) markers for the two SNPs were developed to identify genotypes of each line in the F2 and BC1 populations. Furthermore, these two mutant loci of BnFAD2-1 and BnFAD2-2 genes were positively associated with elevated oleic acid levels and had a similar effect with regard to the increase of oleic acid content. Taken together, these two novel SNPs in two different BnFAD2 genes jointly regulated the high oleic acid trait in this special germplasm. The study provided insight into the genetic regulation involved in oleic acid accumulation and highlighted the use of new alleles of BnFAD2-1 and BnFAD2-2 in breeding high oleic acid rapeseed varieties.

Comparative analysis of miRNAs of two rapeseed genotypes in response to acetohydroxyacid synthase-inhibiting herbicides by high-throughput sequencing.

Acetohydroxyacid synthase (AHAS), also called acetolactate synthase, is a key enzyme involved in the first step of the biosynthesis of the branched-chain amino acids valine, isoleucine and leucine. Acetohydroxyacid synthase-inhibiting herbicides (AHAS herbicides) are five chemical families of herbicides that inhibit AHAS enzymes, including imidazolinones (IMI), sulfonylureas (SU), pyrimidinylthiobenzoates, triazolinones and triazolopyrimidines. Five AHAS genes have been identified in rapeseed, but little information is available regarding the role of miRNAs in response to AHAS herbicides. In this study, an AHAS herbicides tolerant genotype and a sensitive genotype were used for miRNA comparative analysis. A total of 20 small RNA libraries were obtained of these two genotypes at three time points (0h, 24 h and 48 h) after spraying SU and IMI herbicides with two replicates. We identified 940 conserved miRNAs and 1515 novel candidate miRNAs in Brassica napus using high-throughput sequencing methods combined with computing analysis. A total of 3284 genes were predicted to be targets of these miRNAs, and their functions were shown using GO, KOG and KEGG annotations. The differentiation expression results of miRNAs showed almost twice as many differentiated miRNAs were found in tolerant genotype M342 (309 miRNAs) after SU herbicide application than in sensitive genotype N131 (164 miRNAs). In additiond 177 and 296 miRNAs defined as differentiated in sensitive genotype and tolerant genotype in response to SU herbicides. The miR398 family was observed to be associated with AHAS herbicide tolerance because their expression increased in the tolerant genotype but decreased in the sensitive genotype. Moreover, 50 novel miRNAs from 39 precursors were predicted. There were 8 conserved miRNAs, 4 novel miRNAs and 3 target genes were validated by quantitative real-time PCR experiment. This study not only provides novel insights into the miRNA content of AHAS herbicides tolerant rapeseed in response to AHAS herbicides, but also demonstrates that miRNAs may be involved in AHAS herbicides tolerance.