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.

Genetic Diversity Analysis of Brassica Yellows Virus Causing Aberrant Color Symptoms in Oilseed Rape.

The emergence of brassica yellow virus (BrYV) has increasingly damaged crucifer crops in China in recent years. In 2020, a large number of oilseed rape in Jiangsu showed aberrant leaf color. A combined RNA-seq and RT-PCR analysis identified BrYV as the major viral pathogen. A subsequent field survey showed that the average incidence of BrYV was 32.04%. In addition to BrYV, turnip mosaic virus (TuMV) was also frequently detected. As a result, two near full-length BrYV isolates, BrYV-814NJLH and BrYV-NJ13, were cloned. Based on the newly obtained sequences and the reported BrYV and turnip yellow virus (TuYV) isolates, a phylogenetic analysis was performed, and it was found that all BrYV isolates share a common root with TuYV. Pairwise amino acid identity analysis revealed that both P2 and P3 were conserved in BrYV. Additionally, recombination analysis revealed seven recombinant events in BrYV as TuYV. We also attempted to determine BrYV infection by quantitative leaf color index, but no significant correlation was found between the two. Systemic observations indicated that BrYV-infected plants had different symptoms, such as no symptom, purple stem base and red old leaves. Overall, our work proves that BrYV is closely related to TuYV and could be considered as an epidemic strain for oilseed rape in Jiangsu.

Genetic Dissection and Germplasm Selection of the Low Crude Fiber Component in Brassica napus L. Shoots.

BACKGROUND: Brassica napus is one of the most important oil crops in the world, and B. napus shoots are nutrient-rich fresh vegetables. The crude fiber (CF) component is one of the most important factors affecting the taste quality of B. napus shoots, but the factors underlying the desirable low-CF trait remain poorly understood. METHODS: In this study, a high-density single-nucleotide polymorphism (SNP) map was used to map quantitative trait loci (QTLs) for five CF-related traits in a recombinant inbred population. RESULTS: A total of 49 QTLs were obtained in four environments, including eleven, twelve, eight, twelve and six QTLs for content of neutral detergent fiber, acid detergent fiber, acid detergent lignin, hemicellulose and cellulose, respectively. The phenotypic variation explained by single QTL ranged from 4.62% to 14.76%. Eight of these QTLs were further integrated into four unique QTLs, which controlled two different traits simultaneously. Five CF-component-related candidate genes were identified, among which BnaC03g07110D and BnaC07g21271D were considered to be the most likely candidate genes. In addition, five lines with low CF content were selected, which can be used as excellent germplasm resources in breeding. CONCLUSIONS: The QTLs identified in this study will contribute to our understanding of the genetic mechanism of CF and can be used as targets for reducing CF content in B. napus shoots. In addition, this study also provided excellent germplasm resources for low CF content breeding.

The complete mitochondrial genome sequences of Brassica napus varieties NY18 and 088018.

Brassica napus variety NY18 and 088018 are female and male parents of the national registered variety Ningza 1818, respectively. Here, we determined the complete mitochondrial genomes of these two varieties. The genome sizes of NY18 and 088018 were 221,864 bp and 222,015 bp, respectively. Both genomes contained 40 protein-coding genes, 21 tRNA genes, and three rRNA genes. Considerable structural variations existed between the two mitochondrial genomes, which were separated into five syntenic regions. Phylogenetic analysis using the maximum-likelihood method showed that the mitochondrial sequences of B. napus were closely clustered, forming a single clade which had a relatively close relationship with the clade formed by B. rapa, B. juncea, and B. oleracea.

Genome-Wide Identification and Characterization of the IGT Gene Family in Allotetraploid Rapeseed (Brassica napus L.).

IGT family genes function critically to regulate lateral organ orientation in plants. However, little information is available about this family of genes in Brassica napus. In this study, 27 BnIGT genes were identified on 16 chromosomes and divided into seven clades, namely LAZY1∼LAZY6 and TAC1 (Tiller Angle Control 1), based on their phylogenetic relationships. Duplication analysis revealed that 91.1% of the gene pairs were derived from whole-genome duplication. Most BnIGT genes had a similar structural pattern with one or two very short exons followed by a long and a shorter exon. Common and specific motifs were identified among the seven clades, and motif 1, containing the family-specific GφL(A/T)IGT sequence, was observed in all clades except LAZY5. Three types of cis-elements pertinent to transcription factor binding, light responses, and hormone signaling were detected in the BnIGT promoters. Intriguingly, more than half of the BnIGT genes exhibited no or very low expression in various tissues, and the LAZY1 and TAC1 clade members showed distinct tissue expression preferences. Coexpression analysis revealed that the LAZY1 members had strong associations with cell wall biosynthesis genes. This analysis provides a deeper understanding of the BnIGT gene family and will facilitate further deduction of their role in regulating plant architecture in B. napus.

Arabidopsis UBC22, an E2 able to catalyze lysine-11 specific ubiquitin linkage formation, has multiple functions in plant growth and immunity.

Protein ubiquitination is critical for various biological processes in eukaryotes. A ubiquitin (Ub) chain can be linked through one of the seven lysine (K) residues or the N-terminus methionine of the Ub, and the Ub-conjugating enzymes called E2s play a critical role in determining the linkage specificity of Ub chains. Further, while K48-linked polyubiquitin chain is important for protein degradation, much less is known about the functions of other types of polyubiquitin chains in plants. We showed previously that UBC22 is unique in its ability to catalyze K11-dependent Ub dimer formation in vitro and ubc22 knockout mutants had defects in megasporogenesis. In this study, further analyses of the Arabidopsis ubc22 mutants revealed four subtypes of plants based on the phenotypic changes in vegetative growth. These four subtypes appeared consistently in the plants of three independent ubc22 mutants. Transcriptomic analysis showed that transcript levels of genes related to several pathways were altered differently in different subtypes of mutant plants. In one subtype, the mutant plants had increased expression of genes related to plant defenses and showed enhanced resistance to a necrotrophic plant pathogen. These results suggest multiple functions of UBC22 during plant development and stress response.

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.

Quantitative Trait Transcripts Mapping Coupled with Expression Quantitative Trait Loci Mapping Reveal the Molecular Network Regulating the Apetalous Characteristic in Brassica napus L.

The apetalous trait of rapeseed (Brassica napus, AACC, 2n = 38) is important for breeding an ideal high-yield rapeseed with superior klendusity to Sclerotinia sclerotiorum. Currently, the molecular mechanism underlying the apetalous trait of rapeseed is unclear. In this study, 14 petal regulators genes were chosen as target genes (TGs), and the expression patterns of the 14 TGs in the AH population, containing 189 recombinant inbred lines derived from a cross between apetalous "APL01" and normal "Holly," were analyzed in two environments using qRT-PCR. Phenotypic data of petalous degree (PDgr) in the AH population were obtained from the two environments. Both quantitative trait transcript (QTT)-association mapping and expression QTL (eQTL) analyses of TGs expression levels were performed to reveal regulatory relationships among TGs and PDgr. QTT mapping for PDgr determined that PLURIPETALA (PLP) was the major negative QTT associated with PDgr in both environments, suggesting that PLP negatively regulates the petal development of line "APL01." The QTT mapping of PLP expression levels showed that CHROMATIN-REMODELING PROTEIN 11 (CHR11) was positively associated with PLP expression, indicating that CHR11 acts as a positive regulator of PLP expression. Similarly, QTT mapping for the remaining TGs identified 38 QTTs, associated with 13 TGs, and 31 QTTs, associated with 10 TGs, respectively, in the first and second environments. Additionally, eQTL analyses of TG expression levels showed that 12 and 11 unconditional eQTLs were detected in the first and second environment, respectively. Based on the QTTs and unconditional eQTLs detected, we presented a hypothetical molecular regulatory network in which 14 petal regulators potentially regulated the apetalous trait in "APL01" through the CHR11-PLP pathway. PLP acts directly as the terminal signal integrator negatively regulating petal development in the CHR11-PLP pathway. These findings will aid in the understanding the molecular mechanism underlying the apetalous trait of rapeseed.

Phenotypic characterization and genetic analysis of a partially female-sterile mutant in Brassica napus.

mfs is a partially female-sterile Brassica napus mutant derived from a spontaneous mutation. When the mutant is crossed as a female, very poor seed set is obtained, whereas it is fertile as a pollen donor. The floret of the mutant consisted of almost equal-length stamens, a short pistil, a flat style and ovary, and the stigma was chapped. Measures of pollen viability and pollen tube growth in vitro indicated that the mutation enhanced pollen viability. The papillae of mfs consisted of two conjoint bilobed domes, and the papillar cells were sparse, oblate and large at anthesis, but become withered and senesced quickly afterward. Pollen grains could germinate over the papillar cells, but pollen tubes could not penetrate into it. After flower opening, the number of organelles in mfs papillar cell decreased, the structure of it distinctly degenerated, and vacuolization was abnormally high. Genetic analysis of 3 F2 populations and 3 BC1F1 populations suggested that the mutant trait was controlled by two recessive genes.

Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes.

MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.

Detection of quantitative trait loci for yield and drought tolerance traits in soybean using a recombinant inbred line population.

To investigate the genetic basis of drought tolerance in soybean (Glycine max L. Merr.) a recombinant inbred population with 184 F(2:7:11) lines developed from a cross between Kefeng1 (drought tolerant) and Nannong1138-2 (drought sensitive) were tested under water-stressed and well-watered conditions in field and greenhouse trials. Traits measured included leaf wilting coefficient, excised leaf water loss and relative water content as indicators of plant water status and seed yield. A total of 40 quantitative trait loci (QTLs) were identified: 17 for leaf water status traits under drought stress and 23 for seed yield under well-watered and drought-stressed conditions in both field and greenhouse trials. Two seed yield QTLs were detected under both well-watered and drought-stressed conditions in the field on molecular linkage group H and D1b, while two seed yield QTLs on molecular linkage group C2 were found under greenhouse conditions. Several QTLs for traits associated with plant water status were identified in both field and greenhouse trials, including two leaf wilting coefficient QTLs on molecular linkage group A2 and one excised leaf water loss QTL on molecular linkage group H. Phenotypic correlations of traits suggested several QTLs had pleiotropic or location-linked associations. These results will help to elucidate the genetic basis of drought tolerance in soybean, and could be incorporated into a marker-assisted selection breeding program to develop high-yielding soybean cultivars with improved tolerance to drought stress.

Microarray analysis of gene expression in seeds of Brassica napus planted in Nanjing (altitude: 8.9 m), Xining (altitude: 2261.2 m) and Lhasa (altitude: 3658 m) with different oil content.

The regulation of seed oil synthesis in rapeseed is largely unknown. In this study, Arabidopsis microarray was used to analyze the gene differential expression of the immature seeds 30 days after flowering of a high oil Brassica napus, H105, whose oil content was 46.04 +/- 1.42, 53.94 +/- 1.35 and 53.09 +/- 1.35% when planted in Nanjing (altitude: 8.9 m), Xining (altitude: 2261.2 m) and Lhasa (altitude: 3658 m), respectively. Transcript levels of 363 genes and 421 genes were altered twofold or more for H105 planted in Xining and Lhasa compared to that in Nanjing, respectively. Together, there were 53 common up-regulated and 42 common down-regulated expression transcripts shared by H105 planted in Xining and Lhasa compared to that in Nanjing. Some important genes, such as sucrose synthase, pyruvate kinase and 6-phosphogluconate dehydrogenase which related to sugar metabolism were identified common up-regulated in higher oil content H105. These results revealed the expressional disciplinarian of correlative genes, and provided important information of the molecular genetic mechanism of oil content difference of rapeseed. In addition, these differential expression genes could be suitable as targets for genetic improvement of seed oil content.

Mapping QTLs for seed yield and drought susceptibility index in soybean (Glycine max L.) across different environments.

Drought stress has long been a major constraint in maintaining yield stability of soybean (Glycine max (L.) Merr.) in rainfed ecosystems. The identification of consistent quantitative trait loci (QTL) involving seed yield per plant (YP) and drought susceptibility index (DSI) in a population across different environments would therefore be important in molecular marker-assisted breeding of soybean cultivars suitable for rainfed regions. The YP of a recombinant line population of 184 F(2:7:11) lines from a cross of Kefeng1 and Nannong1138-2 was studied under water-stressed (WS) and well-watered (WW) conditions in field (F) and greenhouse (G) trials, and DSI for yield was calculated in two trials. Nineteen QTLs associated with YP-WS and YP-WW, and 10 QTLs associated with DSI, were identified. Comparison of these QTL locations with previous findings showed that the majority of these regions control one or more traits related to yield and other agronomic traits. One QTL on molecular linkage group (MLG) K for YP-F, and two QTLs on MLG C2 for YP-G, remained constant across different water regimes. The regions on MLG C2 for YP-WW-F and MLG H for YP-WS-F had a pleiotropic effect on DSI-F, and MLG A1 for YP-WS-G had a pleiotropic effect on DSI-G. The identification of consistent QTLs for YP and DSI across different environments will significantly improve the efficiency of selecting for drought tolerance in soybean.

[Mapping insect resistance QTLs of soybean with RIL population].

A recombinant inbred line (RIL) population, NJRIKY, which was derived from the cross Kefeng 1 xNannong 1138-2, was used to constructed the genetic linkage map. Larval weight and pupae weight of cotton worm [Prodenia litura (L.) Fabricius] were examined and used as indicators of resistance. Based on the linkage map constructed with SSR markers of this RIL population, one QTL responsible for larval weight was mapped on linkage group G20-O and the position was 31.91 cM. The QTL's additive effect was 0.0408 and explained 11.74 of the total variation of the larval weight. Two QTLs associated with pupae weight were mapped on linkage group G8-D1b+W and G17-L and the positions were 14.71 cM and 0.01 cM, respectively. The QTLs' additive effects were -0.0139 and 0.0103 ,which explained 11.30 and 6.36 of the total variation of larval weight, respectively.

Mapping of SMV resistance gene Rsc-7 by SSR markers in soybean.

Soybean mosaic virus (SMV) is one of the most prevalent pathogens that limit soybean production. In this study, segregation ratios of resistant plants to susceptible plants in P1, P2, F1, F2 populations of Kefeng No. 1 (P1) x Nannong 1138-2 (P2) and derived RIL populations, were used to study the inheritance of resistance to the SMV strain SC-7. Populations Kefeng No. 1 and F1 were found to be completely resistant to this SMV strain while Nannong 1138-2 was susceptible to it. The F2 and RIL populations segregated to fit a ratio of 3:1 and 1:1for resistant plants to susceptible ones, respectively. These results indicated that a single dominant gene, designated as Rsc-7, controlled resistance to the SMV strain SC-7 in Kefeng No.1. SSR markers were used to analyze the RIL population and MAPMAKER/EXP 3.0b was employed to establish linkage between markers and this resistance gene. Combining the data of SSRs and resistance identification, a soybean genetic map was constructed. This map, covering 2625.9 cM of the genome, converged into 24 linkage groups, consisted of 221 SSR markers and the resistance gene Rsc-7. The Rsc-7 gene was mapped to the molecular linkage group G8-D1b+W. SSR markers Satt266, Satt634, Satt558, Satt157, and Satt698 were found linked to Rsc-7 with distances of 43.7, 18.1, 26.6, 36.4 and 37.9 cM, respectively.