Genome-wide characterization of LEA gene family reveals a positive role of BnaA.LEA6.a in freezing tolerance in rapeseed (Brassica napus L.).

BACKGROUND: Freezing stress is one of the major abiotic stresses that causes extensive damage to plants. LEA (Late embryogenesis abundant) proteins play a crucial role in plant growth, development, and abiotic stress. However, there is limited research on the function of LEA genes in low-temperature stress in Brassica napus (rapeseed). RESULTS: Total 306 potential LEA genes were identified in B. rapa (79), B. oleracea (79) and B. napus (148) and divided into eight subgroups. LEA genes of the same subgroup had similar gene structures and predicted subcellular locations. Cis-regulatory elements analysis showed that the promoters of BnaLEA genes rich in cis-regulatory elements related to various abiotic stresses. Additionally, RNA-seq and real-time PCR results indicated that the majority of BnaLEA family members were highly expressed in senescent tissues of rapeseed, especially during late stages of seed maturation, and most BnaLEA genes can be induced by salt and osmotic stress. Interestingly, the BnaA.LEA6.a and BnaC.LEA6.a genes were highly expressed across different vegetative and reproductive organs during different development stages, and showed strong responses to salt, osmotic, and cold stress, particularly freezing stress. Further analysis showed that overexpression of BnaA.LEA6.a increased the freezing tolerance in rapeseed, as evidenced by lower relative electrical leakage and higher survival rates compared to the wild-type (WT) under freezing treatment. CONCLUSION: This study is of great significance for understanding the functions of BnaLEA genes in freezing tolerance in rapeseed and offers an ideal candidate gene (BnaA.LEA6.a) for molecular breeding of freezing-tolerant rapeseed cultivars.

Integrated Analysis of lncRNA-mRNA Regulatory Networks Related to Lipid Metabolism in High-Oleic-Acid Rapeseed.

A high oleic acid content is considered an essential characteristic in the breeding of high-quality rapeseed in China. Long-chain non-coding RNA (lncRNA) molecules play an important role in the plant's growth and its response to stress. To better understand the role of lncRNAs in regulating plant reproductive development, we analyzed whole-transcriptome and physiological data to characterize the dynamic changes in lncRNA expression during the four representative times of seed development of high- and low-oleic-acid rapeseed in three regions. We identified 21 and 14 lncRNA and mRNA modules, respectively. These modules were divided into three types related to region, development stages, and material. Next, we analyzed the key modules related to the oil content and the oleic acid, linoleic acid, and linolenic acid contents with physiological data and constructed the key functional network analysis on this basis. Genes related to lipid metabolism, such as 3-ketoacyl-CoA synthase 16 (KCS16) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), were present in the co-expression network, suggesting that the effect of these genes on lipid metabolism might be embodied by the expression of these lncRNAs. Our results provide a fresh insight into region-, development-stage-, and material-biased changes in lncRNA expression in the seeds of Brassica napus. Some of these lncRNAs may participate in the regulatory network of lipid accumulation and metabolism, together with regulated genes. These results may help elucidate the regulatory system of lncRNAs in the lipid metabolism of high-oleic-acid rapeseed seeds.

Flavonoid Synthesis-Related Genes Determine the Color of Flower Petals in Brassica napus L.

The color of rapeseed (Brassica napus L.) petal is usually yellow but can be milky-white to orange or pink. Thus, the petal color is a popular target in rapeseed breeding programs. In his study, metabolites and RNA were extracted from the yellow (Y), yellow/purple (YP), light purple (LP), and purple (P) rapeseed petals. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), RNA-Seq, and quantitative real-time (qRT-PCR) analyses were performed to analyze the expression correlation of differential metabolites and differential genes. A total of 223 metabolites were identified in the petals of the three purple and yellow rapeseed varieties by UPLC-MS/MS. A total of 20511 differentially expressed genes (DEGs) between P, LP, YP, versus Y plant petals were detected. This study focused on the co-regulation of 4898 differential genes in the three comparison groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and quantitative RT-PCR analysis showed that the expression of BnaA10g23330D (BnF3'H) affects the synthesis of downstream peonidin and delphinidin and is a key gene regulating the purple color of petals in B. napus. L. The gene may play a key role in regulating rapeseed flower color; however, further studies are needed to verify this. These results deepen our understanding of the molecular mechanisms underlying petal color and provide the theoretical and practical basis for flower breeding targeting petal color.

Tissue-Specific Transcriptome and Metabolome Analysis Reveals the Response Mechanism of Brassica napus to Waterlogging Stress.

During the growth period of rapeseed, if there is continuous rainfall, it will easily lead to waterlogging stress, which will seriously affect the growth of rapeseed. Currently, the mechanisms of rapeseed resistance to waterlogging stress are largely unknown. In this study, the rapeseed (Brassica napus) inbred lines G230 and G218 were identified as waterlogging-tolerant rapeseed and waterlogging-sensitive rapeseed, respectively, through a potted waterlogging stress simulation and field waterlogging stress experiments. After six days of waterlogging stress at the seedling stage, the degree of leaf aging and root damage of the waterlogging-tolerant rapeseed G230 were lower than those of the waterlogging-sensitive rapeseed G218. A physiological analysis showed that waterlogging stress significantly increased the contents of malondialdehyde, soluble sugar, and hydrogen peroxide in rape leaves and roots. The transcriptomic and metabolomic analysis showed that the differential genes and the differential metabolites of waterlogging-tolerant rapeseed G230 were mainly enriched in the metabolic pathways, biosynthesis of secondary metabolites, flavonoid biosynthesis, and vitamin B6 metabolism. Compared to G218, the expression levels of some genes associated with flavonoid biosynthesis and vitamin B metabolism were higher in G230, such as CHI, DRF, LDOX, PDX1.1, and PDX2. Furthermore, some metabolites involved in flavonoid biosynthesis and vitamin B6 metabolism, such as naringenin and epiafzelechin, were significantly up-regulated in leaves of G230, while pyridoxine phosphate was only significantly down-regulated in roots and leaves of G218. Furthermore, foliar spraying of vitamin B6 can effectively improve the tolerance to waterlogging of G218 in the short term. These results indicate that flavonoid biosynthesis and vitamin B6 metabolism pathways play a key role in the waterlogging tolerance and hypoxia stress resistance of Brassica napus and provide new insights for improving the waterlogging tolerance and cultivating waterlogging-tolerant rapeseed varieties.

Regional association and transcriptome analysis revealed candidate genes controlling plant height in Brassica napus.

Plant height is a key morphological trait in rapeseed, which not only plays an important role in determining plant architecture, but is also an important characteristic related to yield. Presently, the improvement of plant architecture is a major challenge in rapeseed breeding. This work was carried out to identify genetic loci related to plant height in rapeseed. In this study, a genome-wide association study (GWAS) of plant height was performed using a Brassica 60 K Illumina Infinium SNP array and 203 Brassica napus accessions. Eleven haplotypes containing important candidate genes were detected and significantly associated with plant height on chromosomes A02, A03, A05, A07, A08, C03, C06, and C09. Moreover, regional association analysis of 50 resequenced rapeseed inbred lines was used to further analyze these eleven haplotypes and revealed nucleotide variation in the BnFBR12-A08 and BnCCR1-C03 gene regions related to the phenotypic variation in plant height. Furthermore, coexpression network analysis showed that BnFBR12-A08 and BnCCR1-C03 were directly connected with hormone genes and transcription factors and formed a potential network regulating the plant height of rapeseed. Our results will aid in the development of haplotype functional markers to further improve plant height in rapeseed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01337-1.

Comparative Cytological and Transcriptome Analyses of Anther Development in Nsa Cytoplasmic Male Sterile (1258A) and Maintainer Lines in Brassica napus Produced by Distant Hybridization.

1258A is a new line of B.napus with Nsa cytoplasmic male sterility (CMS) with potential applications in hybrid rapeseed breeding. Sterile cytoplasm was obtained from XinJiang Sinapis arvensis through distant hybridization and then backcrossed with 1258B for many generations. However, the characteristics and molecular mechanisms underlying pollen abortion in this sterile line are poorly understood. In this study, a cytological analysis revealed normal microsporogenesis and uninucleate pollen grain formation. Pollen abortion was due to non-programmed cell death in the tapetum and the inability of microspores to develop into mature pollen grains. Sucrose, soluble sugar, and adenosine triphosphate (ATP) contents during microspore development were lower than those of the maintainer line, along with an insufficient energy supply, reduced antioxidant enzyme activity, and substantial malondialdehyde (MDA) accumulation in the anthers. Transcriptome analysis revealed that genes involved in secondary metabolite biosynthesis, glutathione metabolism, phenylpropane biosynthesis, cyanoamino acid metabolism, starch and sucrose metabolism, and glycerolipid metabolism may contribute to pollen abortion. The down regulation of nine cytochrome P450 monooxygenases genes were closely associated with pollen abortion. These results suggest that pollen abortion in 1258A CMS stems from abnormalities in the chorioallantoic membranes, energy deficiencies, and dysfunctional antioxidant systems in the anthers. Our results provide insight into the molecular mechanism underlying pollen abortion in Nsa CMS and provide a theoretical basis for better heterosis utilization in B.napus.

Comprehensive Analyses of the Histone Deacetylases Tuin (HDT) Gene Family in Brassicaceae Reveals Their Roles in Stress Response.

Histone deacetylases tuin (HDT) is a plant-specific protein subfamily of histone deacetylation enzymes (HDAC) which has a variety of functions in plant development, hormone signaling and stress response. Although the HDT family's genes have been studied in many plant species, they have not been characterized in Brassicaceae. In this study, 14, 8 and 10 HDT genes were identified in Brassica napus, Brassica rapa and Brassica oleracea, respectively. According to phylogenetic analysis, the HDTs were divided into four groups: HDT1(HD2A), HDT2(HD2B), HDT3(HD2C) and HDT4(HD2D). There was an expansion of HDT2 orthologous genes in Brassicaceae. Most of the HDT genes were intron-rich and conserved in gene structure, and they coded for proteins with a nucleoplasmin-like (NPL) domain. Expression analysis showed that B. napus, B. rapa, and B. oleracea HDT genes were expressed in different organs at different developmental stages, while different HDT subgroups were specifically expressed in specific organs and tissues. Interestingly, most of the Bna/Br/BoHDT2 members were expressed in flowers, buds and siliques, suggesting they have an important role in the development of reproductive organs in Brassicaceae. Expression of BnaHDT was induced by various hormones, such as ABA and ethylene treatment, and some subgroups of genes were responsive to heat treatment. The expression of most HDT members was strongly induced by cold stress and freezing stress after non-cold acclimation, while it was slightly induced after cold acclimation. In this study, the HDT gene family of Brassicaceae was analyzed for the first time, which helps in understanding the function of BnaHDT in regulating plant responses to abiotic stresses, especially freezing stresses.

Regional association analysis coupled with transcriptome analyses reveal candidate genes affecting seed oil accumulation in Brassica napus.

KEY MESSAGE: Regional association analysis of 50 re-sequenced Chinese semi-winter rapeseed accessions in combination with co-expression analysis reveal candidate genes affecting oil accumulation in Brassica napus. One of the breeding goals in rapeseed production is to enhance the seed oil content to cater to the increased demand for vegetable oils due to a growing global population. To investigate the genetic basis of variation in seed oil content, we used 60 K Brassica Infinium SNP array along with phenotype data of 203 Chinese semi-winter rapeseed accessions to perform a genome-wide analysis of haplotype blocks associated with the oil content. Nine haplotype regions harbouring lipid synthesis/transport-, carbohydrate metabolism- and photosynthesis-related genes were identified as significantly associated with the oil content and were mapped to chromosomes A02, A04, A05, A07, C03, C04, C05, C08 and C09, respectively. Regional association analysis of 50 re-sequenced Chinese semi-winter rapeseed accessions combined with transcriptome datasets from 13 accessions was further performed on these nine haplotype regions. This revealed natural variation in the BnTGD3-A02 and BnSSE1-A05 gene regions correlated with the phenotypic variation of the oil content within the A02 and A04 chromosome haplotype regions, respectively. Moreover, co-expression network analysis revealed that BnTGD3-A02 and BnSSE1-A05 were directly linked with fatty acid beta-oxidation-related gene BnKAT2-C04, thus forming a molecular network involved in the potential regulation of seed oil accumulation. The results of this study could be used to combine favourable haplotype alleles for further improvement of the seed oil content in rapeseed.

Genome-wide identification and functional analysis of the TIFY gene family in the response to multiple stresses in Brassica napus L.

BACKGROUND: TIFY is a plant-specific protein family with a diversity of functions in plant development and responses to stress and hormones, which contains JASMONATE ZIM-domain (JAZ), TIFY, PPD and ZML subfamilies. Despite extensive studies of TIFY family in many other species, TIFY has not yet been characterized in Brassica napus. RESULTS: In this study, we identified 77, 36 and 39 TIFY family genes in the genome of B. napus, B. rapa and B. oleracea, respectively. Results of the phylogenetic analysis indicated the 170 TIFY proteins from Arabidopsis, B. napus, B. rapa and B. oleracea could be divided into 11 groups: seven JAZ groups, one PPD group, one TIFY group, and two ZIM/ZML groups. The molecular evolutionary analysis showed that TIFY genes were conserved in Brassicaceae species. Gene expression profiling and qRT-PCR revealed that different groups of BnaTIFY members have distinct spatiotemporal expression patterns in normal conditions or following treatment with different abiotic/biotic stresses and hormones. The BnaJAZ subfamily genes were predominantly expressed in roots and up-regulated by NaCl, PEG, freezing, methyl jasmonate (MeJA), salicylic acid (SA) and Sclerotinia sclerotiorum in leaves, suggesting that they have a vital role in hormone signaling to regulate multiple stress tolerance in B. napus. CONCLUSIONS: The extensive annotation and expression analysis of the BnaTIFY genes contributes to our understanding of the functions of these genes in multiple stress responses and phytohormone crosstalk in B. napus.

GWAS and co-expression network combination uncovers multigenes with close linkage effects on the oleic acid content accumulation in Brassica napus.

BACKGROUND: Strong artificial and natural selection causes the formation of highly conserved haplotypes that harbor agronomically important genes. GWAS combination with haplotype analysis has evolved as an effective method to dissect the genetic architecture of complex traits in crop species. RESULTS: We used the 60 K Brassica Infinium SNP array to perform a genome-wide analysis of haplotype blocks associated with oleic acid (C18:1) in rapeseed. Six haplotype regions were identified as significantly associated with oleic acid (C18:1) that mapped to chromosomes A02, A07, A08, C01, C02, and C03. Additionally, whole-genome sequencing of 50 rapeseed accessions revealed three genes (BnmtACP2-A02, BnABCI13-A02 and BnECI1-A02) in the A02 chromosome haplotype region and two genes (BnFAD8-C02 and BnSDP1-C02) in the C02 chromosome haplotype region that were closely linked to oleic acid content phenotypic variation. Moreover, the co-expression network analysis uncovered candidate genes from these two different haplotype regions with potential regulatory interrelationships with oleic acid content accumulation. CONCLUSIONS: Our results suggest that several candidate genes are closely linked, which provides us with an opportunity to develop functional haplotype markers for the improvement of the oleic acid content in rapeseed.

Genome-Wide Differential DNA Methylation and miRNA Expression Profiling Reveals Epigenetic Regulatory Mechanisms Underlying Nitrogen-Limitation-Triggered Adaptation and Use Efficiency Enhancement in Allotetraploid Rapeseed.

Improving crop nitrogen (N) limitation adaptation (NLA) is a core approach to enhance N use efficiency (NUE) and reduce N fertilizer application. Rapeseed has a high demand for N nutrients for optimal plant growth and seed production, but it exhibits low NUE. Epigenetic modification, such as DNA methylation and modification from small RNAs, is key to plant adaptive responses to various stresses. However, epigenetic regulatory mechanisms underlying NLA and NUE remain elusive in allotetraploid B. napus. In this study, we identified overaccumulated carbohydrate, and improved primary and lateral roots in rapeseed plants under N limitation, which resulted in decreased plant nitrate concentrations, enhanced root-to-shoot N translocation, and increased NUE. Transcriptomics and RT-qPCR assays revealed that N limitation induced the expression of NRT1.1, NRT1.5, NRT1.7, NRT2.1/NAR2.1, and Gln1;1, and repressed the transcriptional levels of CLCa, NRT1.8, and NIA1. High-resolution whole genome bisulfite sequencing characterized 5094 differentially methylated genes involving ubiquitin-mediated proteolysis, N recycling, and phytohormone metabolism under N limitation. Hypermethylation/hypomethylation in promoter regions or gene bodies of some key N-metabolism genes might be involved in their transcriptional regulation by N limitation. Genome-wide miRNA sequencing identified 224 N limitation-responsive differentially expressed miRNAs regulating leaf development, amino acid metabolism, and plant hormone signal transduction. Furthermore, degradome sequencing and RT-qPCR assays revealed the miR827-NLA pathway regulating limited N-induced leaf senescence as well as the miR171-SCL6 and miR160-ARF17 pathways regulating root growth under N deficiency. Our study provides a comprehensive insight into the epigenetic regulatory mechanisms underlying rapeseed NLA, and it will be helpful for genetic engineering of NUE in crop species through epigenetic modification of some N metabolism-associated genes.

NRT1.1-Related NH4 + Toxicity Is Associated with a Disturbed Balance between NH4 + Uptake and Assimilation.

A high concentration of ammonium (NH4 +) as the sole source of nitrogen in the growth medium often is toxic to plants. The nitrate transporter NRT1.1 is involved in mediating the effects of NH4 + toxicity; however, the mechanism remains undefined. In this study, wild-type Arabidopsis (Arabidopsis thaliana Columbia-0 [Col-0]) and NRT1.1 mutants (chl1-1 and chl1-5) were grown hydroponically in NH4NO3 and (NH4)2SO4 media to assess the function of NRT1.1 in NH4 + stress responses. All the plants grew normally in medium containing mixed nitrogen sources, but Col-0 displayed more chlorosis and lower biomass and photosynthesis than the NRT1.1 mutants in (NH4)2SO4 medium. Grafting experiments between Col-0 and chl1-5 further confirmed that NH4 + toxicity is influenced by NRT1.1. In (NH4)2SO4 medium, NRT1.1 induced the expression of NH4 + transporters, increasing NH4 + uptake. Additionally, the activities of glutamine synthetase and glutamate synthetase in roots of Col-0 plants decreased and soluble sugar accumulated significantly, whereas pyruvate kinase-mediated glycolysis was not affected, all of which contributed to NH4 + accumulation. By contrast, the NRT1.1 mutants showed reduced NH4 + accumulation and enhanced NH4 + assimilation through glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase. Moreover, the up-regulation of genes involved in ethylene synthesis and senescence in Col-0 plants treated with (NH4)2SO4 suggests that ethylene is involved in NH4 + toxicity responses. This study showed that NH4 + toxicity is related to a nitrate-independent signaling function of NRT1.1 in Arabidopsis, characterized by enhanced NH4 + accumulation and altered NH4 + metabolism, which stimulates ethylene synthesis, leading to plant senescence.

A multiomics approach reveals the pivotal role of subcellular reallocation in determining rapeseed resistance to cadmium toxicity.

Oilseed rape (Brassica napus) has great potential for phytoremediation of cadmium (Cd)-polluted soils due to its large plant biomass production and strong metal accumulation. Enhanced plant Cd resistance (PCR) is a crucial prerequisite for phytoremediation through hyper-accumulation of excess Cd. However, the complexity of the allotetraploid genome of rapeseed hinders our understanding of PCR. To explore rapeseed Cd-resistance mechanisms, we examined two genotypes, 'ZS11' (Cd-resistant) and 'W10' (Cd-sensitive), that exhibit contrasting PCR while having similar tissue Cd concentrations, and characterized their different fingerprints in terms of plant morphophysiology (electron microscopy), ion abundance (inductively coupled plasma mass spectrometry), DNA variation (whole-genome resequencing), transcriptomics (high-throughput mRNA sequencing), and metabolomics (ultra-high performance liquid chromatography-mass spectrometry). Fine isolation of cell components combined with ionomics revealed that more Cd accumulated in the shoot vacuoles and root pectins of the resistant genotype than in the sensitive one. Genome and transcriptome sequencing identified numerous DNA variants and differentially expressed genes involved in pectin modification, ion binding, and compartmentalization. Transcriptomics-assisted gene co-expression networks characterized BnaCn.ABCC3 and BnaA8.PME3 as the central members involved in the determination of rapeseed PCR. High-resolution metabolic profiles revealed greater accumulation of shoot Cd chelates, and stronger biosynthesis and higher demethylation of root pectins in the resistant genotype than in the sensitive one. Our comprehensive examination using a multiomics approach has greatly improved our understanding of the role of subcellular reallocation of Cd in the determination of PCR.

The high-quality genome of Brassica napus cultivar 'ZS11' reveals the introgression history in semi-winter morphotype.

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi-winter oilseed rape cultivar 'ZS11' and its comprehensive genomic comparison with the genomes of the winter-type cultivar 'Darmor-bzh' as well as two progenitors. The integrated BAC-to-BAC and whole-genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high-quality genome assembly of B. napus 'ZS11'. Within a short evolutionary period (~6700 years ago), semi-winter-type 'ZS11' and the winter-type 'Darmor-bzh' maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to 'Darmor-bzh', both two subgenomes of 'ZS11' are closely related to its progenitors, and the 'ZS11' genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi-winter-type 'ZS11' underwent potential genomic introgressions with B. rapa (Ar ). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization-responsive flowering time in 'ZS11' was first experienced HE, and then underwent genomic introgression event with Ar , which potentially has led to genetic differences in controlling vernalization in the semi-winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi-winter oilseed rape in Asia.

Integrated physiologic, genomic and transcriptomic strategies involving the adaptation of allotetraploid rapeseed to nitrogen limitation.

BACKGROUND: Nitrogen (N) is a macronutrient that is essential for optimal plant growth and seed yield. Allotetraploid rapeseed (AnAnCnCn, 2n = 4x = 38) has a higher requirement for N fertilizers whereas exhibiting a lower N use efficiency (NUE) than cereal crops. N limitation adaptation (NLA) is pivotal for enhancing crop NUE and reducing N fertilizer use in yield production. Therefore, revealing the genetic and molecular mechanisms underlying NLA is urgent for the genetic improvement of NUE in rapeseed and other crop species with complex genomes. RESULTS: In this study, we integrated physiologic, genomic and transcriptomic analyses to comprehensively characterize the adaptive strategies of oilseed rape to N limitation stresses. Under N limitations, we detected accumulated anthocyanin, reduced nitrate (NO3-) and total N concentrations, and enhanced glutamine synthetase activity in the N-starved rapeseed plants. High-throughput transcriptomics revealed that the pathways associated with N metabolism and carbon fixation were highly over-represented. The expression of the genes that were involved in efficient N uptake, translocation, remobilization and assimilation was significantly altered. Genome-wide identification and molecular characterization of the microR827-NLA1-NRT1.7 regulatory circuit indicated the crucial role of the ubiquitin-mediated post-translational pathway in the regulation of rapeseed NLA. Transcriptional analysis of the module genes revealed their significant functional divergence in response to N limitations between allotetraploid rapeseed and the model Arabidopsis. Association analysis in a rapeseed panel comprising 102 genotypes revealed that BnaC5.NLA1 expression was closely correlated with the rapeseed low-N tolerance. CONCLUSIONS: We identified the physiologic and genome-wide transcriptional responses of oilseed rape to N limitation stresses, and characterized the global members of the BnamiR827-BnaNLA1s-BnaNRT1.7s regulatory circuit. The transcriptomics-assisted gene co-expression network analysis accelerates the rapid identification of central members within large gene families of plant species with complex genomes. These findings would enhance our comprehensive understanding of the physiologic responses, genomic adaptation and transcriptomic alterations of oilseed rape to N limitations and provide central gene resources for the genetic improvement of crop NLA and NUE.

Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus.

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO3 (-) to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO3 (-) was retained in roots of Xiangyou15. Moreover, NO3 (-) concentration in xylem sap, [(15)N] shoot:root (S:R) and [NO3 (-)] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO3 (-) in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3 (-) long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO3 (-) in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO3 (-) allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.

[Multifractal Analysis of Rapeseed Spectrum for Chlorophyll Diagnosis Modeling].

One of the most important topics in crop information science is how to make use of the crop's information for non-destructive nutrient diagnosis which can be solved with spectrum analysis. The canopy's spectrum feature is a key indicator to describe the nutritional status for the rapeseeds. The original spectrum is to be disturbed with external factors such as environment and climate; however, it is difficult to be directly used for rapeseed biomass diagnosis due to its huge fluctuation. However, the multifractal feature of the spectra remains stable relatively. In order to study the relationship between the canopy's spectrum of the rapeseed and its chlorophyll, based on the multifractal theory, a quantitative model of chlorophyll prediction and a qualitative model of planting pattern identification were proposed in this paper to study the high oleic acid rapeseed samples in 24 transplanting regions and 24 direct planting regions. At first, the generalized Hurst exponent and mass exponents together with other relevant multifractal parameters of the spectra were extracted with popular multifractal detrended fluctuation analysis (MF-DFA) in different six considered wavelength ranges. It shows that all of them possess representative multifractal nature. However, there are some differences of the multifractal characteristics between the two kinds of regions with different planting pattern in some bands. In addition, by correlation analysis and detection between the multifractal parameters of the spectra and the SPAD values in six considered ranges of bands, it demonstrates that there is some difference of the effective information content in the different ranges of bands. In the quantitative model of chlorophyll prediction, for each groups of samples in transplanting regions and direct planting regions and mixed together in each significant bands, a selected multifractal parameter was used to establish the univariate model for predicting the rapeseed leaf's SPAD values, respectively. The results of all the relative root mean square errors are small than 5%. Finally, the qualitative model was proposed to distinguish the samples by the two planting pattern. Youden index, as the identification accuracy was calculated for the six considered ranges of bands by the Fisher's linear discriminant analysis. The best Youden index is 0.902 5 and the corresponding band range is 350~1 350 nm. The significant work provides a theoretical and practical method for predicting rapeseed leaf's SPAD and also provides effective way to find the sensitive bands of the spectra for identification diagnosis.

Cloning of TTG1 gene and PCR identification of genomes A, B and C in Brassica species.

Arabidopsis Transparent Testa Glabra 1 (TTG1) genes were cloned from three diploid Brassica species (B. rapa, B. nigra and B. oleracea) and two amphidiploids species (B. juncea and B. carinata) by homology cloning. TTG1 homologues identified in all the accessions of the investigated species had a coding sequence of 1,014 bp. One copy was obtained from each diploid species and two copies from each amphidiploid species. Combined analysis of the TTG1 sequences cloned in this study with those obtained from public databases demonstrated that three, forty-five and seven nucleotides were specific variations in TTG1 genes from genomes A, B and C, respectively. Primers designed with genome-specific nucleotide variations were able to distinguish among TTG1 genes originating from genomes A, B and C in Brassica. Therefore, the TTG1 gene could serve as a candidate marker gene to detect the pollen flow of Brassica and provide an alternative method for the detection of pollen drift and risk assessment of gene flow in Brassica species.

Identification of environment-insensitive genes for oil content by combination of transcriptome and genome-wide association analysis in rapeseed.

BACKGROUND: The primary objective of rapeseed breeding is to enhance oil content, which is predominantly influenced by environmental factors. However, the molecular mechanisms underlying the impact of these environmental factors on oil accumulation remain inadequately elucidated. In this study, we used transcriptome data from two higher (HOC) and two lower oil content (LOC) inbred lines at 35 days after pollination (DAP) to investigate genes exhibiting stable expression across three different environments. Meanwhile, a genome-wide association study (GWAS) was utilized to detect candidate genes exhibiting significant associations with seed oil content across three distinct environments. RESULTS: The study found a total of 405 stable differentially expressed genes (DEGs), including 25 involved in lipid/fatty acid metabolism and 14 classified as transcription factors. Among these genes, BnBZIP10-A09, BnMYB61-A06, BnAPA1-A08, BnPAS2-A10, BnLCAT3-C05 and BnKASIII-C09 were also found to exhibit significant associations with oil content across multiple different environments based on GWAS of 50 re-sequenced semi-winter rapeseed inbred lines and previously reported intervals. Otherwise, we revealed the presence of additive effects among BnBZIP10-A09, BnKASIII-C09, BnPAS2-A10 and BnAPA1-A08, resulting in a significant increase in seed oil content. Meanwhile, the majority of these stable DEGs are interconnected either directly or indirectly through co-expression network analysis, thereby giving rise to an elaborate molecular network implicated in the potential regulation of seed oil accumulation and stability. CONCLUSIONS: The combination of transcription and GWAS revealed that natural variation in six environment-insensitive gene regions exhibited significant correlations with seed oil content phenotypes. These results provide important molecular marker information for us to further improve oil content accumulation and stability in rapeseed.

Genetic diversity of kenaf (Hibiscus cannabinus) evaluated by inter-simple sequence repeat (ISSR).

Understanding genetic diversity is very useful for scientific utilization for breeding. In this study, we estimated the genetic distances in a panel of 84 kenaf accessions collected from 26 countries and regions using ISSR markers. The results of UPGMA analysis showed that kenaf germplasm had abundant genetic variation, with genetic dissimilarity coefficients ranging from 0.01 to 0.62. The in-group dissimilarity coefficient (0.29) was observed in 84 kenaf accessions, and all the accessions could be divided into three groups: cultivars (L1-1), relatively wild species (L1-2 and L1-3), and wild species (the others). Further in-group analysis in group L1-1 (0.19) revealed that the kenaf cultivars could be divided into five subgroups with distinct regional characteristics. It is imperative that genes be exchanged among all kinds of tested varieties from different origins. The results provide a useful basis for kenaf germplasm research and breeding.