Lipid and sugar metabolism play an essential role in pollen development and male sterility: a case analysis in Brassica napus.

AIMS: The genic male sterility (GMS) system is an important strategy for generating heterosis in plants. To better understand the essential role of lipid and sugar metabolism and to identify additional candidates for pollen development and male sterility, transcriptome and metabolome analysis of a GMS line of 1205AB in B. napus was used as a case study. DATA RESOURCES GENERATED: To characterize the GMS system, the transcriptome and metabolome profiles were generated for 24 samples and 48 samples of 1205AB in B. napus, respectively. Transcriptome analysis yielded a total of 156.52 Gb of clean data and revealed the expression levels of 109,541 genes and 8,501 novel genes. In addition, a total of 1,353 metabolites were detected in the metabolomic analysis, including 784 in positive ion mode and 569 in negative ion mode. KEY RESULTS: A total of 15,635 differentially expressed genes (DEGs) and 83 differential metabolites (DMs) were identified from different comparison groups, most of which were involved in lipid and sugar metabolism. The combination of transcriptome and metabolome analysis revealed 49 orthologous GMS genes related to lipid metabolism and 46 orthologous GMS genes related to sugar metabolism, as well as 45 novel genes. UTILITY OF THE RESOURCE: The transcriptome and metabolome profiles and their analysis provide useful reference data for the future discovery of additional GMS genes and the development of more robust male sterility breeding systems for use in the production of plant hybrids.

BnPLP1 Positively Regulates Flowering Time, Plant Height, and Main Inflorescence Length in Brassica napus.

Protein prenylation mediated by the Arabidopsis thaliana PLURIPETALA (AtPLP) gene plays a crucial role in plant growth, development, and environmental response by adding a 15-carbon farnesyl group or one to two 20-carbon geranylgeranyl groups onto one to two cysteine residues at the C-terminus of the target protein. However, the homologous genes and their functions of AtPLP in rapeseed are unclear. In this study, bioinformatics analysis and gene cloning demonstrated the existence of two homologous genes of AtPLP in the Brassica napus L. genome, namely, BnPLP1 and BnPLP2. Evolutionary analysis revealed that BnPLP1 originated from the B. rapa L. genome, while BnPLP2 originated from the B. oleracea L. genome. Genetic transformation analysis revealed that the overexpression of BnPLP1 in Arabidopsis plants exhibited earlier flowering initiation, a prolonged flowering period, increased plant height, and longer main inflorescence length compared to the wild type. Contrarily, the downregulation of BnPLP1 expression in B. napus plants led to delayed flowering initiation, shortened flowering period, decreased plant height, and reduced main inflorescence length compared to the wild type. These findings indicate that the BnPLP1 gene positively regulates flowering time, plant height, and main inflorescence length. This provides a new gene for the genetic improvement of flowering time and plant architecture in rapeseed.

Exploration into Natural Variation Genes Associated with Determinate and Capitulum-like Inflorescence in Brassica napus.

Brassica napus is a globally important vegetable and oil crop. The research is meaningful for the yield and plant architecture of B. napus. In this study, one natural mutant line with determinate and capitulum-like inflorescence was chosen for further study. Genetic analysis indicated that the segregation patterns of inflorescences in the F2 populations supported a digenic inheritance model, which was further approved via the BSA-Seq technique. The BSA-Seq method detected two QTL regions on C02 (14.27-18.41 Mb) and C06 (32.98-33.68 Mb) for the genetic control of determinate inflorescences in MT plants. In addition, the expression profile in MT compared with WT was analyzed, and a total of 133 candidate genes for regulating the flower development (75 genes, 56.4%), shoot meristem development (29 genes, 21.8%), and inflorescence meristem development (13 genes, 9.8%) were identified. Then one joint analysis combing BSA-Seq and RNA-Seq identified two candidate genes of BnaTFL1 and BnaAP1 for regulating the MT phenotype. Furthermore, the potential utilization of the MT plants was also discussed.

Comparative Transcriptome Analysis Reveals the Molecular Basis of Brassica napus in Response to Aphid Stress.

Rapeseed is a globally important economic crop that can be severely impacted by aphids. However, our understanding of rapeseed resistance to aphid stress is very limited. In this study, we analyzed the resistance characteristics of the low aphid-susceptible variety APL01 and the highly aphid-susceptible variety Holly in response to aphid stress. APL01 had a more significant inhibitory effect on aphid proliferation compared with Holly during the early stage of inoculation, whereas Holly showed stronger tolerance to aphid stress compared with APL01 during the later stage of inoculation. Through transcriptome, physiological, and gene expression analyses, it was revealed that chitinase activity, catalase activity, calcium signal transduction, and activation of systemic acquired resistance might be involved in aphid resistance in B. napus. The degree of inhibition of photosynthesis in plants under aphid stress directly determines the tolerance of B. napus to aphid stress. Furthermore, four promising candidate genes were screened from eight genes related to rapeseed response to biotic stress through RT-qPCR analysis of gene expression levels. These research findings represent an important step forward in understanding the resistance of rapeseed to aphid stress and provide a solid foundation for the cloning of genes responsible for this resistance.

Quantitative Trait Locus Mapping Combined with RNA Sequencing Reveals the Molecular Basis of Seed Germination in Oilseed Rape.

Rapid and uniform seed germination improves mechanized oilseed rape production in modern agricultural cultivation practices. However, the molecular basis of seed germination is still unclear in Brassica napus. A population of recombined inbred lines of B. napus from a cross between the lower germination rate variety 'APL01' and the higher germination rate variety 'Holly' was used to study the genetics of seed germination using quantitative trait locus (QTL) mapping. A total of five QTLs for germination energy (GE) and six QTLs for germination percentage (GP) were detected across three seed lots, respectively. In addition, six epistatic interactions between the QTLs for GE and nine epistatic interactions between the QTLs for GP were detected. qGE.C3 for GE and qGP.C3 for GP were co-mapped to the 28.5-30.5 cM interval on C3, which was considered to be a novel major QTL regulating seed germination. Transcriptome analysis revealed that the differences in sugar, protein, lipid, amino acid, and DNA metabolism and the TCA cycle, electron transfer, and signal transduction potentially determined the higher germination rate of 'Holly' seeds. These results contribute to our knowledge about the molecular basis of seed germination in rapeseed.

Exploring the basis of 2-propenyl and 3-butenyl glucosinolate synthesis by QTL mapping and RNA-sequencing in Brassica juncea.

Brassica juncea is used as a condiment, as vegetables and as an oilseed crop, especially in semiarid areas. In the present study, we constructed a genetic map using one recombinant inbred line (RIL) of B. juncea. A total of 304 ILP (intron length polymorphism) markers were mapped to 18 linkage groups designated LG01-LG18 in B. juncea. The constructed map covered a total genetic length of 1671.13 cM with an average marker interval of 5.50 cM. The QTLs for 2-propenyl glucosinolates (GSLs) colocalized with the QTLs for 3-butenyl GSLs between At1g26180 and BnapPIP1580 on LG08 in the field experiments of 2016 and 2017. These QTLs accounted for an average of 42.3% and 42.6% phenotypic variation for 2-propenyl and 3-butenyl GSLs, respectively. Furthermore, the Illumina RNA-sequencing technique was used to excavate the genes responsible for the synthesis of GSLs in the siliques of the parental lines of the RIL mapping population, because the bulk of the seed GSLs might originate from the siliques. Comparative analysis and annotation by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) revealed that 324 genes were involved in GSL metabolism, among which only 24 transcripts were differentially expressed genes (DEGs). Among those DEGs, 15 genes were involved in the biosynthesis and transport of aliphatic GSLs, and their expression patterns were further validated by qRT-PCR analysis. Joint QTL mapping and RNA-sequencing analyses reveal one candidate gene of IIL1 (LOC106416451) for GSL metabolism in B. juncea. These results will be helpful for further fine mapping, gene cloning and genetic mechanisms of 2-propenyl and 3-butenyl GSLs in B. juncea.

Detection and molecular characterization of two FAD3 genes controlling linolenic acid content and development of allele-specific markers in yellow mustard (Sinapis alba).

Development of yellow mustard (Sinapis alba L.) with superior quality traits (low erucic and linolenic acid contents, and low glucosinolate content) can make this species as a potential oilseed crop. We have recently isolated three inbred lines Y1127, Y514 and Y1035 with low (3.8%), medium (12.3%) and high (20.8%) linolenic acid (C18∶3) content, respectively, in this species. Inheritance studies detected two fatty acid desaturase 3 (FAD3) gene loci controlling the variation of C18∶3 content. QTL mapping revealed that the two FAD3 gene loci responsible for 73.0% and 23.4% of the total variation and were located on the linkage groups Sal02 and Sal10, respectively. The FAD3 gene on Sal02 was referred to as SalFAD3.LA1 and that on Sal10 as SalFAD3.LA2. The dominant and recessive alleles were designated as LA1 and la1 for SalFAD3.LA1, and LA2 and la2 for SalFAD3.LA2. Cloning and alignment of the coding and genomic DNA sequences revealed that the SalFAD3.LA1 and SalFAD3.LA2 genes each contained 8 exons and 7 introns. LA1 had a coding DNA sequence (CDS) of 1143 bp encoding a polypeptide of 380 amino acids, whereas la1 was a loss-of-function allele due to an insertion of 584 bp in exon 3. Both LA2 and la2 had a CDS of 1152 bp encoding a polypeptide of 383 amino acids. Allele-specific markers for LA1, la1, LA2 and la2 co-segregated with the C18∶3 content in the F2 populations and will be useful for improving fatty acid composition through marker assisted selection in yellow mustard breeding.

Synthesis of a Brassica trigenomic allohexaploid (B. carinata × B. rapa) de novo and its stability in subsequent generations.

Allopolyploidy plays an important role in plant evolution and confers obvious advantages on crop growth and breeding compared to low ploidy levels. The present investigation was aimed at synthesising the first known chromosomally stable hexaploid Brassica with the genome constitution AABBCC. More than 2,000 putative hexaploid plants were obtained through large-scale hybridisation from various combinations of crosses between different cultivars of Brassica carinata (BBCC) and B. rapa (AA). The majority of plants after two generations of selfing within selected hexaploid plants (H(2)) were aneuploid, and only 80 plants (4.6%) had the expected hexaploid chromosome number (2n = 54). The hexaploid ratio increased to an average of 23.0 and 26.3% in the H(3) and H(4) generations, respectively, and was accompanied by an increase in pollen fertility. The appearance of aneuploid plants in each generation could be detected having various chromosomal abnormalities at meiosis. The frequency of hexaploid plants varied significantly among different cultivar combinations, from 0 to 56% in the H(4) generation, and it showed a positive correlation with pollen fertility. The frequency of SSR allelic fragments lost or novel alleles gained was significantly lower in H(4) than in H(2) and H(3), which reflects increasing genome stability in H(4). The A and C genomes were significantly less stable than the B genome, which may mainly result from frequent homoeologous pairing and rearrangements between the A and C genomes. Methods to establish a stable hexaploid Brassica crop by intercrossing these lines followed by intensive selection are also discussed.

Development of a population for substantial new type Brassica napus diversified at both A/C genomes.

Intersubgenomic heterosis in rapeseed has been revealed in previous studies by using traditional Brassica napus (A(n)A(n)C(n)C(n)) to cross partial new type B. napus with A(r)/C(c) introgression from the genomes of B. rapa and B. carinata, respectively. To further enlarge the genetic basis of B. napus and to facilitate a sustained heterosis breeding in rapeseed, it is crucial to create a population for substantial new type B. napus diversified at both A/C genomes. In this experiment, hundreds of artificial hexaploid plants (A(r)A(r)B(c)B(c)C(c)C(c)) involving hundreds of B. carinata/B. rapa combinations were first crossed with elite lines of partial new type B. napus. The pentaploid plants (AABCC) were open-pollinated in isolated conditions, and their offspring were successively self-pollinated and intensively selected for two generations. Thereafter, a population of substantial new type B. napus mainly with a genomic composition of A(r)A(r)C(c)C(c) harbouring genetic diversity from 25 original cultivars of B. rapa and 72 accessions of B. carinata was constructed. The population was cytologically verified to have the correct chromosome constitution of AACC and differed genetically from traditional B. napus, in terms of the genome components of A(r)/C(c) and B(c) as well as the novel genetic variations induced by the interspecific hybridisation process. Synchronously, rich phenotypic variation with plenty of novel valuable traits was observed in the population. The origin of the novel variations and the value of the population are discussed.

Broadening the avenue of intersubgenomic heterosis in oilseed Brassica.

Accumulated evidence has shown that each of the three basic Brassica genomes (A, B and C) has undergone profound changes in different species, and has led to the concept of the "subgenome". Significant intersubgenomic heterosis was observed in hybrids between traditional Brassica napus and first generation lines of new type B. napus. The latter were produced by the partial introgression of subgenomic components from different species into B. napus. To increase the proportion of exotic subgenomic components and thus achieve stronger heterosis, lines of first generation new type B. napus were intercrossed with each other, and subjected to intensive marker-assisted selection to develop the second generation of new type B. napus. The second generation showed better agronomic traits and a higher proportion of introgression of subgenomic components than did the first generation. Compared with the commercial hybrid and the hybrids produced with the first generation new type B. napus, the novel hybrids showed stronger heterosis for seed yield during the 2 years of field trials. The extent of heterosis showed a significant positive correlation with the introgressed subgenomic components in the parental new type B. napus. To increase the content of the exotic subgenomic components further and to allow sustainable breeding of novel lines of new type B. napus, we initiated the development of a gene pool for new type B. napus that contained a substantial amount of genetic variation in the A(r) and C(c) genome. We discuss new approaches to broaden the avenue of intersubgenomic heterosis in oilseed Brassica.