A Golden2-like transcription factor, BnGLK1a, improves chloroplast development, photosynthesis, and seed weight in rapeseed

Enhancing photosynthetic efficiency is a major goal for improving crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development.In this study,we demonstrate that Golden2-like 1a(BnGLK1a)plays an important role in regulating chloroplast development and photosynthetic efficiency.Overexpressing BnGLK1a resulted in significant increases in chlorophyll content,the number of thylakoid membrane layers and photosynthetic efficiency in Brassica napus,while knocking down BnGLK1a transcript levels through RNA interference(RNAi)had the opposite effects.A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1-2(BnPYL1-2)and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit(BnCSN5A),which play essential roles in regulating chloroplast development and photosynthesis.Consistent with this,BnGLK1a-RNAi lines of B.napus display hypersensitivity to the abscisic acid(ABA)response.Importantly,overexpression of BnGLK1a resulted in a 10%increase in thousand-seed weight,whereas seeds from BnGLK1a-RNAi lines were 16%lighter than wild type.We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity.Our results not only provide insights into the mechanisms of BnGLK1a function,but also offer a potential approach for improving the productivity of Brassica species.

Two types of cinnamoyl-Co A reductase function divergently in accumulation of lignins,flavonoids and glucosinolates and enhance lodging resistance in Brassica napus

Brassica crops,which are of worldwide importance,provide various oil,vegetable and ornamental products,as well as feedstocks for animal husbandry and biofuel industry.Cinnamoyl-Co A reductase(CCR)is the entry point to the lignin pathway and a crucial locus in manipulation of associated traits,but CCRassociated metabolism and traits in Brassica crops have remained largely unstudied except in Arabidopsis thaliana.We report the identification of 16 CCR genes from Brassica napus and its parental species B.rapa and B.oleracea.The Bn CCR1 and Bn CCR2 subfamilies displayed divergent organ-specificity and participation in the yellow-seed trait.Their functions were dissected via overexpression of representative paralogs in B.napus.Bn CCR1 was expressed preferentially in G-and H-lignin biosynthesis and vascular development,while Bn CCR2 was expressed in S-lignin biosynthesis and interfascicular fiber development.Bn CCR1 showed stronger effects on lignification-related development,lodging resistance,phenylpropanoid flux control,and seed coat pigmentation,whereas Bn CCR2 showed a stronger effect on sinapate biosynthesis.Bn CCR1 upregulation delayed bolting and flowering time,while Bn CCR2 upregulation weakened the leaf vascular system in consequence of suppressed G-lignin accumulation.Bn CCR1 and Bn CCR2 were closely but almost oppositely linked with glucosinolate metabolism via inter-pathway crosstalk.We conclude that Bn CCR1 and Bn CCR2 subfamilies offer great but differing potential for manipulating traits associated with phenylpropanoids and glucosinolates.This study reveals the CCR1–CCR2 divergence in Brassicaceae and offers a resource for rapeseed breeding for lodging resistance,yellowseed traits,and glucosinolate traits.

Genome-Wide Identification and Expression Profiling Suggest that Invertase Genes Function in Silique Development and the Response to Sclerotinia sclerotiorum in Brassica napus

Invertase(INV),a key enzyme in sucrose metabolism,irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose,thus playing important roles in plant growth,development,and biotic and abiotic stress responses.In this study,we identified 27 members of the BnaINV family in Brassica napus.We constructed a phylogenetic tree of the family and predicted the gene structures,conserved motifs,cis-acting elements in promoters,physicochemical properties of encoded proteins,and chromosomal distribution of the BnaINVs.We also analyzed the expression of the BnaINVs in different tissues and developmental stages in the B.napus cultivar Zhongshuang 11 using qRT-PCR.In addition,we analyzed RNA-sequencing data to explore the expression patterns of the BnaINVs in four cultivars with different harvest indices and in plants inoculated with the pathogenic fungus Sclerotinia sclerotiorum.We used WGCNA(weighted coexpression network analysis)to uncover BnaINV regulatory networks.Finally,we explored the expression patterns of several BnaINV genes in cultivars with long(Zhongshuang 4)and short(Ningyou 12)siliques.Our results suggest that BnaINVs play important roles in the growth and development of rapeseed siliques and the defense response against pathogens.Our findings could facilitate the breeding of high-yielding B.napus cultivars with strong disease resistance.

Development of a target capture sequencing SNP genotyping platform for genetic analysis and genomic breeding in rapeseed

Rapeseed(Brassica napus)is an oil crop grown worldwide,making it a key plant species in molecular breeding research.However,the complexity of its polyploid genome increases sequencing costs and reduces sequencing accuracy.Target capture coupled with high-throughput sequencing is an efficient approach for detecting genetic variation at genomic regions or loci of interest.In this study,588 resequenced accessions of rapeseed were used to develop a target capture sequencing SNP genotyping platform named BnaPan50T.The platform comprised 54,765,with 54,058 resequenced markers from the pan-genome,and 855 variant trait-associated markers for 12 agronomic traits.The capture quality of BnaPan50T was demonstrated well in 12 typical accessions.Compared with a conventional genotyping array,BnaPan50T has a high SNP density and a high proportion of SNPs in unique physical positions and in annotated functional genes,promising wide application.Target capture sequencing and wholegenome resequencing in 90 doubled-haploid lines yielded 60%specificity,78%uniformity within tenfold coverage range,and 93%genotyping accuracy for the platform.BnaPan50T was used to construct a genetic map for quantitative trait loci(QTL)mapping,identify 21 unique QTL,and predict several candidate genes for yield-related traits in multiple environments.A set of 132 core SNP loci was selected from BnaPan50T to construct DNA fingerprints and germplasm identification resources.This study provides genomics resources to support target capture sequencing,genetic analysis and genomic breeding of rapeseed.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed,and anthocyanin and proanthocyanidins(PAs)are major seed-coat pigments.Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed.In this study,we identified a transcription factor gene BnbHLH92a(BnaA06T0441000ZS)in rapeseed.Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs.Correspondingly,the expression profiles of anthocyanin and PA biosynthesis genes(TT3,BAN,TT8,TT18,and TTG1)were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds,indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis.BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed.BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression.These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B.napus.

Identification and characterization of a curly-leaf locus CL1 encoding an IAA2 protein in Brassica napus

The leaf is the main organ for rapeseed photosynthesis,and its morphology influences photosynthetic efficiency and supports increased planting density and yield.However,the molecular regulatory mechanism of leaf morphology in Brassica napus is poorly understood,restricting progress in breeding for the trait.We describe a novel dominant mutation,curly leaf 1(cl1),which confers uneven dorsal–ventral axis development,irregular cellular structure and influenced gravitropic response in the seedling stage.The CL1 locus was mapped to a 1.573-Mb interval on chromosome A05 using simple sequence repeat(SSR)markers,and co-segregated with the phenotype of plants in the curly F2 population.A substitution(P62S)was identified in the highly conserved degron motif(GWSPV)of the IAA2 protein in the cl1mutant,and the P62S substitution impaired the interaction between IAA2 and TIR1 in the presence of auxin,influencing auxin signaling.The P62S substitution-induced curly leaf phenotype was verified by ectopic expression of Bna A05.iaa2 in Arabidopsis and B.napus.Our findings explain the function of IAA2 in rapeseed,providing a foundation for future investigation of auxin signaling and the mechanisms underlying leaf development in B.napus.

Genome-wide association study identifies novel loci and candidate genes for drought stress tolerance in rapeseed

Rapeseed(Brassica napus)is one of the most important oil crops worldwide;however,drought seriously curtails its growth and productivity.Identifying drought-tolerant germplasm is an efficient strategy for addressing water shortages.Here,we phenotyped a panel of 264 B.napus accessions at full-bloom stage using water loss ratio(WLR)as drought-tolerant index.It identified 8 low-WLR and 6 high-WLR accessions,regarded as droughttolerant and drought-sensitive,respectively.Comparing with drought-sensitive accessions at the seedling stage,drought-tolerant accessions had shown better performance in maintaining fresh and dry weights,and performed the higher expression of drought-induced marker genes under drought stress.Subsequently,a total of 139 SNPs(single nucleotide polymorphisms)were identified associated with the WLR using a genome-wide association study(GWAS)among 264 B.napus accessions,with the largest number SNPs at chromosome A10,and 13 SNPs significantly were associated with the WLR(-log_(10)(p-value)>6).Furthermore,4 putative candidate genes(BnaC09.RPS6,BnaC09.MATE,BnaA10.PPD5 and BnaC09.Histone)were screened involving in drought tolerance in B.napus.Together,our results highlight the WLR's importance in drought tolerance and establish the foundation for improving WLR-associated drought tolerance in rapeseed.

Overexpression of BnKCS1-1,BnKCS1-2,and BnCER1-2 promotes cuticular wax production and increases drought tolerance in Brassica napus

Higher amounts of cuticular wax in plants have been associated with improved plant stress tolerance and increased potential for industrial use.In this study,orthologs of KCS1 and CER1 in Arabidopsis,designated BnKCS1-1,BnKCS1-2,and BnCER1-2,were isolated from Brassica napus.Transcription of BnKCS1-1 and BnKCS1-2 in B.napus were induced by abscisic acid(ABA)and drought treatment,while transcription of BnCER1-2 was induced only by drought treatment.All three gene transcripts decreased significantly when plants were treated with methyl jasmonate(MeJA)or subjected to cold stress.Overexpression of BnKCS1-1,BnKCS1-2,and BnCER1-2 under the control of the CaMV35S promoter led to a significant increase in cuticular wax on transgenic B.napus leaves.BnKCS1-1 and BnKCS1-2 overexpression led to similar differences from non-transformed plants,with significantly higher levels of aldehydes(C29 and C30),alkanes(C28,C29,and C31)and secondary alcohols(C28 and C29),and a significantly lower level of C29 ketone.Overexpression of BnCER1-2 led to an increase in alkanes(C27,C28,C29,and C31),a decrease in secondary alcohols(C28 and C29),and insignificant changes in other wax components.Scanning electron microscopy revealed that overexpression of BnKCS1-1,BnKCS1-2,and BnCER1-2 in B.napus resulted in a higher density of wax crystals on the leaf surface than observed in non-transformed plants.Transgenic plants showed a reduced rate of water loss and increased drought tolerance compared to non-transformed plants.These results suggest that BnKCS1-1,BnKCS1-2,and BnCER1-2 gene products can modify the cuticular wax of B.napus.Changing cuticular waxes using transgenic approaches is a new strategy for genetic improvement of plant drought tolerance and provides an opportunity for development of B.napus as a surface-wax crop.

Broadening the genetic base of Brassica juncea by introducing genomic components from B.rapa and B.nigra via digenomic allohexaploid bridging

A narrow genetic base has hindered improvement of Brassica juncea(A^(j)A^(j)B^(j)B^(j)).In this study,large-scale genomic components were introduced from diploid ancestor species into modern B.juncea using a digenomic hexaploid strategy.The hexaploids A^(j)A^(j)A^(r)A^(r)B^(j)B^(j) and A^(j)A^(j)B^(j)B^(j)B^(n)B^(n) were first developed from B.juncea×B.rapa(A^(r)A^(r))and B.juncea×B.nigra(B^(n)B^(n)),and then crossed with dozens of B.nigra and B.rapa,respectively.Both types of hexaploid showed high pollen fertility and moderate seed set throughout the S_(1) to S_(3) generations,and could be crossed with diploid progenitor species under field conditions,in particular for the combination of A^(j)A^(j)B^(j)B^(j)B^(n)B^(n)×B.rapa.Thirty A^(j)A^(r)B^(n)B^(j)-type and 31 A^(j)A^(r)B^(n)B^(j)-type B.juncea resources were generated,of which the A^(j)A^(r)B^(n)B^(j) type showed higher fertility.Of these new-type B.juncea resources,97 individual plants were genotyped with 42 simple sequence repeat markers,together with 16 current B.juncea accessions and 30 hexaploid plants.Based on 180 polymorphic loci,the new-type B.juncea resources and current B.juncea were separated clearly into distinct groups,with large genetic distance between the new-type B.juncea resources and current B.juncea.Our study provides a novel approach to introducing large-scale genomic components from diploid ancestor species into B.juncea.

Genome-wide identi?cation of loci affecting seed glucosinolate contents in Brassica napus L.

Glucosinolates are amino acid-derived secondary metabolites that act as chemical defense agents against pests.However,the presence of high levels of glucosinolates severely diminishes the nutritional value of seed meals made from rapeseed(Brassica napus L.).To identify the loci affecting seed glucosinolate cont ent(SGC),we con ducted genome-wide resequencing in a population of 307 diverse B.napus accessions from the three B.napus ecotype groups,namely,spring,winter,and semi-winter.These resequencing data were used for a genome-wide association study(GWAS)to identify the loci affecting SGC.In the three ecotype groups,four comm on and four ecotype-specific haplotype blocks(HBs)were significantly associated with SGC.To identify candidate genes controlling SGC,transcriptome analysis was carried out in 36 accessions showing extreme SGC values.Analyses of haplotypes,genomic variation,and candidate gene expression pointed to five and three candidate genes in the common and spring group-specific HBs,respectively.Our expression analyses dem on strated that additive effects of the three candidate genes in the spring group-specific HB play important roles in the SGC of B.napus.

New Insights into Nested Long Terminal Repeat Retrotransposons in Brassica Species

Long terminal repeat （LTR） retrotransposons, one of the foremost types of transposons, continually change or modify gene function and reorganize the genome through bursts of dramatic proliferation. Many LTR-TEs preferen-tially insert within other LTR-TEs, but the cause and evolutionary significance of these nested LTR-TEs are not well under-stood. In this study, a total of 1.52 Gb of Brassica sequence containing 2020 bacterial artificial chromosomes （BACs） was scanned, and six bacterial artificial chromosome （BAC） clones with extremely nested LTR-TEs （LTR-TEs density： 7.24/kb） were selected for further analysis. The majority of the LTR-TEs in four of the six BACs were found to be derived from the rapid proliferation of retrotransposons originating within the BAC regions, with only a few LTR-TEs originating from the proliferation and insertion of retrotransposons from outside the BAC regions approximately 5-23 Mya. LTR-TEs also pref-erably inserted into TA-rich repeat regions. Gene prediction by Genescan identified 207 genes in the 0.84Mb of total BAC sequences. Only a few genes （3/207） could be matched to the Brassica expressed sequence tag （EST） database, indicating that most genes were inactive after retrotransposon insertion. Five of the six BACs were putatively centromeric. Hence, nested LTR-TEs in centromere regions are rapidly duplicated, repeatedly inserted, and act to suppress activity of genes and to reshuffle the structure of the centromeric sequences. Our results suggest that LTR-TEs burst and proliferate on a local scale to create nested LTR-TE regions, and that these nested LTR-TEs play a role in the formation of centromeres.