Targeted mutagenesis of BnTTG1 homologues generated yellow-seeded rapeseed with increased oil content and seed germination under abiotic stress.

Yellow seed is one desirable trait with great potential to improve seed oil quality and yield. The present study surveys the redundant role of BnTTG1 genes in the proanthocyanidins (PA) biosynthesis, oil content and abiotic stress resistance. Stable yellow seed mutants were generated after mutating BnTTG1 by CRISPR/Cas9 genome editing system. Yellow seed phenotype could be obtained only when both functional homologues of BnTTG1 were simultaneously knocked out. Homozygous mutants of BnTTG1 homologues showed decreased thickness and PA accumulation in seed coat. Transcriptome and qRT-PCR analysis indicated that BnTTG1 mutation inhibited the expression of genes involved in phenylpropanoid and flavonoid biosynthetic pathways. Increased seed oil content and alteration of fatty acid (FA) composition were observed in homozygous mutants of BnTTG1 with enriched expression of genes involved in FA biosynthesis pathway. In addition, target mutation of BnTTG1 accelerated seed germination rate under salt and cold stresses. Enhanced seed germination capacity in BnTTG1 mutants was correlated with the change of expression level of ABA responsive genes. Overall, this study elucidated the redundant role of BnTTG1 in regulating seed coat color and established an efficient approach for generating yellow-seeded oilseed rape genetic resources with increase oil content, modified FA composition and resistance to multiple abiotic stresses.

Establishment of new convenient two-line system for hybrid production by targeting mutation of OPR3 in allopolyploid Brassica napus.

The two-line pollination control system, which usually depends on the utilization of thermosensitive or photoperiod genic male-sterile lines, has been widely used in various crops. However, this system is susceptible to instability issues caused by uncontrollable weather fluctuations. A stable and handy two-line pollination control system is highly desirable in many crop species for heterosis exploitation. Oxophytodienoic acid reductase 3 (OPR3) was proven to be involved in jasmonate biosynthesis. In the present study, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat) was utilized to mutate two OPR3 homologs in Brassica napus. After two OPR3 homologs were simultaneously mutated, mutants exhibited complete male sterility, and fertility could be easily restored by exogenous MeJA treatment. Hybrids produced from crosses between the opr3 sterile lines and normal varieties exhibited heterosis. This new two-line system based on OPR3 mutation provides higher stability and convenience than traditional systems. By using exogenous MeJA treatment to restore fertility, the system enables more precise control of male fertility transition, which has great potential to significantly contribute to the maneuverable production of hybrid seeds in rapeseed as well as other Brassica species crops.

Identification and Phylogenetic Analysis of the R2R3-MYB Subfamily in Brassica napus.

The R2R3-MYB sub-family proteins are composed of most members of MYB (v-Myb avian myeloblastosis viral oncogene homolog) protein, a plant-specific transcription factor (TF) that is classified into four classes depending on the number of MYB repeats. R2R3-MYB TFs are involved in physiological and biochemical processes. However, the functions of the Brassica napus R2R3-MYB genes are still mainly unknown. In this study, 35 Brassica napus MYB (BnaMYB) genes were screened in the genome of Brassica napus, and details about their physical and chemical characteristics, evolutionary relationships, chromosome locations, gene structures, three-dimensional protein structures, cis-acting promoter elements, and gene duplications were uncovered. The BnaMYB genes have undergone segmental duplications and positive selection pressure, according to evolutionary studies. The same subfamilies have similar intron-exon patterns and motifs, according to the genes' structure and conserved motifs. Additionally, through cis-element analysis, many drought-responsive and other stress-responsive cis-elements have been found in the promoter regions of the BnaMYB genes. The expression of the BnaMYB gene displays a variety of tissue-specific patterns. Ten lignin-related genes were chosen for drought treatment. Our research screened four genes that showed significant upregulation under drought stress, and thus may be important drought-responsive genes. The findings lay a new foundation for understanding the complex mechanisms of BnaMYB in multiple developmental stages and pathways related to drought stress in rapeseed.

Auxin Biosynthesis Genes in Allotetraploid Oilseed Rape Are Essential for Plant Development and Response to Drought Stress.

Crucial studies have verified that IAA is mainly generated via the two-step pathway in Arabidopsis, in which tryptophan aminotransferase (TAA) and YUCCA (YUC) are the two crucial enzymes. However, the role of the TAA (or TAR) and YUC genes in allotetraploid oilseed rape underlying auxin biosynthesis and development regulation remains elusive. In the present study, all putative TAR and YUC genes were identified in B. napus genome. Most TAR and YUC genes were tissue that were specifically expressed. Most YUC and TAR proteins contained trans-membrane regions and were confirmed to be endoplasmic reticulum localizations. Enzymatic activity revealed that YUC and TAR protein members were involved in the conversion of IPA to IAA and Trp to IPA, respectively. Transgenic plants overexpressing BnaYUC6a in both Arabidopsis and B. napus displayed high auxin production and reduced plant branch angle, together with increased drought resistance. Moreover, mutation in auxin biosynthesis BnaTARs genes by CRISPR/Cas9 caused development defects. All these results suggest the convergent role of BnaYUC and BnaTAR genes in auxin biosynthesis. Different homoeologs of BnaYUC and BnaTAR may be divergent according to sequence and expression variation. Auxin biosynthesis genes in allotetraploid oilseed rape play a pivotal role in coordinating plant development processes and stress resistance.

Functional Differentiation of BnVTE4 Gene Homologous Copies in α-Tocopherol Biosynthesis Revealed by CRISPR/Cas9 Editing.

Tocopherols are essential nutrients for human health known as vitamin E. Vitamin E deficiency can have a profound effect on human health, including the central nervous system and cardiovascular and immune protection. Multiple enzymatic steps are involved in the conversion between different forms of tocopherols. Among them, γ-tocopherol methyltransferase encoded by gene VTE4 catalyzes the conversion of γ- to α-tocopherol or δ- to ß-tocopherol isoforms. However, the gene copies and their functional contribution of VTE4 homologs in Brassica napus were not elucidated. To this end, different mutation combinations of four putative BnVTE4 homologous copies were generated by using CRISPR/Cas9 genome editing technology. Editing of those BnVTE4 homologs led to a significant change of the α-tocopherol content and the ratio between α- and γ-tocopherol compared with wide-type control. Analysis of the different combinations of BnVTE4-edited homologs revealed that the contribution of the BnVTE4 individual gene displayed obvious functional differentiation in α-tocopherol biosynthesis. Their contribution could be in order of VTE4.C02-2 (BnaC02G0331100ZS) > VTE4.A02-1 (BnaA02G0247300ZS) > VTE4.A02-2 (BnaA02G0154300ZS). Moreover, the VTE4.A02-1 and VTE4.A02-2 copies might have severe functional redundancies in α-tocopherol biosynthesis. Overall, this study systemically studied the different effects of BnVTE4 homologs, which provided a theoretical basis for breeding high α-tocopherol content oilseed rape.

Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system.

CRISPR/Cas-base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A-PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base-editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base-edited plants of ALS conferred high herbicide resistance, while base-edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T0 to T1 generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off-target sites, large-scale SNP variations were determined through whole-genome sequencing between some base-edited and wild-type plants. These results revealed that A3A-PBE base-editing system could effectively convert C to T substitution with high-editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.

A copia-like retrotransposon insertion in the upstream region of the SHATTERPROOF1 gene, BnSHP1.A9, is associated with quantitative variation in pod shattering resistance in oilseed rape.

Seed loss resulting from pod shattering is a major constraint in production of oilseed rape (Brassica napus L.). However, the molecular mechanisms underlying pod shatter resistance are not well understood. Here, we show that the pod shatter resistance at quantitative trait locus qSRI.A9.1 is controlled by one of the B. napus SHATTERPROOF1 homologs, BnSHP1.A9, in a doubled haploid population generated from parents designated R1 and R2 as well as in a diverse panel of oilseed rape. The R1 maternal parental line of the doubled haploid population carried the allele for shattering at qSRI.A9.1, while the R2 parental line carried the allele for shattering resistance. Quantitative RT-PCR showed that BnSHP1.A9 was expressed specifically in flower buds, flowers, and developing siliques in R1, while it was not expressed in any tissue of R2. Transgenic plants constitutively expressing either of the BnSHP1.A9 alleles from the R1 and R2 parental lines showed that both alleles are responsible for pod shattering, via a mechanism that promotes lignification of the enb layer. These findings indicated that the allelic differences in the BnSHP1.A9 gene per se are not the causal factor for quantitative variation in shattering resistance at qSRI.A9.1. Instead, a highly methylated copia-like long terminal repeat retrotransposon insertion (4803 bp) in the promotor region of the R2 allele of BnSHP1.A9 repressed the expression of BnSHP1.A9, and thus contributed to pod shatter resistance. Finally, we showed a copia-like retrotransposon-based marker, BnSHP1.A9R2, can be used for marker-assisted breeding targeting the pod shatter resistance trait in oilseed rape.

QTL and Candidate Gene Identification for Silique Length Based on High-Dense Genetic Map in Brassica napus L.

Silique length (SL) is an important yield trait and positively correlates with seeds per silique and seed weight. In the present study, two double haploid (DH) populations, established from crosses Zhongshuang11 × R11 (ZR) and R1 × R2 (RR), containing 280 and 95 DH lines, respectively, were used to map quantitative trait loci (QTL) for SL. A high-dense genetic map from ZR population was constructed comprising 14,658 bins on 19 linkage groups, with map length of 2,198.85 cM and an average marker distance of 0.15 cM. Genetic linkage map from RR population was constructed by using 2,046 mapped markers anchored to 19 chromosomes with 2,217-cM map length and an average marker distance of 1.08 cM. Major QTL qSL_ZR_A09 and qSL_RR_A09b on A09 were identified from ZR and RR populations, respectively. Both QTL could be stably detected in four environments. QTL qSL_RR_A09b and qSL_ZR_A09 were located on 68.5-70.8 cM and 91.33-91.94 cM interval with R2 values of 14.99-39.07% and 15.00-20.36% in RR and ZR populations, respectively. Based on the physical positions of single nucleotide polymorphism (SNP) markers flanking qSL_ZR_A09 and gene annotation in Arabidopsis, 26 genes were identified with SNP/Indel variation between parents and two genes (BnaA09g41180D and BnaA09g41380D) were selected as the candidate genes. Expression analysis further revealed BnaA09g41180D, encoding homologs of Arabidopsis fasciclin-like arabinogalactan proteins (FLA3), as the most promising candidate gene for qSL_ZR_A09. The QTL identification and candidate gene analysis will provide new insight into the genomic regions controlling SL in Brassica napus as well as candidate genes underlying the QTL.

Organelle genome composition and candidate gene identification for Nsa cytoplasmic male sterility in Brassica napus.

BACKGROUND: Nsa cytoplasmic male sterility (CMS) is a novel alloplasmic male sterility system derived from somatic hybridization between Brassica napus and Sinapis arvensis. Identification of the CMS-associated gene is a prerequisite for a better understanding of the origin and molecular mechanism of this CMS. With the development of genome sequencing technology, organelle genomes of Nsa CMS line and its maintainer line were sequenced by pyro-sequencing technology, and comparative analysis of the organelle genomes was carried out to characterize the organelle genome composition of Nsa CMS as well as to identify the candidate Nsa CMS-associated genes. RESULTS: Nsa CMS mitochondrial genome showed a higher collinearity with that of S. arvensis than B. napus, indicating that Nsa CMS mitochondrial genome was mainly derived from S. arvensis. However, mitochondrial genome recombination of parental lines was clearly detected. In contrast, the chloroplast genome of Nsa CMS was highly collinear with its B. napus parent, without any evidence of recombination of the two parental chloroplast genomes or integration from S. arvensis. There were 16 open reading frames (ORFs) specifically existed in Nsa CMS mitochondrial genome, which could not be identified in the maintainer line. Among them, three ORFs (orf224, orf309, orf346) possessing chimeric and transmembrane structure are most likely to be the candidate CMS genes. Sequences of all three candidate CMS genes in Nsa CMS line were found to be 100% identical with those from S. arvensis mitochondrial genome. Phylogenetic and homologous analysis showed that all the mitochondrial genes were highly conserved during evolution. CONCLUSIONS: Nsa CMS contains a recombined mitochondrial genome of its two parental species with the majority form S. arvensis. Three candidate Nsa CMS genes were identified and proven to be derived from S. arvensis other than recombination of its two parental species. Further functional study of the candidate genes will help to identify the gene responsible for the CMS and the underlying molecular mechanism.

CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.

Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum-valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.

Transcriptome and Hormone Comparison of Three Cytoplasmic Male Sterile Systems in Brassica napus.

The interaction between plant mitochondria and the nucleus markedly influences stress responses and morphological features, including growth and development. An important example of this interaction is cytoplasmic male sterility (CMS), which results in plants producing non-functional pollen. In current research work, we compared the phenotypic differences in floral buds of different Brassica napus CMS (Polima, Ogura, Nsa) lines with their corresponding maintainer lines. By comparing anther developmental stages between CMS and maintainer lines, we identified that in the Nsa CMS line abnormality occurred at the tetrad stage of pollen development. Phytohormone assays demonstrated that IAA content decreased in sterile lines as compared to maintainer lines, while the total hormone content was increased two-fold in the S2 stage compared with the S1 stage. ABA content was higher in the S1 stage and exhibited a two-fold decreasing trend in S2 stage. Sterile lines however, had increased ABA content at both stages compared with the corresponding maintainer lines. Through transcriptome sequencing, we compared differentially expressed unigenes in sterile and maintainer lines at both (S1 and S2) developmental stages. We also explored the co-expressed genes of the three sterile lines in the two stages and classified these genes by gene function. By analyzing transcriptome data and validating by RT-PCR, it was shown that some transcription factors (TFs) and hormone-related genes were weakly or not expressed in the sterile lines. This research work provides preliminary identification of the pollen abortion stage in Nsa CMS line. Our focus on genes specifically expressed in sterile lines may be useful to understand the regulation of CMS.

An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape.

With the rapid development of sequence specific nucleases (SSNs) for genome targeting, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is now considered the most promising method for functional genetic researches, as well as genetic improvement in crop plants. However, the gene redundancy feature within the allotetraploid rapeseed genome is one of the major obstacles for simultaneous modification of different homologs in the first generation. In addition, large scale screening to identify mutated transgenic plants is very time-and labor-consuming using the conventional restriction enzyme-based approaches. In this study, a streamlined rapeseed CRISPR-Cas9 genome editing platform was developed through synthesizing a premade U6-26 driven sgRNA expression cassette and optimizing polyacrylamide gel electrophoresis (PAGE)-based screening approach. In our experiment, a sgRNA was constructed to target five rapeseed SPL3 homologous gene copies, BnSPL3-A5/BnSPL3-A4/BnSPL3-C3/BnSPL3-C4/BnSPL3-Cnn. High-throughput sequencing analysis demonstrated that the editing frequency of CRISPR/Cas9-induced mutagenesis ranged from 96.8 to 100.0% in plants with obvious heteroduplexed PAGE bands, otherwise this proportion was only 0.00-60.8%. Consistent with those molecular analyses, Bnspl3 mutants exhibited developmental delay phenotype in the first generation. In summary, our data suggest that this set of CRISPR/Cas9 platform is qualified for rapidly generating and identifying simultaneous mutagenesis of multiple gene homologs in allotetraploid rapeseed.

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.

Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus.

Oilseed rape (Brassica napus L.) is the second largest oilseed crop worldwide and one of the most important oil crops in China. As a component of plant architecture, branch angle plays an important role in yield performance, especially under high-density planting conditions. However, the mechanisms underlying the regulation of branch angle are still largely not understood. Two oilseed rape lines with significantly different branch angles were used to conduct RNA- and miRNA-profiling at two developmental stages, identifying differential expression of a large number of genes involved in auxin- and brassinosteroid (BR)-related pathways. Many auxin response genes, including AUX1, IAA, GH3, and ARF, were enriched in the compact line. However, a number of genes involved in BR signaling transduction and biosynthesis were down-regulated. Differentially expressed miRNAs included those involved in auxin signaling transduction. Expression patterns of most target genes were fine-tuned by related miRNAs, such as miR156, miR172, and miR319. Some miRNAs were found to be differentially expressed at both developmental stages, including three miR827 members. Our results provide insight that auxin- and BR-signaling may play a pivotal role in branch angle regulation.

Identification of BnaYUCCA6 as a candidate gene for branch angle in Brassica napus by QTL-seq.

Oilseed rape (Brassica napus L.) is one of the most important oil crops in China as well as worldwide. Branch angle as a plant architecture component trait plays an important role for high density planting and yield performance. In this study, bulked segregant analysis (BSA) combined with next generation sequencing technology was used to fine map QTL for branch angle. A major QTL, designated as branch angle 1 (ba1) was identified on A06 and further validated by Indel marker-based classical QTL mapping in an F2 population. Eighty-two genes were identified in the ba1 region. Among these genes, BnaA0639380D is a homolog of AtYUCCA6. Sequence comparison of BnaA0639380D from small- and big-branch angle oilseed rape lines identified six SNPs and four amino acid variation in the promoter and coding region, respectively. The expression level of BnaA0639380D is significantly higher in the small branch angle line Purler than in the big branch angle line Huyou19, suggesting that the genomic mutations may result in reduced activity of BnaA0639380D in Huyou19. Phytohormone determination showed that the IAA content in Purler was also obviously increased. Taken together, our results suggested BnaA0639380D is a possible candidate gene for branch angle in oilseed rape.

Genomic identification, characterization and differential expression analysis of SBP-box gene family in Brassica napus.

BACKGROUND: SBP-box genes belong to one of the largest families of transcription factors. Though members of this family have been characterized to be important regulators of diverse biological processes, information of SBP-box genes in the third most important oilseed crop Brassica napus is largely undefined. RESULTS: In the present study, by whole genome bioinformatics analysis and transcriptional profiling, 58 putative members of SBP-box gene family in oilseed rape (Brassica napus L.) were identified and their expression pattern in different tissues as well as possible interaction with miRNAs were analyzed. In addition, B. napus lines with contrasting branch angle were used for investigating the involvement of SBP-box genes in plant architecture regulation. Detailed gene information, including genomic organization, structural feature, conserved domain and phylogenetic relationship of the genes were systematically characterized. By phylogenetic analysis, BnaSBP proteins were classified into eight distinct groups representing the clear orthologous relationships to their family members in Arabidopsis and rice. Expression analysis in twelve tissues including vegetative and reproductive organs showed different expression patterns among the SBP-box genes and a number of the genes exhibit tissue specific expression, indicating their diverse functions involved in the developmental process. Forty-four SBP-box genes were ascertained to contain the putative miR156 binding site, with 30 and 14 of the genes targeted by miR156 at the coding and 3'UTR region, respectively. Relative expression level of miR156 is varied across tissues. Different expression pattern of some BnaSBP genes and the negative correlation of transcription levels between miR156 and its target BnaSBP gene were observed in lines with different branch angle. CONCLUSIONS: Taken together, this study represents the first systematic analysis of the SBP-box gene family in Brassica napus. The data presented here provides base foundation for understanding the crucial roles of BnaSBP genes in plant development and other biological processes.

Multigenic Control of Pod Shattering Resistance in Chinese Rapeseed Germplasm Revealed by Genome-Wide Association and Linkage Analyses.

The majority of rapeseed cultivars shatter seeds upon maturity especially under hot-dry and windy conditions, reducing yield and gross margin return to growers. Here, we identified quantitative trait loci (QTL) for resistance to pod shatter in an unstructured diverse panel of 143 rapeseed accessions, and two structured populations derived from bi-parental doubled haploid (DH) and inter-mated (IF2) crosses derived from R1 (resistant to pod shattering) and R2 (prone to pod shattering) accessions. Genome-wide association analysis identified six significant QTL for resistance to pod shatter located on chromosomes A01, A06, A07, A09, C02, and C05. Two of the QTL, qSRI.A09 delimited with the SNP marker Bn-A09-p30171993 (A09) and qSRI.A06 delimited with the SNP marker Bn-A06-p115948 (A06) could be repeatedly detected across environments in a diversity panel, DH and IF2 populations, suggesting that at least two loci on chromosomes A06 and A09 were the main contributors to pod shatter resistance in Chinese germplasm. Significant SNP markers identified in this study especially those that appeared repeatedly across environments provide a cost-effective and an efficient method for introgression and pyramiding of favorable alleles for pod shatter resistance via marker-assisted selection in rapeseed improvement programs.

Characterizing Variation of Branch Angle and Genome-Wide Association Mapping in Rapeseed (Brassica napus L.).

Changes in the rapeseed branch angle alter plant architecture, allowing more efficient light capture as planting density increases. In this study, a natural population of rapeseed was grown in three environments and evaluated for branch angle trait to characterize their phenotypic patterns and genotype with a 60K Brassica Infinium SNP array. Significant phenotypic variation was observed from 20 to 70°. As a result, 25 significant quantitative trait loci (QTL) associated with branch angle were identified on chromosomes A2, A3, A7, C3, C5, and C7 by the MLM model in TASSEL 4.0. Orthologs of the functional candidate genes involved in branch angle were identified. Among the key QTL, the peak SNPs were close to the key orthologous genes BnaA.Lazy1 and BnaC.Lazy1 on A3 and C3 homologous genome blocks. With the exception of Lazy (LA) orthologous genes, SQUMOSA PROMOTER BINDING PROTEIN LIKE 14 (SPL14) and an auxin-responsive GRETCHEN HAGEN 3 (GH3) genes from Arabidopsis thaliana were identified close to two clusters of SNPs on the A7 and C7 chromosomes. These findings on multiple novel loci and candidate genes of branch angle will be useful for further understanding and genetic improvement of plant architecture in rapeseed.

Down-regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil.

High oleic oil is an important industrial feedstock that has been one of the main targets for oil improvement in a number of oil crops. Crambe (Crambe abyssinica) is a dedicated oilseed crop, suitable for industrial oil production. In this study, we down-regulated the crambe fatty acid desaturase (FAD) and fatty acid elongase (FAE) genes for creating high oleic seed oil. We first cloned the crambe CaFAD2, CaFAD3 and CaFAE1 genes. Multiple copies of each of these genes were isolated, and the highly homologous sequences were used to make RNAi constructs. These constructs were first tested in Arabidopsis, which led to the elevated oleic or linoleic levels depending on the genes targeted, indicating that the RNAi constructs were effective in regulating the expression of the target genes in nonidentical but closely related species. Furthermore, down-regulation of CaFAD2 and CaFAE1 in crambe with the FAD2-FAE1 RNAi vector resulted in even more significant increase in oleic acid level in the seed oil with up to 80% compared to 13% for wild type. The high oleic trait has been stable in subsequent five generations and the GM line grew normally in greenhouse. This work has demonstrated the great potential of producing high oleic oil in crambe, thus contributing to its development into an oil crop platform for industrial oil production.