Genome-wide association study of stem structural characteristics that extracted by a high-throughput phenotypic analysis "LabelmeP rice" in rice.

Stem is important for assimilating transport and plant strength; however, less is known about the genetic basis of its structural characteristics. In this study, a high-throughput method, "LabelmeP rice" was developed to generate 14 traits related to stem regions and vascular bundles, which allows the establishment of a stem cross-section phenotype dataset containing anatomical information of 1738 images from hand-cut transections of stems collected from 387 rice germplasm accessions grown over two successive seasons. Then, the phenotypic diversity of the rice accessions was evaluated. Genome-wide association studies identified 94, 83, and 66 significant single nucleotide polymorphisms (SNPs) for the assayed traits in 2 years and their best linear unbiased estimates, respectively. These SNPs can be integrated into 29 quantitative trait loci (QTL), and 11 of them were common in 2 years, while correlated traits shared 19. In addition, 173 candidate genes were identified, and six located at significant SNPs were repeatedly detected and annotated with a potential function in stem development. By using three introgression lines (chromosome segment substitution lines), four of the 29 QTLs were validated. LOC_Os01g70200, located on the QTL uq1.4, is detected for the area of small vascular bundles (SVB) and the rate of large vascular bundles number to SVB number. Besides, the CRISPR/Cas9 editing approach has elucidated the function of the candidate gene LOC_Os06g46340 in stem development. In conclusion, the results present a time- and cost-effective method that provides convenience for extracting rice stem anatomical traits and the candidate genes/QTL, which would help improve rice.

Functional genomics of Brassica napus: Progresses, challenges, and perspectives.

Brassica napus, commonly known as rapeseed or canola, is a major oil crop contributing over 13% to the stable supply of edible vegetable oil worldwide. Identification and understanding the gene functions in the B. napus genome is crucial for genomic breeding. A group of genes controlling agronomic traits have been successfully cloned through functional genomics studies in B. napus. In this review, we present an overview of the progress made in the functional genomics of B. napus, including the availability of germplasm resources, omics databases and cloned functional genes. Based on the current progress, we also highlight the main challenges and perspectives in this field. The advances in the functional genomics of B. napus contribute to a better understanding of the genetic basis underlying the complex agronomic traits in B. napus and will expedite the breeding of high quality, high resistance and high yield in B. napus varieties.

Targeted mutagenesis of flavonoid biosynthesis pathway genes reveals functional divergence in seed coat colour, oil content and fatty acid composition in Brassica napus L.

Yellow-seed is widely accepted as a good-quality trait in Brassica crops. Previous studies have shown that the flavonoid biosynthesis pathway is essential for the development of seed colour, but its function in Brassica napus, an important oil crop, is poorly understood. To systematically explore the gene functions of the flavonoid biosynthesis pathway in rapeseed, several representative TRANSPARENT TESTA (TT) genes, including three structural genes (BnaTT7, BnaTT18, BnaTT10), two regulatory genes (BnaTT1, BnaTT2) and a transporter (BnaTT12), were selected for targeted mutation by CRISPR/Cas9 in the present study. Seed coat colour, lignin content, seed quality and yield-related traits were investigated in these Bnatt mutants together with Bnatt8 generated previously. These Bnatt mutants produced seeds with an elevated seed oil content and decreased pigment and lignin accumulation in the seed coat without any serious defects in the yield-related traits. In addition, the fatty acid (FA) composition was also altered to different degrees, i.e., decreased oleic acid and increased linoleic acid and α-linolenic acid, in all Bnatt mutants except Bnatt18. Furthermore, gene expression analysis revealed that most of BnaTT mutations resulted in the down-regulation of key genes related to flavonoid and lignin synthesis, and the up-regulation of key genes related to lipid synthesis and oil body formation, which may contribute to the phenotype. Collectively, our study generated valuable resources for breeding programs, and more importantly demonstrated the functional divergence and overlap of flavonoid biosynthesis pathway genes in seed coat colour, oil content and FA composition of rapeseed.

Fine mapping and candidate gene analysis of a major QTL for oil content in the seed of Brassica napus.

KEY MESSAGE: By integrating QTL fine mapping and transcriptomics, a candidate gene responsible for oil content in rapeseed was identified. The gene is anticipated to primarily function in photosynthesis and photosystem metabolism pathways. Brassica napus is one of the most important oil crops in the world, and enhancing seed oil content is an important goal in its genetic improvement. However, the underlying genetic basis for the important trait remains poorly understood in this crop. We previously identified a major locus, OILA5 responsible for seed oil content on chromosome A5 through genome-wide association study. To better understand the genetics of the QTL, we performed fine mapping of OILA5 with a double haploid population and a BC3F2 segregation population consisting of 6227 individuals. We narrowed down the QTL to an approximate 43 kb region with twelve annotated genes, flanked by markers ZDM389 and ZDM337. To unveil the potential candidate gene responsible for OILA5, we integrated fine mapping data with transcriptome profiling using high and low oil content near-isogenic lines. Among the candidate genes, BnaA05G0439400ZS was identified with high expression levels in both seed and silique tissues. This gene exhibited homology with AT3G09840 in Arabidopsis that was annotated as cell division cycle 48. We designed a site-specific marker based on resequencing data and confirmed its effectiveness in both natural and segregating populations. Our comprehensive results provide valuable genetic information not only enhancing our understanding of the genetic control of seed oil content but also novel germplasm for advancing high seed oil content breeding in B. napus and other oil crops.

A novel type of Brassica napus with higher stearic acid in seeds developed through genome editing of BnaSAD2 family.

KEY MESSAGE: Modifications of multiple copies of the BnaSAD2 gene family with genomic editing technology result in higher stearic acid content in the seed of polyploidy rapeseed. Solid fats from vegetable oils are widely used in food processing industry. Accumulating data showed that stearic acid is more favorite as the major composite among the saturate fatty acids in solid fats in considerations of its effects on human health. Rapeseed is the third largest oil crop worldwide, and has potential to be manipulated to produce higher saturated fatty acids as raw materials of solid fats. Toward that end, we identified four SAD2 gene family members in B. napus genome and established spatiotemporal expression pattern of the BnaSAD2 members. Genomic editing technology was applied to mutate all the copies of BnaSAD2 in this allopolyploid species and mutants at multiple alleles were generated and characterized to understand the effect of each BnaSAD2 member on blocking desaturation of stearic acid. Mutations occurred at BnaSAD2.A3 resulted in more dramatic changes of fatty acid profile than ones on BnaSAD2.C3, BnaSAD2.A5 and BnaSAD2.C4. The content of stearic acid in mutant seeds with single locus increased dramatically with a range of 3.1-8.2%. Furthermore, combination of different mutated alleles of BnaSAD2 resulted in more dramatic changes in fatty acid profiles and the double mutant at BnaSAD2.A3 and BnaSAD2.C3 showed the most dramatic phenotypic changes compared with its single mutants and other double mutants, leading to 11.1% of stearic acid in the seeds. Our results demonstrated that the members of BnaSAD2 have differentiated in their efficacy as a Δ9-Stearoyl-ACP-Desaturase and provided valuable rapeseed germplasm for breeding high stearic rapeseed oil.

Targeted mutagenesis of BnaSTM leads to abnormal shoot apex development and cotyledon petiole fusion at the seedling stage in Brassica napus L.

The Arabidopsis homeodomain transcription factor SHOOT MERISTEMLESS (STM) is crucial for shoot apical meristem (SAM) function, which cooperates with CLAVATA3 (CLV3)/WUSCHEL (WUS) feedback regulation loops to maintain the homeostasis of stem cells in SAM. STM also interacts with the boundary genes to regulate the tissue boundary formation. However, there are still few studies on the function of STM in Brassica napus, an important oil crop. There are two homologs of STM in B. napus (BnaA09g13310D and BnaC09g13580D). In the present study, CRISPR/Cas9 technology was employed to create the stable site-directed single and double mutants of the BnaSTM genes in B. napus. The absence of SAM could be observed only in the BnaSTM double mutants at the mature embryo of seed, indicating that the redundant roles of BnaA09.STM and BnaC09.STM are vital for regulating SAM development. However, different from Arabidopsis, the SAM gradually recovered on the third day after seed germination in Bnastm double mutants, resulting in delayed true leaves development but normal late vegetative and reproductive growth in B. napus. The Bnastm double mutant displayed a fused cotyledon petiole phenotype at the seedling stage, which was similar but not identical to the Atstm in Arabidopsis. Further, transcriptome analysis showed that targeted mutation of BnaSTM caused significant changes for genes involved in the SAM boundary formation (CUC2, CUC3, LBDs). In addition, Bnastm also caused significant changes of a sets of genes related to organogenesis. Our findings reveal that the BnaSTM plays an important yet distinct role during SAM maintenance as compared to Arabidopsis.

Precise A∙T to G∙C base editing in the allotetraploid rapeseed (Brassica napus L.) genome.

Rapeseed is an important source of oilseed crop in the world. Achieving genetic improvement has always been the major goal in rapeseed production. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Nowadays, Cas-base editing acts as an efficient tool to mediate single-base substitution at the target site. In this study, four adenine base editors (ABE) were modified to achieve adenosine base editing at different genome sites in allotetraploid Brassica napus. We designed 18 small guide RNAs to target phytoene desaturase (PDS), acetolactate synthase (ALS), CLAVATA3 (CLV3), CLV2, TRANSPARENT TESTA12 (TT12), carotenoid isomerase (CRTISO), designated de-etiolated-2 (DET2), BRANCHED1 (BRC1), zeaxanthin epoxidase (ZEP) genes, respectively. Among the four ABE systems, pBGE17 had the highest base-editing efficiency, with an average editing efficiency of 3.51%. Target sequencing results revealed that the editing window ranged from A5 to A8 of the protospacer-adjacent motif (PAM) sequence. Moreover, the ABEmax-nCas9NG system with NG PAM was developed, with a base-editing efficiency of 1.22%. These results revealed that ABE system developed in this study could efficiently induce A to G substitution and the ABE-nCas9NG system could broaden editing window in oilseed rape.

Global identification of quantitative trait loci and candidate genes for cold stress and chilling acclimation in rice through GWAS and RNA-seq.

MAIN CONCLUSION: Associated analysis of GWAS with RNA-seq had detected candidate genes responsible for cold stress and chilling acclimation in rice. Haplotypes of two candidate genes and geographic distribution were analyzed. To explore new candidate genes and genetic resources for cold tolerance improvement in rice, genome-wide association study (GWAS) mapping experiments with 351 rice core germplasms was performed for three traits (survival rate, shoot length and chlorophyll content) under three temperature conditions (normal temperature, cold stress and chilling acclimation), yielding a total of 134 QTLs, of which 54, 59 and 21 QTLs were responsible for normal temperature, cold stress and chilling acclimation conditions, respectively. Integrated analysis of significant SNPs in 134 QTLs further identified 116 QTLs for three temperature treatments, 53, 43 and 18 QTLs responsible for normal temperature, cold stress and chilling acclimation, respectively, and 2 QTLs were responsible for both cold stress and chilling acclimation. Matching differentially expressed genes from RNA-seq to 43 and 18 QTLs for cold stress and chilling acclimation, we identified 69 and 44 trait-associated candidate genes, respectively, to be classified into six and five groups, particularly involved in metabolisms, reactive oxygen species scavenging and hormone signaling. Interestingly, two candidate genes LOC_Os01g04814, encoding a vacuolar protein sorting-associating protein 4B, and LOC_Os01g48440, encoding glycosyltransferase family 43 protein, showed the highest expression levels under chilling acclimation. Haplotype analysis revealed that both genes had a distinctive differentiation with subpopulation. Haplotypes of both genes with more japonica accessions have higher latitude distribution and higher chilling tolerance than the chilling sensitive indica accessions. These findings reveal the new insight into the molecular mechanism and candidate genes for cold stress and chilling acclimation in rice.

Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

The diversity of petal and leaf color can improve the ornamental value of rapeseed and promote the development of agriculture and tourism. The two copies of carotenoid isomerase gene (BnaCRTISO) in Brassica napus (BnaA09.CRTISO and BnaC08.CRTISO) was edited using the CRISPR/Cas9 system in the present study. The mutation phenotype of creamy white petals and yellowish leaves could be recovered only in targeted mutants of both BnaCRTISO functional copies, indicating that the redundant roles of BnaA09.CRTISO and BnaC08.CRTISO are vital for the regulation of petal and leaf color. The carotenoid content in the petals and leaves of the BnaCRTISO double mutant was significantly reduced. The chalcone content, a vital substance that makes up the yellow color, also decreased significantly in petals. Whereas, the contents of some carotenes (lycopene, α-carotene, γ-carotene) were increased significantly in petals. Further, transcriptome analysis showed that the targeted mutation of BnaCRTISO resulted in the significant down-regulation of important genes BnaPSY and BnaC4H in the carotenoid and flavonoid synthesis pathways, respectively; however, the expression of other genes related to carotenes and xanthophylls synthesis, such as BnaPDS3, BnaZEP, BnaBCH1 and BCH2, was up-regulated. This indicates that the molecular mechanism regulating petal color variation in B. napus is more complicated than those reported in Arabidopsis and other Brassica species. These results provide insight into the molecular mechanisms underlying flower color variation in rapeseed and provides valuable resources for rapeseed breeding.

Advances and Challenges for QTL Analysis and GWAS in the Plant-Breeding of High-Yielding: A Focus on Rapeseed.

Yield is one of the most important agronomic traits for the breeding of rapeseed (Brassica napus L), but its genetic dissection for the formation of high yield remains enigmatic, given the rapid population growth. In the present review, we review the discovery of major loci underlying important agronomic traits and the recent advancement in the selection of complex traits. Further, we discuss the benchmark summary of high-throughput techniques for the high-resolution genetic breeding of rapeseed. Biparental linkage analysis and association mapping have become powerful strategies to comprehend the genetic architecture of complex agronomic traits in crops. The generation of improved crop varieties, especially rapeseed, is greatly urged to enhance yield productivity. In this sense, the whole-genome sequencing of rapeseed has become achievable to clone and identify quantitative trait loci (QTLs). Moreover, the generation of high-throughput sequencing and genotyping techniques has significantly enhanced the precision of QTL mapping and genome-wide association study (GWAS) methodologies. Furthermore, this study demonstrates the first attempt to identify novel QTLs of yield-related traits, specifically focusing on ovule number per pod (ON). We also highlight the recent breakthrough concerning single-locus-GWAS (SL-GWAS) and multi-locus GWAS (ML-GWAS), which aim to enhance the potential and robust control of GWAS for improved complex traits.

Fine mapping and candidate gene analysis of a major locus controlling ovule abortion and seed number per silique in Brassica napus L.

KEY MESSAGE: A major QTL controlling ovule abortion and SN was fine-mapped to a 80.1-kb region on A8 in rapeseed, and BnaA08g07940D and BnaA08g07950D are the most likely candidate genes. The seed number per silique (SN), an important yield determining trait of rapeseed, is the final consequence of a complex developmental process including ovule initiation and the subsequent ovule/seed development. To explore the genetic mechanism regulating the natural variation of SN and its related components, quantitative trait locus (QTL) mapping was conducted using a doubled haploid (DH) population derived from the cross between C4-146 and C4-58B, which showed significant differences in SN and aborted ovule number (AON), but no obvious differences in ovule number (ON). QTL analysis identified 19 consensus QTLs for six SN-related traits across three environments. A novel QTL on chromosome A8, un.A8, which associates with multiple traits, except for ON, was stably detected across the three environments. This QTL explained more than 50% of the SN, AON and percentage of aborted ovules (PAO) variations as well as a moderate contribution on silique length (SL) and thousand seed weight (TSW). The C4-146 allele at the locus increases SN and SL but decreases AON, PAO and TSW. Further fine mapping narrowed down this locus into an 80.1-kb interval flanked by markers BM1668 and BM1672, and six predicted genes were annotated in the delimited region. Expression analyses and DNA sequencing showed that two homologs of Arabidopsis photosystem I subunit F (BnaA08g07940D) and zinc transporter 10 precursor (BnaA08g07950D) were the most promising candidate genes underlying this locus. These results provide a solid basis for cloning un.A8 to reduce the ovule abortion and increase SN in the yield improvement of rapeseed.

QTL Mapping and Candidate Gene Identification of Swollen Root Formation in Turnip.

The swollen root is an important agronomic trait and is a determinant of yield for turnips, which are cultivated as both vegetables and fodder. However, the genetic mechanism of swollen root formation is poorly understood. In this study, we analyzed the F2 and BC1P2 populations derived from a cross between "10601" (European turnip with swollen root, Brassica rapa ssp. rapifera, AA, 2n = 2× = 20) and "10603" (Chinese cabbage with normal root, Brassica rapa ssp. pekinensis, AA, 2n = 2× = 20), and suggested that the swollen root is a quantitative trait. Two major quantitative trait loci (QTLs), FR1.1 (Fleshy root 1.1) and FR7.1 (Fleshy root 7.1), were identified by QTL-seq analysis and further confirmed by QTL mapping in F2 and BC1P2 populations. The QTL FR1.1 with a likelihood of odd (LOD) of 7.01 explained 17.2% of the total phenotypic variations for root diameter and the QTL FR7.1 explained 23.0% (LOD = 9.38) and 31.0% (LOD = 13.27) of the total phenotypic variations in root diameter and root weight, respectively. After a recombinant screening, the major QTL FR7.1 was further narrowed down to a 220 kb region containing 47 putative genes. A candidate gene, Bra003652, which is a homolog of AT1G78240 that plays an essential role in cell adhesion and disorganized tumor-like formation in Arabidopsis thaliana, was identified in this region. In addition, expression and parental allele analysis supported that Bra003652 was a possible candidate gene of QTL FR7.1 for swollen root formation in turnip. Our research may provide new insight into the molecular mechanism of swollen root formation in root crops.

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production.

Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T1 and T2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.

Comprehensive study and multipurpose role of the CLV3/ESR-related (CLE) genes family in plant growth and development.

The CLAVATA3/endosperm surrounding region-related (CLE) is one of the most important signaling peptides families in plants. These peptides signaling are common in the cell to cell communication and control various physiological and developmental processes, that is cell differentiation and proliferation, self-incompatibility, and the defense response. The CLE signaling systems are conserved across the plant kingdom but have a diverse mode of action in various developmental processes in different species. In this review, we concise various methods of peptides identification, structure, and molecular identity of the CLE family, the developmental role of CLE genes/peptides in plants, environmental stimuli, and CLE family and some other novel progress in CLE genes/peptides in various crops, and so forth. According to previous literature, about 1,628 CLE genes were identified in land plants, which deeply explained the tale of plant development. Nevertheless, some important queries need to be addressed to get clear insights into the CLE gene family in other organisms and their role in various physiological and developmental processes. Furthermore, we summarized the power of the CLE family around the environment as well as bifunctional activity and the crystal structure recognition mechanism of CLE peptides by their receptors and CLE clusters functions. We strongly believed that the discovery of the CLE family in other organisms would provide a significant breakthrough for future revolutionary and functional studies.

BnA10.RCO, a homeobox gene, positively regulates leaf lobe formation in Brassica napus L.

KEY MESSAGE: BnA10.RCO positively regulates the development of leaf lobes in Brassica napus, and cis-regulatory divergences cause the different allele effects. The functional importance of lobed leaves in rapeseed (Brassica napus L.) has been identified with potential advantages for high-density planting and hybrid production. Our previous studies indicated that the tandemly duplicated LMI1-like genes BnA10. RCO and BnA10.LMI1 are candidate genes of an incompletely dominant locus, which is responsible for the lobed-leaf shape in rapeseed. We provided strong evidence that BnA10.LMI1 positively regulates leaf lobe formation. Here, we show that BnA10.RCO is a nucleus-specific protein, encoding an HD-ZIP I transcription factor, which is responsible for the lobed-leaf shape in rapeseed. Sequence analysis of parental alleles revealed that no vital sequence variation was detected in the coding sequence of BnA10.RCO, whereas abundant variations were identified in the regulatory regions. Consistent with this finding, the expression level of BnRCO was substantially elevated in the lobed-leaved parent HY compared with its near-isogenic line. Moreover, the altered expression of BnA10.RCO in transgenic lines showed a positive connection with leaf complexity without a substantial change in BnLMI1 transcript level. Furthermore, CRISPR/Cas9-induced null mutations of BnA10.RCO in the lobed-leaved parent HY were sufficient to produce an unlobed leaf without alteration in BnLMI1 transcript level. Our results indicate that BnA10.RCO functions together with BnA10.LMI1 to positively determine the lobed-leaf development, providing a fundamental basis for crop improvement by targeting leaf shape in rapeseed.

Identification of QTLs Containing Resistance Genes for Sclerotinia Stem Rot in Brassica napus Using Comparative Transcriptomic Studies.

Sclerotinia stem rot is a major disease in Brassica napus that causes yield losses of 10-20% and reaching 80% in severely infected fields. SSR not only causes yield reduction but also causes low oil quality by reducing fatty acid content. There is a need to identify resistant genetic sources with functional significance for the breeding of SSR-resistant cultivars. In this study, we identified 17 QTLs involved in SSR resistance in three different seasons using SNP markers and disease lesion development after artificial inoculation. There were no common QTLs in all 3 years, but there were three QTLs that appeared in two seasons covering all seasons with a shared QTL. The QTLs identified in the 2 years were SRA9a, SRC2a and SRC3a with phenotypic effect variances of 14.75 and 11.57% for SRA9a, 7.49 and 10.38% for SRC3a and 7.73 and 6.81% for SRC2a in their 2 years, respectively. The flowering time was also found to have a negative correlation with disease resistance, i.e., early-maturing lines were more susceptible to disease. The stem width has shown a notably weak effect on disease development, causing researchers to ignore its effect. Given that flowering time is an important factor in disease resistance, we used comparative RNA-sequencing analysis of resistant and susceptible lines with consistent performance in 3 years with almost the same flowering time to identify the resistance genes directly involved in resistance within the QTL regions. Overall, there were more genes differentially expressed in resistant lines 19,970 than in susceptible lines 3936 compared to their mock-inoculated lines, demonstrating their tendency to cope with disease. We identified 36 putative candidate genes from the resistant lines that were upregulated in resistant lines compared to resistant mock and susceptible lines that might be involved in resistance to SSR.

Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus.

KEY MESSAGE: Genomic editing with CRISPR/Cas9 system can simultaneously modify multiple copies of theBnaFAD2 gene to develop novel variations in fatty acids profiles in polyploidy rapeseed. Fatty acid composition affects edible and processing quality of vegetable oil and has been one of the primary targets for genetic modification in oilseed crops including rapeseed (Brassica napus). Fatty acid desaturase 2 gene, FAD2, is a key player that affects three major fatty acids, namely oleic, linoleic and linolenic acid, in oilseed plants. Previously, we showed that there are four copies of BnaFAD2 in allotetraploid rapeseed. In this study, we further established spatiotemporal expression pattern of each copy of BnaFAD2 using published RNA-seq data. Genomic editing technology based on CRISPR/Cas9 system was used to mutate all the copies of BnaFAD2 to create novel allelic variations in oleic acid and other fatty acid levels. A number of mutants at two targeting sites were identified, and the phenotypic variation in the mutants was systematically evaluated. The oleic acid content in the seed of the mutants increased significantly with the highest exceeding 80% compared with wild type of 66.43%, while linoleic and linolenic acid contents decreased accordingly. Mutations on BnaFAD2.A5 caused more dramatic changes of fatty acid profile than the mutations on BnaFAD2.C5 alleles that were identified with gene editing technique for the first time. Moreover, combining different mutated alleles of BnaFAD2 can even broaden the variation more dramatically. It was found that effects of different mutation types at BnaFAD2 alleles on oleic levels varied, indicating a possibility to manipulate fatty acid levels by precise mutation at specific region of a gene.

Nonspecific phospholipase C6 increases seed oil production in oilseed Brassicaceae plants.

Plant oils are valuable commodities for food, feed, renewable industrial feedstocks and biofuels. To increase vegetable oil production, here we show that the nonspecific phospholipase C6 (NPC6) promotes seed oil production in the Brassicaceae seed oil species Arabidopsis, Camelina and oilseed rape. Overexpression of NPC6 increased seed oil content, seed weight and oil yield both in Arabidopsis and Camelina, whereas knockout of NPC6 decreased seed oil content and seed size. NPC6 is associated with the chloroplasts and microsomal membranes, and hydrolyzes phosphatidylcholine and galactolipids to produce diacylglycerol. Knockout and overexpression of NPC6 decreased and increased, respectively, the flux of fatty acids from phospholipids and galactolipids into triacylglycerol production. Candidate-gene association study in oilseed rape indicates that only BnNPC6.C01 of the four homeologues NPC6s is associated with seed oil content and yield. Haplotypic analysis indicates that the BnNPC6.C01 favorable haplotype can increase both seed oil content and seed yield. These results indicate that NPC6 promotes membrane glycerolipid turnover to accumulate TAG production in oil seeds and that NPC6 has a great application potential for oil yield improvement.

Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L).

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L.

Yellow seed is a desirable trait with great potential for improving seed quality in Brassica crops. Unfortunately, no natural or induced yellow seed germplasms have been found in Brassica napus, an important oil crop, which likely reflects its genome complexity and the difficulty of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we demonstrate the first application of CRISPR/Cas9 for creating yellow-seeded mutants in rapeseed. The targeted mutations of the BnTT8 gene were stably transmitted to successive generations, and a range of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The yellow-seeded phenotype could be recovered only in targeted mutants of both BnTT8 functional copies, indicating that the redundant roles of BnA09.TT8 and BnC09.TT8b are vital for seed colour. The BnTT8 double mutants produced seeds with elevated seed oil and protein content and altered fatty acid (FA) composition without any serious defects in the yield-related traits, making it a valuable resource for rapeseed breeding programmes. Chemical staining and histological analysis showed that the targeted mutations of BnTT8 completely blocked the proanthocyanidin (PA)-specific deposition in the seed coat. Further, transcriptomic profiling revealed that the targeted mutations of BnTT8 resulted in the broad suppression of phenylpropanoid/flavonoid biosynthesis genes, which indicated a much more complex molecular mechanism underlying seed colour formation in rapeseed than in Arabidopsis and other Brassica species. In addition, gene expression analysis revealed the possible mechanism through which BnTT8 altered the oil content and fatty acid composition in seeds.