Plant Functional Genomics Based on High-Throughput CRISPR Library Knockout Screening: A Perspective.

Plant biology studies in the post-genome era have been focused on annotating genome sequences' functions. The established plant mutant collections have greatly accelerated functional genomics research in the past few decades. However, most plant genome sequences' roles and the underlying regulatory networks remain substantially unknown. Clustered, regularly interspaced short palindromic repeat (CRISPR)-associated systems are robust, versatile tools for manipulating plant genomes with various targeted DNA perturbations, providing an excellent opportunity for high-throughput interrogation of DNA elements' roles. This study compares methods frequently used for plant functional genomics and then discusses different DNA multi-targeted strategies to overcome gene redundancy using the CRISPR-Cas9 system. Next, this work summarizes recent reports using CRISPR libraries for high-throughput gene knockout and function discoveries in plants. Finally, this work envisions the future perspective of optimizing and leveraging CRISPR library screening in plant genomes' other uncharacterized DNA sequences.

Multi-Omics Analysis Revealed the AGR-FC.C3 Locus of Brassica napus as a Novel Candidate for Controlling Petal Color.

Variations in the petal color of Brassica napus are crucial for ornamental value, but the controlled loci for breeding remain to be unraveled. Here, we report a candidate locus, AGR-FC.C3, having conducted a bulked segregant analysis on a segregating population with different petal colors. Our results showed that the locus covers 9.46 Mb of the genome, harboring 951 genes. BnaC03.MYB4, BnaC03.MYB85, BnaC03.MYB73, BnaC03.MYB98, and BnaC03.MYB102 belonging to MYB TFs families that might regulate the petal color were observed. Next, a bulk RNA sequencing of white and orange-yellow petals on three development stages was performed to further identify the possible governed genes. The results revealed a total of 51 genes by overlapping the transcriptome data and the bulked segregant analysis data, and it was found that the expression of BnaC03.CCD4 was significantly up-regulated in the white petals at three development stages. Then, several novel candidate genes such as BnaC03.ENDO3, BnaC03.T22F8.180, BnaC03.F15C21.8, BnaC03.Q8GSI6, BnaC03.LSD1, BnaC03.MAP1Da, BnaC03.MAP1Db, and BnaC03G0739700ZS putative to controlling the petal color were identified through deeper analysis. Furthermo re, we have developed two molecular markers for the reported functional gene BnaC03.CCD4 to discriminate the white and orange-yellow petal colors. Our results provided a novel locus for breeding rapeseed with multi-color petals.

3D genome structural variations play important roles in regulating seed oil content of Brassica napus.

Dissecting the complex regulatory mechanism of seed oil content (SOC) is one of the main research goals in Brassica napus. Increasing evidence suggests that genome architecture is linked to multiple biological functions. However, the effect of genome architecture on SOC regulation remains unclear. Here, we used high-throughput chromatin conformation capture to characterize differences in the three-dimensional (3D) landscape of genome architecture of seeds from two B. napus lines, N53-2 (with high SOC) and Ken-C8 (with low SOC). Bioinformatics analysis demonstrated that differentially accessible regions and differentially expressed genes between N53-2 and Ken-C8 were preferentially enriched in regions with quantitative trait loci (QTLs)/associated genomic regions (AGRs) for SOC. A multi-omics analysis demonstrated that expression of SOC-related genes was tightly correlated with genome structural variations in QTLs/AGRs of B. napus. The candidate gene BnaA09g48250D, which showed structural variation in a QTL/AGR on chrA09, was identified by fine-mapping of a KN double-haploid population derived from hybridization of N53-2 and Ken-C8. Overexpression and knockout of BnaA09g48250D led to significant increases and decreases in SOC, respectively, in the transgenic lines. Taken together, our results reveal the 3D genome architecture of B. napus seeds and the roles of genome structural variations in SOC regulation, enriching our understanding of the molecular mechanisms of SOC regulation from the perspective of spatial chromatin structure.

Transcriptome Shock in Developing Embryos of a Brassica napus and Brassica rapa Hybrid.

Interspecific crosses that fuse the genomes of two different species may result in overall gene expression changes in the hybrid progeny, called 'transcriptome shock'. To better understand the expression pattern after genome merging during the early stages of allopolyploid formation, we performed RNA sequencing analysis on developing embryos of Brassica rapa, B. napus, and their synthesized allotriploid hybrids. Here, we show that the transcriptome shock occurs in the developing seeds of the hybrids. Of the homoeologous gene pairs, 17.1% exhibit expression bias, with an overall expression bias toward B. rapa. The expression level dominance also biases toward B. rapa, mainly induced by the expression change in homoeologous genes from B. napus. Functional enrichment analysis revealed significant differences in differentially expressed genes (DEGs) related to photosynthesis, hormone synthesis, and other pathways. Further study showed that significant changes in the expression levels of the key transcription factors (TFs) could regulate the overall interaction network in the developing embryo, which might be an essential cause of phenotype change. In conclusion, the present results have revealed the global changes in gene expression patterns in developing seeds of the hybrid between B. rapa and B. napus, and provided novel insights into the occurrence of transcriptome shock for harnessing heterosis.

Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library.

The recently constructed mutant libraries of diploid crops by the CRISPR-Cas9 system have provided abundant resources for functional genomics and crop breeding. However, because of the genome complexity, it is a big challenge to accomplish large-scale targeted mutagenesis in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve genome-scale targeted editing in an allotetraploid crop of Brassica napus A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088 sgRNAs were obtained. Editing interrogation results revealed that 93 of the 178 genes were identified as mutated, thus representing an editing efficiency of 52.2%. Furthermore, we have discovered that Cas9-mediated DNA cleavages tend to occur at all the target sites guided by the same individual sgRNA, a novel finding in polyploid plants. Finally, we show the strong capability of reverse genetic screening for various traits with the postgenotyped plants. Several genes, which might dominate the fatty acid profile and seed oil content and have yet to be reported, were unveiled from the forward genetic studies. Our research provides valuable resources for functional genomics, elite crop breeding, and a good reference for high-throughput targeted mutagenesis in other polyploid plants.

New insight into the genetic basis of oil content based on noninvasive three-dimensional phenotyping and tissue-specific transcriptome in Brassica napus.

BACKGROUND: Increasing seed oil content is the most important breeding goal in Brassica napus, and phenotyping is crucial to dissect its genetic basis in crops. To date, QTL mapping for oil content has been based on whole seeds, and the lipid distribution is far from uniform in different tissues of seeds in B. napus. In this case, the phenotype based on whole seeds was unable to sufficiently reveal the complex genetic characteristics of seed oil content. RESULTS: Here, the three-dimensional (3D) distribution of lipid was determined for B. napus seeds by magnetic resonance imaging (MRI) and 3D quantitative analysis, and ten novel oil content-related traits were obtained by subdividing the seeds. Based on a high-density genetic linkage map, 35 QTLs were identified for 4 tissues, the outer cotyledon (OC), inner cotyledon (IC), radicle (R) and seed coat (SC), which explained up to 13.76% of the phenotypic variation. Notably, 14 tissue-specific QTLs were reported for the first time, 7 of which were novel. Moreover, haplotype analysis showed that the favorable alleles for different seed tissues exhibited cumulative effects on oil content. Furthermore, tissue-specific transcriptomes revealed that more active energy and pyruvate metabolism influenced carbon flow in the IC, OC and R than in the SC at the early and middle seed development stages, thus affecting the distribution difference in oil content. Combining tissue-specific QTL mapping and transcriptomics, 86 important candidate genes associated with lipid metabolism were identified that underlie 19 unique QTLs, including the fatty acid synthesis rate-limiting enzyme-related gene CAC2, in the QTLs for OC and IC. CONCLUSIONS: The present study provides further insight into the genetic basis of seed oil content at the tissue-specific level.

Dissecting the Meiotic Recombination Patterns in a Brassica napus Double Haploid Population Using 60K SNP Array.

Meiotic recombination not only maintains the stability of the chromosome structure but also creates genetic variations for adapting to changeable environments. A better understanding of the mechanism of crossover (CO) patterns at the population level is useful for crop improvement. However, there are limited cost-effective and universal methods to detect the recombination frequency at the population level in Brassica napus. Here, the Brassica 60K Illumina Infinium SNP array (Brassica 60K array) was used to systematically study the recombination landscape in a double haploid (DH) population of B. napus. It was found that COs were unevenly distributed across the whole genome, and a higher frequency of COs existed at the distal ends of each chromosome. A considerable number of genes (more than 30%) in the CO hot regions were associated with plant defense and regulation. In most tissues, the average gene expression level in the hot regions (CO frequency of greater than 2 cM/Mb) was significantly higher than that in the regions with a CO frequency of less than 1 cM/Mb. In addition, a bin map was constructed with 1995 recombination bins. For seed oil content, Bin 1131 to 1134, Bin 1308 to 1311, Bin 1864 to 1869, and Bin 2184 to 2230 were identified on chromosomes A08, A09, C03, and C06, respectively, which could explain 8.5%, 17.3%, 8.6%, and 3.9% of the phenotypic variation. These results could not only deepen our understanding of meiotic recombination in B. napus at the population level, and provide useful information for rapeseed breeding in the future, but also provided a reference for studying CO frequency in other species.

Characterization of Oil Body and Starch Granule Dynamics in Developing Seeds of Brassica napus.

Brassica napus is the most important oilseed crop in the world, and the lipid was stored in the oil body (OB) in the form of triacylglycerol. At present, most of studies on the relationship between oil body morphology and seed oil content in B. napus was focused on mature seeds. In the present study, the OBs in different developing seeds of B. napus with relatively high oil content (HOC) of about 50% and low oil content (LOC) of about 39% were analyzed. It was revealed that the size of OBs was first increased and then decreased in both materials. And in late seed developmental stages, the average OB size of rapeseed with HOC was higher than that of LOC, while it was reversed in the early seed developmental stages. No significant difference was observed on starch granule (SG) size in HOC and LOC rapeseed. Further results indicated that the expression of genes that involved in malonyl-CoA metabolism, fatty acid carbon chain extension, lipid metabolism, and starch synthesis in the rapeseed with HOC was higher than that of rapeseed with LOC. These results give some new insight for understanding the dynamics of OBs and SGs in embryos of B. napus.

Class A lysophosphatidic acid acyltransferase 2 from Camelina sativa promotes very long-chain fatty acids accumulation in phospholipid and triacylglycerol.

Very long-chain fatty acids (VLCFAs) are important industrial raw materials and can be produced by genetically modified oil plants. For a long time, class A lysophosphatidic acid acyltransferase (LPAT) was considered unable to promote the accumulation of VLCFA in oil crops. The bottlenecks that the transgenic high VLCFA lines have an oil content penalty and the low amount of VLCFA in phosphatidylcholine remains intractable. In the present study, a class A LPAT2 from Camelina sativa (CsaLPAT2) promoting VLCFAs accumulation in phospholipid was found. Overexpression of CsaLPAT2 alone in Arabidopsis seeds significantly increased the VLCFA content in triacylglycerol, including C20:0, C20:2, C20:3, C22:0, and C22:1. The proportion of phosphatidic acid molecules containing VLCFAs in transgenic seeds reached up to 45%, which was 2.8-fold greater than that in wild type. The proportion of phosphatidylcholine and diacylglycerol molecules containing VLCFAs also increased significantly. Seed size in CsaLPAT2 transgenic lines showed a slight increase without an oil content penalty. The total phospholipid content in the seed of the CsaLPAT2 transgenic line was significantly increased. Furthermore, the function of class A LPAT in promoting the accumulation of VLCFAs is conserved in the representative oil crops of Brassicaceae, such as C. sativa, Arabidopsis thaliana, Brassica napus, Brassica rapa, and Brassica oleracea. The findings of this study provide a promising gene resource for the production of VLCFAs.

Lysophosphatidic acid acyltransferase 2 and 5 commonly, but differently, promote seed oil accumulation in Brassica napus.

BACKGROUND: Increasing seed oil content (SOC) of Brassica napus has become one of the main plant breeding goals over the past decades. Lysophosphatidic acid acyltransferase (LPAT) performs an important molecular function by regulating the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane and storage lipids. However, the mechanism underlying the effect of LPAT on the SOC of B. napus remains unclear. RESULTS: In the present study, significant elevation of SOC was achieved by overexpressing BnLPAT2 and BnLPAT5 in B. napus. RNAi and CRISPR-Cas9 were also successfully used to knock down and knock out these two genes in B. napus where SOC significantly decreased. Meanwhile, we found an accumulation of lipid droplets and oil bodies in seeds of BnLPAT2 and BnLPAT5 overexpression lines, whereas an increase of sugar and protein in Bnlpat2 and Bnlpat5 mutant seeds. Sequential transcriptome analysis was further performed on the developing seeds of the BnLPAT2 and BnLPAT5 overexpression, knockdown, and knockout rapeseed lines. Most differentially expressed genes (DEGs) that were expressed in the middle and late stages of seed development were enriched in photosynthesis and lipid metabolism, respectively. The DEGs involved in fatty acid and lipid biosynthesis were active in the overexpression lines but were relatively inactive in the knockdown and knockout lines. Further analysis revealed that the biological pathways related to fatty acid/lipid anabolism and carbohydrate metabolism were specifically enriched in the BnLPAT2 overexpression lines. CONCLUSIONS: BnLPAT2 and BnLPAT5 are essential for seed oil accumulation. BnLPAT2 preferentially promoted diacylglycerol synthesis to increase SOC, whereas BnLPAT5 tended to boost PA synthesis for membrane lipid generation. Taken together, BnLPAT2 and BnLPAT5 can jointly but differently promote seed oil accumulation in B. napus. This study provides new insights into the potential mechanisms governing the promotion of SOC by BnLPAT2 and BnLPAT5 in the seeds of B. napus.

Further insight into decreases in seed glucosinolate content based on QTL mapping and RNA-seq in Brassica napus L.

KEY MESSAGE: The QTL hotspots determining seed glucosinolate content instead of only four HAG1 loci and elucidation of a potential regulatory model for rapeseed SGC variation. Glucosinolates (GSLs) are amino acid-derived, sulfur-rich secondary metabolites that function as biopesticides and flavor compounds, but the high seed glucosinolate content (SGC) reduces seed quality for rapeseed meal. To dissect the genetic mechanism and further reduce SGC in rapeseed, QTL mapping was performed using an updated high-density genetic map based on a doubled haploid (DH) population derived from two parents that showed significant differences in SGC. In 15 environments, a total of 162 significant QTLs were identified for SGC and then integrated into 59 consensus QTLs, of which 32 were novel QTLs. Four QTL hotspot regions (QTL-HRs) for SGC variation were discovered on chromosomes A09, C02, C07 and C09, including seven major QTLs that have previously been reported and four novel major QTLs in addition to HAG1 loci. SGC was largely determined by superimposition of advantage allele in the four QTL-HRs. Important candidate genes directly related to GSL pathways were identified underlying the four QTL-HRs, including BnaC09.MYB28, BnaA09.APK1, BnaC09.SUR1 and BnaC02.GTR2a. Related differentially expressed candidates identified in the minor but environment stable QTLs indicated that sulfur assimilation plays an important rather than dominant role in SGC variation. A potential regulatory model for rapeseed SGC variation constructed by combining candidate GSL gene identification and differentially expressed gene analysis based on RNA-seq contributed to a better understanding of the GSL accumulation mechanism. This study provides insights to further understand the genetic regulatory mechanism of GSLs, as well as the potential loci and a new route to further diminish the SGC in rapeseed.

Anthocyanins identification and transcriptional regulation of anthocyanin biosynthesis in purple Brassica napus.

KEY MESSAGE: The main anthocyanin components were identified, and the transcriptional regulation pattern of anthocyanin related genes in leaves and stem bark was elucidated in a purple B. napus. Brassica napus is one of the most important oil crops planted worldwide, and developing varieties of dual-purpose for oil and vegetable is beneficial to improve economic benefits. Anthocyanins are a class of secondary metabolites that not only make plants present beautiful colors, but have a variety of important physiological functions and biological activities. Therefore, increasing the accumulation of anthocyanin in vegetative organs can improve vegetable value of rapeseed. However, anthocyanin enriched varieties in vegetative organs are rare, and there are few studies on category identification and accumulation mechanism of anthocyanin, which limits the utilization of anthocyanins in B. napus. In this study, 157 anthocyanin biosynthesis related genes (ABGs) were identified in B. napus genome by homology comparison and collinearity analysis of genes related to anthocyanin synthesis and regulation in Arabidopsis. Moreover, five anthocyanins were identified in the stem bark and leaves of the purple B. napus PR01 by high performance liquid chromatography-mass spectrometry (HPLC-MS), and the expression characteristics of ABGs in the leaves and stem bark of PR01 were analyzed and compared with the green cultivar ZS11 by RNA-Seq. Combining further weighted gene co-expression network analysis (WGCNA), the up-regulation of transcript factors BnaA07. PAP2 and BnaC06. PAP2 were identified as the key to the up-regulation of most of anthocyanin synthesis genes that promoted anthocyanin accumulation in PR01. This study is helpful to understand the transcriptional regulation of anthocyanin biosynthesis in B. napus and provides the theoretical basis for breeding novel varieties of dual-purpose for oil and vegetable.

The Transcriptome and Metabolome Reveal the Potential Mechanism of Lodging Resistance in Intergeneric Hybrids between Brassica napus and Capsella bursa-pastoris.

Lodging is one of the main reasons for the reduction in seed yield and is the limitation of mechanized harvesting in B. napus. The dissection of the regulatory mechanism of lodging resistance is an important goal in B. napus. In this study, the lodging resistant B. napus line, YG689, derived from the hybridization between B. napus cv. Zhongyou 821 (ZY821) and Capsella bursa-pastoris, was used to dissect the regulation mechanism of hard stem formation by integrating anatomical structure, transcriptome and metabolome analyses. It was shown that the lignocellulose content of YG689 is higher than that of ZY821, and some differentially expressed genes (DEGs) involved in the lignocellulose synthesis pathway were revealed by transcriptome analyses. Meanwhile, GC-TOF-MS and UPLC-QTOF-MS identified 40, 54, and 31 differential metabolites in the bolting stage, first flower stage, and the final flower stage. The differential accumulation of these metabolites might be associated with the lignocellulose biosynthesis in B. napus. Finally, some important genes that regulate the metabolic pathway of lignocellulose biosynthesis, such as BnaA02g18920D, BnaA10g15590D, BnaC05g48040D, and NewGene_216 were identified in B. napus through the combination of transcriptomics and metabolomics data. The present results explored the potential regulatory mechanism of lignocellulose biosynthesis, which provided a new clue for the breeding of B. napus with lodging resistance in the future.

Construction of a Quantitative Genomic Map, Identification and Expression Analysis of Candidate Genes for Agronomic and Disease-Related Traits in Brassica napus.

Rapeseed is the second most important oil crop in the world. Improving seed yield and seed oil content are the two main highlights of the research. Unfortunately, rapeseed development is frequently affected by different diseases. Extensive research has been made through many years to develop elite cultivars with high oil, high yield, and/or disease resistance. Quantitative trait locus (QTL) analysis has been one of the most important strategies in the genetic deciphering of agronomic characteristics. To comprehend the distribution of these QTLs and to uncover the key regions that could simultaneously control multiple traits, 4,555 QTLs that have been identified during the last 25 years were aligned in one unique map, and a quantitative genomic map which involved 128 traits from 79 populations developed in 12 countries was constructed. The present study revealed 517 regions of overlapping QTLs which harbored 2,744 candidate genes and might affect multiple traits, simultaneously. They could be selected to customize super-rapeseed cultivars. The gene ontology and the interaction network of those candidates revealed genes that highly interacted with the other genes and might have a strong influence on them. The expression and structure of these candidate genes were compared in eight rapeseed accessions and revealed genes of similar structures which were expressed differently. The present study enriches our knowledge of rapeseed genome characteristics and diversity, and it also provided indications for rapeseed molecular breeding improvement in the future.

A major yellow-seed QTL on chromosome A09 significantly increases the oil content and reduces the fiber content of seed in Brassica napus.

KEY MESSAGE: A major yellow-seed QTL on chromosome A09 significantly increases the oil content and reduces the fiber content of seed in Brassica napus. The yellow-seed trait (YST) has always been a main breeding objective for rapeseed because yellow-seeded B. napus generally contains higher oil contents, fewer pigments and polyphenols and lower fiber content than black-seeded B. napus, although the mechanism controlling this correlation remains unclear. In this study, QTL mapping was implemented for YST based on a KN double haploid population derived from the hybridization of yellow-seeded B. napus N53-2 with a high oil content and black-seeded Ken-C8 with a relatively low oil content. Ten QTLs were identified, including four stable QTLs that could be detected in multiple environments. A major QTL, cqSC-A09, on chromosome A09 was identified by both QTL mapping and BSR-Seq technology, and explained more than 41% of the phenotypic variance. The major QTL cqSC-A09 for YST not only controls the seed color but also affects the oil and fiber contents in seeds. More importantly, the advantageous allele could increase the oil content and reduce the pigment and fiber content at the same time. This is the first QTL reported to control seed color, oil content and fiber content simultaneously with a large effect and has great application value for breeding high oil varieties with high seed quality. Important candidate genes, including BnaA09. JAZ1, BnaA09. GH3.3 and BnaA09. LOX3, were identified for cqSC-A09 by combining sequence variation annotation, expression differences and an interaction network, which lays a foundation for further cloning and breeding applications in the future.

Characterization of non-specific lipid transfer protein (nsLtp) gene families in the Brassica napus pangenome reveals abundance variation.

BACKGROUND: Brassica napus is an important agricultural species, improving stress resistance was one of the main breeding goals at present. Non-specific lipid transfer proteins (nsLTPs) are small, basic proteins which are involved in some biotic or abiotic stress responses. B. napus is susceptible to a variety of fungal diseases, so identify the BnLTPs and their expression in disease responses is very important. The common reference genome of B. napus does not contain all B. napus genes because of gene presence/absence variations between individuals. Therefore, it was necessary to search for candidate BnLTP genes in the B. napus pangenome. RESULTS: In the present study, the BnLTP genes were identified throughout the pangenome, and different BnLTP genes were presented among varieties. Totally, 246 BnLTP genes were identified and could be divided into five types (1, 2, C, D, and G). The classification, phylogenetic reconstruction, chromosome distribution, functional annotation, and gene expression were analyzed. We also identified potential cis-elements that respond to biotic and abiotic stresses in the 2 kb upstream regions of all BnLTP genes. RNA sequencing analysis showed that the BnLTP genes were involved in the response to Sclerotinia sclerotiorum infection. We identified 32 BnLTPs linked to blackleg resistance quantitative trait locus (QTL). CONCLUSION: The identification and analysis of LTP genes in the B. napus pangenome could help to elucidate the function of BnLTP family members and provide new information for future molecular breeding in B. napus.

The genome wide analysis of Tryptophan Aminotransferase Related gene family, and their relationship with related agronomic traits in Brassica napus.

Tryptophan Aminotransferase of Arabidopsis1/Tryptophan Aminotransferase-Related (TAA1/TAR) proteins are the enzymes that involved in auxin biosynthesis pathway. The TAA1/TAR gene family has been systematically characterized in several plants but has not been well reported in Brassica napus. In the present study, a total of 102 BnTAR genes with different number of introns were identified. It was revealed that these genes are distributed unevenly and occurred as clusters on different chromosomes except for A4, A5, A10 and C4 in B. napus. Most of the these BnTAR genes are conserved despite of existing of gene loss and gene gain. In addition, the segmental replication and whole-genome replication events were both play an important role in the BnTAR gene family formation. Expression profiles analysis indicated that the expression of BnTAR gene showed two patterns, part of them were mainly expressed in roots, stems and leaves of vegetative organs, and the others were mainly expressed in flowers and seeds of reproductive organs. Further analysis showed that many of BnTAR genes were located in QTL intervals of oil content or seed weight, for example BnAMI10 was located in cqOC-C5-4 and cqSW-A2-2, it indicated that some of the BnTAR genes might have relationship with these two characteristics. This study provides a multidimensional analysis of the TAA1/TAR gene family and a new insight into its biological function in B. napus.

Genome-wide unbalanced expression bias and expression level dominance toward Brassica oleracea in artificially synthesized intergeneric hybrids of Raphanobrassica.

Raphanobrassica (RrRrCrCr, 2n = 4x = 36), which is generated by distant hybridization between the maternal parent Raphanus sativus (RsRs, 2n = 2x = 18) and the paternal parent Brassica oleracea (C°C°, 2n = 2x = 18), displays intermediate silique phenotypes compared to diploid progenitors. However, the hybrid shares much more similarities in silique phenotypes with those of B. oleracea than those of R. sativus. Strikingly, the silique of Raphanobrassica is obviously split into two parts. To investigate the gene expression patterns behind these phenomena, transcriptome analysis was performed on the upper, middle, and lower sections of pods (RCsiu, RCsim, and RCsil), seeds in the upper and lower sections of siliques (RCseu and RCsel) from Raphanobrassica, whole pods (Rsi and Csi) and all seeds in the siliques (Rse and Cse) from R. sativus and B. oleracea. Transcriptome shock was observed in all five aforementioned tissues of Raphanobrassica. Genome-wide unbalanced biased expression and expression level dominance were also discovered, and both of them were toward B. oleracea in Raphanobrassica, which is consistent with the observed phenotypes. The present results reveal the global gene expression patterns of different sections of siliques of Raphanobrassica, pods, and seeds of B. oleracea and R. sativus, unraveling the tight correlation between global gene expression patterns and phenotypes of the hybrid and its parents.

In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa, and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology.

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.