Abstract

Background

   Brassica napus is of substantial economic value for vegetable oil,
   biofuel, and animal fodder production. The breeding of yellow-seeded B.
   napus to improve seed quality with higher oil content, improved oil and
   meal quality with fewer antinutrients merits attention. Screening the
   genes related to this phenotype is valuable for future rapeseed
   breeding.

Results

   A total of 85,407 genes, including 4317 novel genes, were identified in
   the developing seeds of yellow- and black-seeded B. napus, and yellow
   rapeseed was shown to be an introgression line between black-seeded B.
   napus and yellow-seeded Sinapis alba. A total of 15,251 differentially
   expressed genes (DEGs) were identified among all the libraries, and 563
   and 397 common DEGs were identified throughout black and yellow seed
   development, including 80 upregulated and 151 downregulated genes
   related to seed development and fatty acid accumulation. In addition,
   11 up-DEGs and 31 down-DEGs were identified in all developmental stages
   of yellow rapeseed compared with black seed. Enrichment analysis
   revealed that many DEGs were involved in biosynthetic processes,
   pigment metabolism, and oxidation-reduction processes, such as
   flavonoid and phenylpropanoid biosynthesis, phenylalanine metabolism,
   flavone and flavonol biosynthesis, and fatty acid biosynthesis and
   metabolism. We found that more than 77 DEGs were related to flavonoid
   and lignin biosynthesis, including 4CL, C4H, and PAL, which
   participated in phenylalanine metabolism, and BAN, CHI/TT5, DFR, F3H,
   FLS, LDOX, PAP, CHS/TT4, TT5, bHLH/TT8, WD40, MYB, TCP, and CYP, which
   were involved in flavonoid biosynthesis. Most of these DEGs were
   downregulated in yellow rapeseed and were consistent with the decreased
   flavonoid and lignin contents. Both up- and down-DEGs related to fatty
   acid biosynthesis and metabolism were also analyzed, which could help
   to explain the improved oil content of yellow rapeseed.

Conclusion

   This research provided comprehensive transcriptome data for
   yellow-seeded B. napus with a unique genetic background, and all the
   DEGs in comparison with the black-seeded counterpart could help to
   explain seed quality differences, such as lower pigmentation and lignin
   contents, and higher oil content.

Electronic supplementary material

   The online version of this article (10.1186/s12870-019-1821-z) contains
   supplementary material, which is available to authorized users.

   Keywords: Brassica napus, Seed coat color, Flavonoid biosynthesis,
   Fatty acid, Gene expression

Background

   Brassica napus, as the third leading oilseed crop grown worldwide,
   greatly contributes to providing edible oils, biofuels, and animal
   fodder [[37]1]. Rapeseed with increased oil content, better oil and
   meal quality, and improved yield has been the main breeding drive
   during the past decades [[38]2]. Yellow-seeded B. napus was improved
   with better seed oil and meal quality due to fewer pigments and
   polyphenols; thus, breeding yellow rapeseed has been preferred for
   decades, and studies on the molecular mechanism of this phenotype have
   been reported as well [[39]3]. Fewer antinutrients, including phenolic
   compounds, tannins, proanthocyanins (PAs), and lignin, in yellow
   rapeseed were correlated with the flavonoid biosynthetic,
   phenylpropanoid, and phenylalanine/tyrosine metabolic pathways with
   common substrates, such as coumaroyl CoA, caffeoyl CoA, and feruloyl
   CoA [[40]4–[41]6]. Hitherto, yellow-seeded B. napus has been selected
   from interspecific hybridization of Brassica species and intergeneric
   hybridization [[42]7, [43]8]. As reported in Arabidopsis thaliana,
   mutations in transparent testa (TT) genes are responsible for seed coat
   color variations, including early (EBGs) and late (LBGs) biosynthesis
   genes [[44]9]. The EBGs include TT4/CHS, TT5/CHI, TT6/F3H, and
   TT7/F3’H, and LBGs include TT3/DFR, TT18/LDOX/ANS, BAN/ANR, TT12,
   TT19/GSTF12/GST26, and AHA10. In addition, the MYB-bHLH-WD40 (MBW)
   complex has been reported to have important regulatory functions in
   anthocyanin accumulation. Four MBW have been reported, including
   TT2-TT8-TTG1, MYB5-TT8-TTG1, TT2-EGL3-TTG1, and TT2-GL3-TTG1 [[45]10].
   Appelhagen et al. (2011) found that PAP1/MYB75 and PAP2/MYB90
   participated in the formation of MBW [[46]11]. Homologous TTs related
   to seed coat variation in Brassicaceae have been delineated, including
   95 copies of 21 TTs in B. napus [[47]3]. Previously, F3’H, TT2, PAL,
   BAN, TTG1, TT10, and TT1 were cloned and shown to have functions in
   flavonoid biosynthesis [[48]12–[49]19]. Yu (2013) reviewed the
   molecular mechanism of manupulating seed coat color in Brassica
   species, including the homologous TTs cloned in Brassicas [[50]20].
   Besides, molecular markers have also been developed for yellow-seeded
   B. rapa [[51]21–[52]23], B. juncea [[53]24, [54]25] and B. napus. Liu
   et al. (2012) reported a lignin biosynthesis gene, BnCCR1, associated
   with yellow seed character, indicating a strong correlation between
   seed color and lignin content [[55]26]. Stein et al. (2013) found that
   BnAHA10 had effects on seed color and lignin content [[56]27]. Wang et
   al. (2017) identified 22 single nucleotide polymorphisms (SNPs) on 7
   chromosomes associated with seed coat color using a genome wide
   association study (GWAS) [[57]28]. Functional analysis of BnTT10 and
   BnTT1 revealed that they are involved in PA metabolism, lignin
   synthesis, seed coat pigmentation, and fatty acid (FA) biosynthesis
   [[58]18, [59]19]. However, due to the genome complexity of B. napus and
   the sensitivity of yellow seed color to environmental influences (e.g.,
   light, temperature, fertilizers, and harvesting time), the molecular
   mechanisms of this phenotype remained unclear until now [[60]29,
   [61]30].

   The short domestication history of B. napus after interspecific
   hybridization between B. rapa and B. oleracea and the artificial
   selection during rapeseed breeding have greatly narrowed the genetic
   background [[62]31]. No yellow-seed germplasm has been found in natural
   B. napus, and most of the yellow-seeded B. napus were created by
   crossing between Brassica species, such as hybrids between B. napus and
   B. juncea, B. napus and B. carinata, B. juncea and B. oleracea [[63]32,
   [64]33]. Wild species in Brassicaceae, such as Sinapis alba, S.
   arvensis, Camelina sativa, Crambe abyssinica, and Descurainia sophia,
   exhibit many desirable characteristics, such as yellow seed coat, high
   erucic acid, pod shattering resistance, high unsaturated FA contents,
   and resistance to various diseases and abiotic stresses
   [[65]34–[66]36]. Introducing gene resources from these wild species
   would help to enrich the genetic background of B. napus, accompanying
   desirable traits. Zhang et al. (2009) obtained yellow-seeded rapeseed
   from intergeneric hybrids between B. napus and D. sophia [[67]37].
   Previously, Wang et al. (2005) created somatic hybrids between B. napus
   and S. alba [[68]38] and selected yellow-seeded B. napus from hybrid
   progenies with improved oil content and decreased antinutrients [[69]8,
   [70]39, [71]40]. In the present study, we used RNA-Seq to identify the
   expression differences at different seed developmental stages of
   yellow- and black-seeded B. napus, revealing the expression patterns of
   genes involved in various biological pathways related to seed coat
   pigmentation, FA biosynthesis and metabolism.

Results

Transcriptome sequencing and read mapping of yellow- and black-seeded B.
napus

   RNA-Seq was performed at three weeks after flowering (3 WAF), 4 WAF, 5
   WAF, 6 WAF, and mature stage of yellow- and black-seeded B. napus to
   investigate the transcriptome difference that might be related to the
   quality variation between two rapeseeds. After quality control, ~ 30.3
   to ~ 37.6 million reads from the libraries were uniquely mapped to the
   B. napus genome (Table [72]1). A total of 85,407 genes, including 4317
   novel genes, were expressed in the developing seeds of two rapeseeds
   (Fig. [73]1, Additional file [74]1: Table S1). We found 52,452 and
   51,777 common genes were expressed in the developing seeds of yellow
   and black rapeseed, respectively. In addition, 67,629 (3 WAF), 64,070
   (4 WAF), 64,775 (5 WAF), 62,143 (6 WAF), and 54,257 (mature stage)
   overlapping genes were expressed at each stage in two rapeseed lines.
   Besides, 47,912 genes were coexpressed in all the developmental stages
   of yellow and black rapeseed lines.

Table 1.

   Summary of read mapping for RNA-seq
   Sample name B3 B4 B5 B6 BM Y3 Y4 Y5 Y6 YM
   Raw reads 47,536,866 42,753,820 44,341,556 51,507,518 51,272,946
   48,704,282 45,694,038 44,797,562 45,996,584 52,804,244
   Clean reads 45,555,018 41,801,216 40,620,518 43,833,034 48,665,348
   46,813,976 43,798,322 42,843,336 43,736,106 50,268,482
   Total mapped 36,499,205 (80.12%) 34,214,293 (81.85%) 32,844,328
   (80.86%) 35,032,820 (79.92%) 39,325,179 (80.81%) 38,116,991 (81.42%)
   35,893,586 (81.95%) 35,376,347 (82.57%) 36,029,106 (82.38%) 40,839,634
   (81.24%)
   Multiple mapped 2,175,877 (4.78%) 2,332,136 (5.58%) 2,561,972 (6.31%)
   3,271,514 (7.46%) 3,151,365 (6.48%) 2,327,452 (4.97%) 2,287,911 (5.22%)
   2,274,889 (5.31%) 2,451,932 (5.61%) 3,198,280 (6.36%)
   Uniquely mapped 34,323,328 (75.34%) 31,882,157 (76.27%) 30,282,356
   (74.55%) 31,761,306 (72.46%) 36,173,814 (74.33%) 35,789,539 (76.45%)
   33,605,675 (76.73%) 33,101,458 (77.26%) 33,577,174 (76.77%) 37,641,354
   (74.88%)
   Reads map to ‘+’ 17,137,064 (37.62%) 15,873,247 (37.97%) 15,138,033
   (37.27%) 15,900,632 (36.28%) 18,123,297 (37.24%) 17,872,927 (38.18%)
   16,769,787 (38.29%) 16,508,902 (38.53%) 16,764,771 (38.33%) 18,866,098
   (37.53%)
   Reads map to ‘-’ 17,186,264 (37.73%) 16,008,910 (38.3%) 15,144,323
   (37.28%) 15,860,674 (36.18%) 18,050,517 (37.09%) 17,916,612 (38.27%)
   16,835,888 (38.44%) 16,592,556 (38.73%) 16,812,403 (38.44%) 18,775,256
   (37.35%)
   Non-splice reads 21,266,684 (46.68%) 21,368,313 (51.12%) 21,381,177
   (52.64%) 22,323,651 (50.93%) 24,534,251 (50.41%) 22,491,873 (48.05%)
   22,291,894 (50.9%) 24,114,006 (56.28%) 24,559,779 (56.15%) 26,061,989
   (51.85%)
   Splice reads 13,056,644 (28.66%) 10,513,844 (25.15%) 8,901,179 (21.91%)
   9,437,655 (21.53%) 11,639,563 (23.92%) 13,297,666 (28.41%) 11,313,781
   (25.83%) 8,987,452 (20.98%) 9,017,395 (20.62%) 11,579,365 (23.04%)
   [75]Open in a new tab

   B3, B4, B5, B6 and BM indicate 3 ~ 6 WAF and mature black seed. Y3, Y4,
   Y5, Y6 and YM indicate 3 ~ 6 WAF and mature yellow seed

Fig. 1.

   [76]Fig. 1
   [77]Open in a new tab

   Genes commonly and specifically expressed in developing seeds of
   yellow- and black-seeded B. napus. a-e Venn diagram of genes expressed
   between each developmental stage of yellow and black seed. f Venn
   diagram of genes in black seed. g Venn diagram of genes in yellow seed.
   B3, B4, B5, B6 and BM indicate 3 ~ 6 WAF and mature black seed. Y3, Y4,
   Y5, Y6 and YM indicate 3 ~ 6 WAF and mature yellow seed

Identification of differentially expressed genes

   Global changes in differentially expressed genes were identified using
   DESeq, and a total of 15,251 DEGs (including 523 novel genes) were
   identified among all the libraries. We found 563 common DEGs in B4 (4
   WAF of black seed), B5 (5 WAF of black seed), B6 (6 WAF of black seed),
   and BM (mature stage of black seed) compared with B3 (3 WAF of black
   seed), including 141 upregulated and 331 downregulated genes at each
   developmental stage of the black seed. A total of 397 common DEGs were
   identified in Y4 (4 WAF of yellow seed), Y5 (5 WAF of yellow seed), Y6
   (6 WAF of yellow seed), and YM (mature stage of yellow seed) compared
   with Y3 (3 WAF of yellow seed), including 92 upregulated and 220
   downregulated genes throughout yellow seed development (Fig. [78]2a, b;
   Additional file [79]2: Table S2). Generally, 80 upregulated and 151
   downregulated genes were identified during the seed development of two
   B. napus lines, which might be related to seed development and FA
   accumulation (Additional file [80]3: Table S3). Those DEGs related to
   cruciferin, oleosin, caleosin, late embryogenesis abundant (LEA)
   hydroxyproline, lipid transfer protein, myrosinase-binding protein,
   embryo-specific protein, and alpha-tonoplast intrinsic protein were
   upregulated, and those related to galactosyltransferase, glycosyl
   hydrolase, lipid transfer protein, basic leucine zipper protein,
   cytochrome P450 (CYP), laccase, chalcone synthase, delta vacuolar
   processing enzyme, jasmonic acid carboxyl methyltransferase, and
   senescence were downregulated with seed development. In addition, 42
   DEGs were identified between yellow and black rapeseeds at each stage,
   including 11 upregulated and 31 downregulated genes in yellow-seeded B.
   napus (Fig. [81]2c, Additional file [82]4: Table S4). These common DEGs
   were mainly related to the RNA-binding family protein, nascent
   polypeptide-associated complex (NAC), calcium binding EF-hand family
   protein, and metallothionein protein that were upregulated and
   dormancy/auxin associated protein, S-adenosylmethionine synthetase,
   pectin lyase-like superfamily protein, insulinase family protein that
   were downregulated in all stages of yellow seed compared with black
   seed. The number of DEGs increased with seed development in both
   rapeseed lines, whereas the downregulated DEGs at the mature stage
   dramatically increased to ~ 8000 (Fig. [83]2d, e). The up-regulated and
   downregulated DEGs between Y4 and B4 were higher than those in the
   other stages of the two rapeseed lines (Fig. [84]2f). Hierarchical
   cluster analysis of all the DEGs was performed using the
   log[10](RPKM+ 1) value (Fig. [85]2g). H-cluster and SOM-cluster of the
   DEGs from five developmental stages of two rapeseed lines were also
   performed using the log[2](Fold change), and the DEGs in all the
   clusters showed similar patterns between two rapeseed lines, except for
   subcluster_1_1, which includes DEGs with significant differences
   between Y4 and B4 (Fig. [86]2h, Additional file [87]5: Figure S1).

Fig. 2.

   [88]Fig. 2
   [89]Open in a new tab

   Expressional analysis of DEGs in developing seeds of yellow- and
   black-seeded B. napus. (a) DEGs with black seed development. (b) DEGs
   with yellow seed development. (c) DEGs between yellow and black
   rapeseed. (d-f) Number of up- and down-DEGs. (g-h) Clustering of DEGs

Enrichment analysis of DEGs

   To acknowledge the putative functions and pathways of the DEGs, GO and
   KEGG enrichment analysis of DEGs was proceeded with GOseq and KOBAS
   2.0. Among all the identified DEGs, 11,772 were annotated with GO terms
   and assigned to three categories: cellular component, biological
   process, and molecular function. Most of the up- and downregulated DEGs
   between the same stage of yellow and black seeds were assigned to
   catalytic activity, binding, metabolic process, cellular process,
   followed by cell, cell part, membrane, organelle part, macromolecular
   complex, regulation of biological process, and biological regulation
   (Fig. [90]3, Additional file [91]6: Table S5). In detail, among all the
   downregulated genes (1435 out of 1945 with GO annotations) between
   yellow and black rapeseed, 425, 39, 211, 89, and 65 DEGs were assigned
   to biosynthetic process (GO: 0009058), pigment metabolic process (GO:
   0042440), oxidation-reduction process (GO: 0055114), cellular response
   to stimulus (GO: 0051716), and signal transduction (GO: 0007165),
   respectively. In contrast, 181 upregulated genes between yellow and
   black seed were assigned to biosynthetic process (GO: 0009058), 95
   up-DEGs related to oxidation-reduction process (GO: 0055114), and 181
   up-DEGs related to biosynthetic process (GO: 0009058).

Fig. 3.

   [92]Fig. 3
   [93]Open in a new tab

   GO enrichment of up- and down-DEGs between yellow- and black-seeded B.
   napus

   KEGG pathway analysis showed that flavonoid biosynthesis was most
   significantly enriched with downregulated DEGs in comparisons of Y3 vs
   B3, Y4 vs B4, Y5 vs B5, and Y6 vs B6. Phenylpropanoid biosynthesis was
   also enriched with downregulated genes between the same stages of the
   two rapeseed lines, except for the mature stage. Phenylalanine
   metabolism, flavone and flavonol biosynthesis was enriched with
   downregulated DEGs in comparisons of Y3 vs B3 and Y4 vs B4
   (Additional file [94]7: Figure S2). In contrast, the upregulated DEGs
   between yellow and black rapeseed were most significantly enriched in
   photosynthesis, followed by carbon metabolism, FA biosynthesis and
   metabolism (Additional file [95]8: Figure S3, Additional file [96]9:
   Table S6). The pathway enrichment analysis helps to determine the
   functions of DEGs and complemented secondary metabolisms that were not
   specified by GO terms. Using the log[2](Fold change) values between
   yellow and black rapeseed (Y3 vs. B3, Y4 vs. B4, Y5 vs. B5, Y6 vs. B6,
   and YM vs. BM), we assigned all the DEGs to all the metabolism pathways
   using the Arabidopsis TAIR9 version as mapping reference. We also found
   many DEGs were enriched in metabolism (flavonoids, terpenes,
   phenylpropanoids and phenolics) and lipids (Additional file [97]10:
   Figure S4).

DEGs associated with flavonoid and lignin biosynthesis

   As mentioned above, yellow seed color is associated with flavonoid and
   lignin biosynthesis. We found 77 DEGs related to phenylpropanoid and
   flavonoid biosynthesis and the phenylalanine metabolic pathway. As
   shown in Fig. [98]4, genes encoding 4-coumarate CoA ligase (4CL),
   cinnamate 4-hydroxylase (C4H), and phenylalanine ammonia-lyase (PAL)
   were downregulated in yellow rapeseed compared with black seed, except
   for a homolog of PAL (BnaC05g42780D). These genes play important roles
   in lignin biosynthesis, and the expression changes should be related to
   the reduced lignin in yellow seed. Genes involved in the flavonoid
   pathway were also downregulated in yellow seed compared to black seed,
   including BAN, CHI/TT5, DFR, F3H, FLS, LDOX, PAP, CHS/TT4, TT5,
   bHLH/TT8, WD40, and MYB, which participate in the biosynthesis of
   chalcones, flavanones, flavonols, anthocyanins, and PAs. However, we
   found a MATE (BnaC01g40630D) and UGT84A1 (BnaA01g18540D) were
   upregulated at the early developmental stages of yellow seed compared
   with black seed. Comparing with other two down-regulated MATEs in
   yellow-seeded B. napus, BnaA01g24940D (a homologous of AT3G03620) and
   BnaA07g18120D (a homologous of AtTT12), we found BnaC01g40630D with
   lower expression throughout rapeseed development than that of
   BnaA01g24940D and BnaA07g18120D. Expressional differences of TCPs and
   CYPs were also identified, which might be related to flavonoid
   biosynthesis, since TCP could interact with R2R3-MYB [[99]41], and CYPs
   (e.g., CYP71/75/93/81) have been reported to function in the
   biosynthesis of anthocyanin pigments and condensed tannins [[100]42].
   In addition, we compared the expression pattern of all the genes
   involved in secondary metabolism and found DEGs encoding peroxidase
   superfamily protein (BnaA02g02010D, BnaC06g32660D, BnaA02g14050D, and
   BnaC06g21080D), glycosyl hydrolase family protein (BnaUnng02770D), beta
   glucosidase 19/29/25 (BnaA06g09700D, BnaA05g04040D, BnaC05g11190D,
   BnaC01g43700D, and BnaC01g40610D), thioglucoside glucohydrolase 1
   (BnaCnng53320D) were also assigned to phenylpropanoid biosynthesis and
   the phenylalanine metabolic pathway (Additional file [101]11: Figure
   S5). Generally, most of the genes involved in secondary metabolism were
   downregulated in yellow seed compared with black seed.

Fig. 4.

   Fig. 4
   [102]Open in a new tab

   Heatmap of DEGs related to flavonoid and lignin biosynthesis

DEGs associated with fatty acid biosynthesis and metabolism

   We found that genes involved in FA biosynthesis, metabolism, and
   degradation were also changed (Fig. [103]5). For instance, five up-DEGs
   in yellow seed were involved in FA biosynthesis, encoding an
   NAD(P)-binding Rossmann-fold superfamily protein (BnaC01g40290D), a
   thioesterase superfamily protein (BnaA03g02830D), acetyl Coenzyme A
   carboxylase carboxyltransferase (BnaA03g17570D),
   stearoyl-acyl-carrier-protein desaturase (BnaA03g28050D), and
   chloroplastic acetylcoenzyme A carboxylase 1 (BnaC02g06560D). However,
   another homolog of the thioesterase superfamily protein (BnaC03g04180D)
   was downregulated in yellow seed compared with black seed. Five up-DEGs
   (BnaA03g02830D, BnaA03g17570D, BnaA03g28050D, BnaC01g40290D, and
   BnaC02g06560D) and six down-DEGs (BnaA02g30470D, BnaA05g30630D,
   BnaA08g28600D, BnaC02g38800D, BnaC03g04180D, and BnaC03g04350D) were
   involved in FA metabolism. BnaC03g04350D was also related to FA
   elongation. In addition, four down-DEGs (BnaC06g21150D, BnaA02g30470D,
   BnaA05g30630D, and BnaC02g38800D) were related to FA degradation. These
   DEGs might be related to the different oil content between yellow- and
   black-seeded B. napus.

Fig. 5.

   Fig. 5
   [104]Open in a new tab

   Heatmap of DEGs related to fatty acid biosynthesis and metabolism

Real-time qPCR validates gene expression profiles

   To verify the sequencing results, DEGs involved in flavonoid and lignin
   biosynthesis, FA biosynthesis and metabolism were randomly selected for
   qPCR detection, including BnaC01g40290D encoding NAD(P)-binding
   Rossmann-fold superfamily protein/short-chain dehydrogenase/reductase
   (SDR), BnaA03g17570D encoding acetyl CoA carboxylase
   carboxyltransferase alpha subunit (CAC3), BnaC03g04350D encoding
   3-hydroxyacyl-CoA dehydratase PASTICCINO 2 (PAS2), 4CL (BnaC03g42000D),
   C4H (BnaC04g14330D, BnaA05g11950D), LBD41 (BnaC03g33010D), PAL
   (BnaC04g08190D), BAN (BnaA01g36200D), CHI (BnaC09g50050D), LDOX
   (BnaC01g14310D) and CHS (BnaA10g19670D). The results showed that the
   qPCR data of the DEGs were in accordance with the sequencing results
   throughout seed development of yellow- and black-seeded B. napus (Fig.
   [105]6).

Fig. 6.

   [106]Fig. 6
   [107]Open in a new tab

   qPCR confirmation of DEGs between yellow- and black-seeded B. napus

Discussion

   Due to the important economic value of rapeseed worldwide, research on
   improving seed quality has been carried out over the past half century,
   including reducing the erucic acid and glucosinolate contents and
   increasing the oleic acid content [[108]43, [109]44]. However, breeding
   of rapeseed has been greatly hindered by the narrow genetic background
   either due to the short history or the artificial selection [[110]31].
   Previously, Wang et al. created yellow-seeded B. napus from the
   backcrossing progenies of B. napus-S. alba hybrids, including S. alba
   specific fragments in the 38 chromosomes [[111]8, [112]34, [113]45].
   RNA-Seq is a reliable method in analyzing the gene expressional level
   on the whole transcriptome level, and it also helps to reveal the
   expressional differences among different samples, which could somehow
   explain the different characteristics among samples. In the past
   decade, RNA-Seq has been broadly applied in expressional comparison
   among plant samples, and in finding alternative splicing, novel
   transcripts, SNP and InDels [[114]46–[115]48]. To identify the gene
   expression differences related to yellow seed character and
   accompanying quality variations, we carried out comparative
   transcriptome analysis between different developmental stages of
   yellow- and black-seeded B. napus. As reported by Qu et al. (2013),
   accumulation of polymeric phenolic compounds were similar in yellow and
   black rapeseeds, but the flavonoids were more accumulated in black seed
   since 3 WAF [[116]49]. Thus, we collected the seeds at 3 WAF, 4 WAF, 5
   WAF, 6 WAF and mature stage for RNA-Seq analysis. We found a total of
   39,632 SNPs in ten libraries, including 1142 SNPs common in five
   developmental stages of black seed and 1543 SNPs common in five
   developmental stages of yellow seed, indicating that genomic
   differences exist between yellow- and black-seeded B. napus. We found a
   total of 15,251 DEGs among all the libraries, including 80 upregulated
   and 151 downregulated genes identified throughout yellow and black seed
   development, which might be related to seed development and FA
   accumulation (Additional file [117]2: Table S2). Regarding the DEGs
   between yellow and black rapeseed, we found that most were annotated to
   biosynthetic, pigment metabolic, and oxidation-reduction processes,
   such as flavonoid and phenylpropanoid biosynthesis, phenylalanine
   metabolism, flavone and flavonol biosynthesis, and FA biosynthesis and
   metabolism (Additional file [118]8: Figure S3, Additional file [119]9:
   Table S6). This agreed with Hong et al. who found that the
   downregulated DEGs in yellow seed coats were enriched in
   phenylpropanoid and flavonoid biosynthesis using yellow- and
   brown-seeded near-isogenic lines (NILs) of rapeseed as research
   materials [[120]50]. Similar expression changes in TT genes have been
   reported in B. juncea seed coat [[121]51].

   Flavonoid biosynthesis has been considered to be the main pathway
   related to plant pigmentation, and TT genes related to it have been
   clarified in A. thaliana. Recently, homologs of TTs in Brassica have
   been comprehensively identified by Qu et al. [[122]3], as well as the
   expression patterns in different yellow- and black-seeded B. napus
   inbred lines. In the present study, we compared the expression pattern
   of genes related to flavonoid biosynthesis in developing seeds of B.
   napus cv. ‘Yangyou 6’ and yellow-seeded B. napus selected from somatic
   hybrids of B. napus-S. alba (Fig. [123]4). Four homologs of BnBAN
   (BnaA01g36200D, BnaA03g60670D, BnaC01g29820D and BnaC04g18950D) were
   downregulated at two to four consecutive developmental stages of yellow
   rapeseed compared with black rapeseed. Two homologs of BnDFR
   (BnaA09g15710D and BnaC09g17150D) were also expressed at lower levels
   in yellow seed, which encodes an enzyme catalyzing dihydroflavanones to
   leucoanthocyanidins that was then converted to anthocyanidin [[124]52].
   BnLDOX (BnaC01g14310D, BnaA03g45610D and BnaC07g37670D) was
   downregulated in yellow seed, which encodes leucoanthocyanidin
   reductase and catalyzes the formation of anthocyanidins. Genes encoding
   chalcone synthase (BnaA10g19670D, BnaC09g43250D, BnaC03g06120D,
   BnaC02g05070D and BnaC08g26020D) were also downregulated throughout
   yellow seed development, which catalyzes the first step of flavonoid
   biosynthesis. BnTT5 (BnaC08g26020D) was also downregulated in yellow
   seed compared with black seed at specific stages, encoding chalcone
   isomerase that is redundant and responsive to lights in Brassicaceae
   [[125]53]. BnTT8 (BnaC09g24870D and BnaA09g22810D) was also
   downregulated in yellow seed compared with black seed at specific
   stages, encoding the transcription factor bHLH as an important
   regulator throughout flavonoid biosynthesis. Another two bHLHs
   (BnaA01g17420D and BnaA01g24020D) were also downregulated, which have
   been proven to have functions in regulating jasmonate responses
   [[126]54] and glucose homeostasis, and may affect ABA or salinity
   response in Arabidopsis [[127]55]. In the present study, 21 CYPs were
   differentially expressed between yellow and black rapeseeds, although
   they were not shown to have functions in the secondary metabolism of B.
   napus. Previously, CYP71/75/93/81 have been reported to have functions
   in the biosynthesis of anthocyanin pigments and condensed tannins
   [[128]42]. Lam et al. found that CYP75B4 and CYP93G1 promote tricin
   accumulation in Arabidopsis and O. sativa, which functions in
   generating 3′-hydroxylated flavonoids and flavone aglycones [[129]56,
   [130]57]. Recently, Lei et al. found that a group of CYPs, including
   members of CYP71/72/77/78/81/85/86/90/93, were differentially expressed
   in Dendrobium catenatum from different locations and with different
   outlooks, which might be related to flavonoid biosynthesis [[131]58].
   CYP86 has been reported to be involved in suberin monomer biosynthesis
   in Arabidopsis [[132]59]. Expressional differences of five TCPs were
   also identified, which might be related to flavonoid biosynthesis since
   TCP3 could interact with R2R3-MYB [[133]41]. In addition, TCP1 directly
   activates DWARF4 and promotes brassinosteroid biosynthesis [[134]60].

   Asides from flavonoids, lignin has been revealed to have a positive
   correlation with seed coat color [[135]26]. We found that 4CL, C4H and
   PAL were downregulated in yellow rapeseed compared with black seed,
   which play important roles in the less lignin in yellow seed (Fig.
   [136]4). PAL is involved in the first step of the phenylalanine
   metabolic pathway by catalyzing phenylalanine into cinnamic acid.
   Cinnamic acid was then transformed into coumaric acid by C4H. 4CL plays
   an important role in several steps of lignin biosynthesis and
   participates in the formation of coumaroyl-CoA, caffeoyl-CoA and
   feruloyl-CoA. Previously, Jiang et al. (2013) and Wang et al. (2018)
   reported that sinapic acid was more accumulated in yellow seed than
   black seed [[137]39, [138]61]. Down-regulation of 4CL in yellow
   rapeseed indicated that caffeic acid and ferulic acid might be less
   transformed into caffeoyl-CoA and feruloyl-CoA. Thus, sinapis acid as
   another downstream chemical of caffeic acid and ferulic acid was more
   accumulated (Fig. [139]7). All these compounds are necessary
   intermediates of lignin biosynthesis [[140]5]. Hong et al. found that
   the expression of genes involved in lignin biosynthesis were slightly
   but not markedly changed in yellow-seeded NILs [[141]50]. As to the
   other DEGs related to secondary metabolism, we found genes encoding
   S-adenosylmethionine synthetase 2 (BnaA09g00390D), peroxidase
   (BnaA02g14050D, BnaC06g32660D, BnaC06g21080D), branched-chain amino
   acid transaminase 1 (BnaA01g36200D), methionine synthase 2
   (BnaC03g33530D), aspartate aminotransferase 5 (BnaC01g06460D),
   phosphoglycerate kinase (BnaA07g34980D, BnaC02g46710D), lactate/malate
   dehydrogenase (BnaA09g16400D), glucose-1-phosphate adenylyltransferase
   family protein (BnaC04g33380D), beta glucosidase 19/28 (BnaA05g04030D,
   BnaC01g43700D, BnaC05g11190D, BnaA06g09700D), and pyruvate kinase
   (BnaA03g36910D) were downregulated in yellow rapeseed
   (Additional file [142]11: Figure S5). Peroxidase is one of three major
   enzymes involved in flavonoid oxidation through the associated
   reduction of hydrogen peroxide in the peroxidative cycle [[143]62].
   Hong et al. assigned peroxidase superfamily protein (e.g., peroxidase
   4/7/25/52/53) to phenylpropanoid and flavonoid pathways, which were
   differentially expressed between yellow and black rapeseed NILs
   [[144]50]. The DEGs related to flavonoid and lignin biosynthesis
   confirmed that they contribute to the different testa color.

Fig. 7.

   Fig. 7
   [145]Open in a new tab

   Proposed model for DEGs related to yellow seed quality. Blue font,
   downregulated genes in yellow seed compared with black seed. Red frame,
   increased chemical in yellow seed. Green frame, decreased chemical in
   yellow seed

   Since the oil content of yellow seed was higher than that of black
   seed, we found 12 DEGs related to FA biosynthesis, metabolism,
   elongation and degradation (Fig. [146]5). In addition, we found that
   BnaA09g51510D (encoding pyruvate dehydrogenase E1 alpha) was
   upregulated throughout yellow seed development compared with black seed
   (Additional file [147]11: Figure S5). Its homolog in A. thaliana is
   important for seed oil biosynthesis. Plastidic pyruvate kinase (PKP)
   provides most of the pyruvate for plastidic FA synthesis, and the
   mutation of PKP severely impairs seed storage lipid synthesis
   [[148]63]. BnaA03g17570D (acetyl Coenzyme A carboxylase
   carboxyltransferase alpha subunit) was upregulated in yellow seed. Its
   homolog in A. thaliana has been confirmed to be a subunit of
   heteromeric acetyl-CoA carboxylase (ACCase), which catalyzes the
   carboxyltransferase (CT) reaction. ACCase is responsible for the first
   step of FA synthesis [[149]64]. Additionally, homolog of UDP-glucose
   pyrophosphorylase (UGP) (BnaA05g32480D), a sucrose-regulated protein,
   was upregulated in yellow seed, which is required for fumonisin
   B1-induced cell death [[150]65]. A homolog of beta glucosidase 25
   (BnaC01g40610D) was also upregulated in yellow seed, which is involved
   in the carbohydrate metabolic process [[151]66]. Upregulation of
   cytochrome B5 reductase 1 (CB5R1) (BnaC02g02110D) in yellow seed might
   also be related to the FA variation, since CB5R is a microsomal
   membrane-bound protein that functions as part of the microsomal
   electron transfer system in FA desaturation [[152]67]. CB5R can
   interact with ankyrin repeat-containing protein 2A (AKR2A), which
   interacts with ascorbate peroxidase 3 (APX3). APX3 can target
   peroxisomes [[153]68]. This might be related to the above-mentioned
   expressional changes in genes encoding peroxidases. The expression
   changes of these genes may be helpful in explaining the FA variation in
   yellow seed and its adaptability to environments after flavonoid
   reduction.

Conclusions

   In the present study, whole transcriptome gene expression was analyzed
   in developing seeds of yellow-seeded B. napus derived from hybrids of
   B. napus-S. alba, and black-seeded B. napus. We identified the DEGs
   with seed development, which might be related to the development and
   biosynthetic process. In addition, DEGs related to the quality
   difference between yellow and black rapeseed have been identified,
   which mainly participate in flavonoid biosynthesis, phenylpropanoid
   biosynthesis, phenylalanine metabolism, flavone and flavonol
   biosynthesis, fatty acid biosynthesis and metabolism. These
   down-regulated genes are helpful to explain the less pigmentation (e.g.
   CHS, CHI, F3H, FLS, DFR, LDOC, BAN) and lignin (PAL, C4H and 4CL), and
   higher oil content in yellow rapeseed compared with black seed (Fig.
   [154]7). Future functional analysis of these genes would contribute to
   the molecular dissection of yellow seed character in B. napus.

Methods

Plant material

   Yellow-seeded B. napus (line W82) was preserved in our lab and was
   selected from back-crossing progenies of somatic hybrids of B. napus-S.
   alba. The black-seeded rapeseed (B. napus cv. ‘Yangyou 6’) was obtained
   from the Jiangsu Lixiahe Region Agricultural Research Institute, China
   [[155]45]. Both rapeseed lines were grown in the experimental field of
   Yangzhou University, Yangzhou, China. In addition to the visible
   difference in seed coat color, the flavonoid content in yellow seed was
   lower than that in black seed, and the seed FA composition and content
   were different between yellow- and black-seeded B. napus. The oil
   content of W82 was 6% higher than that of Yangyou 6. Higher protein and
   sucrose contents, less dietary fiber and crude fiber, and fewer
   glucosinolates accumulated in the seed meal of yellow rapeseed compared
   with black rapeseed (Additional file [156]12: Table S7) [[157]8,
   [158]39, [159]61, [160]69]. The developing seeds at 3 WAF, 4 WAF, 5
   WAF, 6 WAF, and mature seeds were collected from three pods each of ten
   individual plants for comparative analysis. During the seed
   development, differentially accumulated pigments were visual since 5
   WAF of yellow and black rapeseeds. Proanthocyanidins (PAs) were less
   accumulated throughout yellow seed development than black seed
   [[161]39, [162]70].

RNA extraction, library construction, and RNA sequencing

   For each developmental stage of each rapeseed line, five RNA samples
   were separately isolated from polled seeds of ten plants using Trizol
   Reagent Solution (Invitrogen, USA). Each library was pooled by mixing
   equal quantities of five RNA samples. The RNA quality was validated
   using agarose gel electrophoresis, Nanodrop, Qubit, and Agilent 2100 to
   confirm the purity, concentration, and integrity, respectively. mRNA
   was purified using beads with Oligo (dT), and cDNA was then synthesized
   with random hexamers after fragmentation of mRNA. After purification of
   cDNA with AMPure XP beads, unique adaptors and indexes were ligated.
   Certain fragments were selected with beads and enriched using PCR
   amplification. Finally, ten cDNA libraries for five developmental
   stages (3 WAF, 4 WAF, 5 WAF, 6 WAF and mature seeds) of yellow- and
   black-seeded B. napus were normalized based on a Qubit assessment, and
   the insert size was validated by Agilent 2100. Then, the polled
   libraries were sequenced by the Illumina HiSeq™ 2000 platform.

Bioinformatics analysis

   After removing adaptor sequences and low-quality sequences, clean reads
   were mapped to the B. napus cv. Darmor-bzh genome (version 5) using
   TopHat2 [[163]71, [164]72]. Based on the predicted gene models of
   Cufflinks, classification and statistics of alternative splicing were
   carried out using AS profile [[165]73, [166]74]. Novel transcripts were
   predicted using Cufflinks to assemble the mapped reads on the genome
   and compare it with the known gene models [[167]75]. The abundance of
   reads mapped to reference was estimated and normalized using Reads Per
   Kilo bases per Million reads (RPKM), and all the transcripts with RPKM
   value> 1 were used for further analysis. Differential gene expression
   analysis was performed using DESeq [[168]76, [169]77], and DEGs between
   libraries were screened with a threshold of | (FoldChange)| > 1 and q
   value< 0.005. All the DEGs among different libraries were clustered
   base on the log[10](RPKM+ 1) and log[2](Fold Change) value. Gene
   ontology (GO) enrichment of the DEGs was performed using GOseq with a
   corrected p-value < 0.05 [[170]78], and KEGG pathway enrichment of DEGs
   was performed using KOBAS 2.0 with a corrected p-value < 0.05
   [[171]79]. An overview of pathways related to these DEGs was predicted
   by MapMan (version 3.6.0) analysis [[172]80].

cDNA synthesis and qRT-PCR validation

   Subsamples of RNA-Seq were reverse transcribed into cDNA for real-time
   qPCR validation using the Revert Aid First Strand cDNA Synthesis Kit
   and SYBR Green Real-Time PCR Master Mixes (Thermo, USA). qRT-PCR was
   performed on a fluorescence quantitative system Mx3005P (Agilent, USA).
   Genes, primers and size of the amplicon are listed in
   Additional file [173]13: Table S8. B. napus β-actin (NCBI
   [174]AF111812) was used as an endogenous control to generate the △Ct
   for three technological replicates.

Additional files

   [175]Additional file 1:^ (33.8MB, xlsx)

   Table S1. Summary of read mapping in RNA-Seq analysis of yellow and
   black rapeseeds. B3, B4, B5, B6 and BM indicate 3 ~ 6 WAF and mature
   black seed. Y3, Y4, Y5, Y6 and YM indicate 3 ~ 6 WAF and mature yellow
   seed. (XLSX 34577 kb)
   [176]Additional file 2:^ (10.8MB, xlsx)

   Table S2. DEGs identified with black or yellow seed development and
   between same developmental stages of two rapeseeds. (XLSX 11030 kb)
   [177]Additional file 3:^ (94KB, xlsx)

   Table S3. DEGs related to seed development. (XLSX 94 kb)
   [178]Additional file 4:^ (25.9KB, xlsx)

   Table S4. Up- and downregulated genes among all the developmental
   stages of yellow- and black-seeded B. napus. (XLSX 25 kb)
   [179]Additional file 5^ (173.6KB, xlsx)

   Figure S1. The SOM clusters of DEGs from five developmental stages of
   two rapeseed lines. (XLSX 173 kb)
   [180]Additional file 6:^ (63.7KB, xlsx)

   Table S5. All the up- and down-DEGs with GO annotation. (XLSX 63 kb)
   [181]Additional file 7:^ (24.6KB, xlsx)

   Figure S2. KEGG enrichment of DEGs (Y3 vs. B3) in flavonoid and
   phenylpropanoid biosynthesis. (XLSX 24 kb)
   [182]Additional file 8:^ (31.5KB, xls)

   Figure S3. KEGG enrichment of up- and down-DEGs between yellow- and
   black-seeded B. napus. (XLS 31 kb)
   [183]Additional file 9:^ (519.3KB, pdf)

   Table S6. KEGG pathways of DEGs between yellow- and black-seeded B.
   napus. (PDF 519 kb)
   [184]Additional file 10:^ (982KB, pdf)

   Figure S4. Overview of pathways related to the DEGs between yellow- and
   black-seeded B. napus. (PDF 981 kb)
   [185]Additional file 11:^ (6MB, jpg)

   Figur S5. Heatmap of DEGs involved in secondary metabolism. (JPG 6164
   kb)
   [186]Additional file 12:^ (1.9MB, jpg)

   Table S7. Quality differences between yellow- and black-seeded B.
   napus. (JPG 1896 kb)
   [187]Additional file 13:^ (3.2MB, jpg)

   Table S8. Primers for qPCR validation of DEGs (JPG 3234 kb)

Acknowledgements