Identification and sequence analysis of shattering genes

A set of 32 individual shattering genes orthologues from B. napus and B. juncea genome was retrieved and their annotations were checked using keyword gene id to search Swissport annotations at the Brassica database (BRAD) (http://brassicadb.org/brad/). These genes were in greater number than those of model plant Arabidopsis thaliana as shown in Tables 2 and 3. The domain of these shattering genes was also identified using EMBL (http://smart.embl.de/smart/set_mode.cgi). The first six shattering genes of B. napus (Br-nS1-Br-nS6) contain the MADS-box domain whereas, 7–10 contain HLH, 11, 12, Pfam, 13, 14 pox/Hox 15–17 contain PbH1 domain. In B. juncea, 18–22 contain MADS-box domain while 23–26 HLH, 27, 28 Pfam, 29, 30 pox/Hox and 31, 32 PbH1 domain. We selected 32 annotated shattering genes of B. napus and B. juncea as Br-nS1 and Br-jS1 followed by Arabic numbers 1–32.In total shattering genes, 11 were mad box genes having mad box domain whereas the other 21 genes did not possess mad box domain. The genes that lacked mad box domain shared a large sequence resemblance with mad box protein of other crop varieties that also lack this domain and are considered to be mad box or shattering genes. Sequence analysis showed that all shattering genes of B. napus and B. juncea have introns except the gene IND. The maximum numbers of introns were identified in MADS-box shattering genes. These appearances are persistent with shattering genes previously determined in Arabidopsis thaliana and Brassica rapa.

Table 2 In silico study of 17 shattering genes identified in B. napus with their closest Arabidopsis homologs and sequence feature
Table 3 In silico study of 15 shattering genes identified in B. juncea with their closest Arabidopsis homologs and sequence feature

Phylogenetic analysis of shattering genes

The identified shattering genes protein sequences were used to analyze the phylogenetic relationship of the shattering gene family in B. napus, B. juncea, Arabidopsis, citrus, tomato, wheat, cotton, and rice. The unrooted phylogenetic tree characterizes the length of clades and the level of the evolutionary relationship with well-supported bootstrap values. The sequences of shattering genes SHP1, SHP2, FUL, IND, ALC, NAC, RPL, PG and their orthologous determined into B. juncea, B. napus, Arabidopsis, citrus, tomato, wheat, cotton and rice were aligned to generate the NJ phylogenetic tree (Fig. 1). Every individual shattering gene organized in a distinct clade with various color, characterize their functional and sequential conservation. The light green and dark brown colour in the tree is Clade I which contains a duplication of SHP1 and SHP2 in various plant like B. napus, B. juncea, citrus, tomato, wheat, cotton and rice except SHP2 gene where no duplication was observed in B. napus and B. juncea plants. However, clade II with yellow color consists of FUL genes where duplications were observed. This shows that clades I and II are closely related to each other as compared to other clades. Clade III indicated with blue color, shows duplication and triplication of IND and ALC in B. juncea, B. napus and other plants like citrus, tomato, wheat, cotton and rice that indicates divergence in sequences and clade IV with red colour, duplication of NAC genes was observed. It is clear from the resulting tree that clade III and clade IV are closely related to clade I and II. Similarly, clade V with purple colour and clade IV with dark blue colour contains PG and RPL genes in a duplicated form in B. napus, B. juncea, and all other plants. The clade comprising SHP1 and SHP2 genes contains a greater number of genes as compared to others. Genes from these two clades are present on different chromosomes indicate that every individual gene bear duplication and whole genome triplication events before reaching this level. Environmental, physiological, and chromosomal rearrangement at the development level brought changes in the genome. These results authenticate that every individual gene of B. napus, B. juncea, citrus, tomato, wheat, cotton, and rice under observation are shattering genes having a close resemblance to each other and with a model plant Arabidopsis thaliana as shown in Fig. 1.

Fig. 1
figure 1

Neighbor joining consensus phylogenetic tree of 181 shattering genes from B. napus (17), B. juncea (15), Arabidopsis (At 8), citrus (Ct 30), cotton (Cn 29), rice (Ri 27), wheat (Wt 35), and tomato (TO 20)

Gene structure organization and conserved motifs analysis of shattering proteins

We compared the coding DNA sequences of exons and introns to their genomic DNA sequences to facilitate phylogenetic reconstruction. As shown in Fig. 2a, the distribution, number, and length of exons and introns were not highly diverse among all genes. Br-nS1 was the longest sequence and Br-nS10 was the shortest among all shattering genes. Br-nS1 contain 8 introns. While Br-nS2, Br-jS18, and Br-jS19 contain six introns. Br-nS4, Br-nS5, and Br-nS6 contain seven introns while in B. juncea divergence is found Br-jS21 contains 4 and Br-jS22 contains 7 introns for the same gene. Br-nS9 contain a single intron, whereas the same gene Br-nS10, Br-jS25 and Br-jS26 didn’t contain any introns and showed a divergence in the gene sequence. Br-nS13 and Br-nS14 contain four introns whereas the same genes in B. juncea, Br-jS29, and Br-jS30 contain three introns showing a clear difference among B. napus and B. juncea. Similarly, Br-nS15, Br-nS16, and Br-nS17 genes contain three introns, while in other species B. juncea Br-jS31 contain three and Br-jS32 contains two introns for the same genes, which also showed some variance in genes sequences.

Fig. 2
figure 2

a Exon–intron structure of shattering genes in B. napus and B. juncea. Exons are shown as yellow boxes, introns are shown as a thin black line, and UTRs are shown as blue boxes. b Motif distribution analysis, 10 motifs are shown as colored boxes

MEME (Multiple Em for Motif Elicitation) motif search tool was used to identify 10 conserved motifs of 32 shattering protein sequences of B. napus and B. juncea (Fig. 2b). Motifs 1 and 2 exhibit the MADS-box domain which was found in 11 genes whereas other shattering genes did not show motif 1 or 2 features. The genes which exhibit the characteristics of motifs 1 or 2 were Br-nS1-Br-nS6 and Br-jS18-Br-jS22. These genes did not contain other representative motifs of Mads-box family such as motifs 4, 5, 6, 7, 8, 9, and 10. Motif 4 and 5 comprised of PbH1 domain found in 5 genes which were Br-nS15, Br-nS16, Br-nS17, Br-jS31, and Br-jS32. Br-nS7, Br-nS9, Br-nS10, Br-jS21, Br-jS23 Br-jS24, Br-jS25, and Br-jS26 genes consists of single motif whereas Br-nS8 gene did not contain any motif. Motif 8 and 10 showed pox/Hox domain which was found in Br-nS13, Br-nS14, Br-jS29, and Br-jS30 gene. Br-nS15, Br-nS16, Br-nS17, Br-jS31, and Br-jS32 comprised PbH1 domain with motif 5 and 6 features. Motif 1 and Motif 2 were conserved among genes which is the characteristic feature of shattering genes. The different motifs are represented by different colors that showed similarities among B. napus and B. juncea as shown in (Fig. 2b). The number of motifs found in both species is similar except for Br-nS7, Br-nS9, Br-nS10, Br-jS21, Br-jS23 Br-jS24, Br-jS25, and Br-jS26 which shows single motif and revealed similarities and differences with other shattering genes among brassica species.

Chromosomal distributions of shattering genes

According to the available Brassica genome database, 32 shattering genes were mapped onto 10 chromosomes of B. napus and B. juncea. The chromosome localization of Brassica gene ID was confirmed by ensemble Plant Browser. There were 17 shattering genes distributed on both the A and C subgenome of B. napus while on the B and A sub-genome of B. juncea, 15 shattering genes were observed. Similarly, on the A subgenome of both plants, a total of 13 genes were identified and on the B sub-genome of B. juncea, 11 genes were observed, while on the C genome of B. napus, 8 genes were observed.

According to our results, Br-nS4 and Br-nS10 genes were observed on similar chromosome A03, whereas Br-nS3 lies on chromosome A05. Similarly, Br-nS1 and Br-jS24 were identified on the same chromosome A07, while Br-nS17 and Br-jS31 were located on chromosome A08. Genes like Br-nS5 and Br-nS16 were located on the A09 chromosome, whereas Br-nS11, Br-nS13, Br-jS27, and Br-jS30 were observed on chromosome A10. Similarly, on chromosome B01, gene Br-jS20 was observed while on the B02 chromosome, gene Br-jS22 was located. Br-jS28 and Br-jS32 genes were identified on the same chromosome B03, whereas on B04 chromosome Br-jS21 gene was located. Hence genes Br-jS18, Br-jS19, and Br-jS23 were observed on similar chromosome B06. Similarly, on the B08 chromosome, genes like Br-jS25, Br-jS26, and Br-jS29 were identified. The genes observed on chromosome C02 were Br-nS6, Br-nS8 and Br-nS14, whereas on other chromosomes like C03, C05, C06, C07, and C08, genes located were Br-nS9, Br-nS12, Br-nS2, Br-nS7, and Br-nS15 respectively. Hence, all the shattering genes were scattered on Brassica chromosomes as shown in Figs. 3 and 4.

Fig. 3
figure 3

Logos of tens motifs discovered in shattering genes

Fig. 4
figure 4

Gene localization of shattering genes on B. napus and B. juncea chromosomes

Syntenic relationship among shattering genes of B. napus and B. juncea

Comparative genomic synteny analysis was performed by circoletto tool (tools.bat.inspire.org/circoletto/) for genome conservation visualization. The orthologs’ relationship and conservation were determined for the shattering gene family in B. napus and B. juncea. Synteny diagram represents a remarkable relationship among these species in the context of duplication, triplication, evolution, function, and expression (Fig. 5) showed a unique relationship among B. juncea and B. napus. It was observed that B. napus Br-nS13 and Br-nS14 gene sequence showed synteny with B. juncea sequence Br-jS29 and Br-jS30, while B. napus gene sequence Br-nS15, 16, and 17 showed synteny with B. juncea gene sequence Br-jS31, 32 and gene sequence Br-nS11 and 12 showed synteny with Br-jS27 and Br-jS28. In Addition, Br-nS7 and Br-nS8 gene sequence showed synteny with Br-jS23 and Br-jS24 gene sequences while Br-nS9 and Br-nS10 showed synteny with Br-jS25 and Br-jS26 gene sequences. Similarly, Br-nS1 and Br-nS2 showed synteny with Br-jS18 and Br-jS19 gene sequences, while Br-nS3 showed synteny with Br-jS20. B. napus gene Br-nS4, 5, 6 sequences showed synteny with Br-jS21 and Br-jS22. In comparative synteny analysis inward tangling ribbons color intensity exhibited the rate of conservation while outward tangling ribbons showed duplication events. Genomic dynamicity and evolutionary improvement along mobile elements in the genome of B. napus and B. juncea were determined in syntenic circles. In chromosomal shuffling, duplication, and triplication mobile elements play an important role. A permanent position was adopted by the blocks at a specific position in genome initiate expression that involve another biological pathway disturbance (Fig. 5).

Fig. 5
figure 5

Representation of synteny of B. napus and B. juncea identifying the level of conservation at the sequence level in 4 colors. The red, green, orange, and blue colors signify the level and intensity of evolutionary conservation among distinct shattering genes, e.g. maximum intensity is from orange to green

qRT-PCR expression of shattering genes in fresh and mature siliques

The expression levels of shattering genes in fresh and mature siliques of B. napus and B. juncea were confirmed by qRT-PCR. Our results inferred that the expression level of shattering genes was higher in B. juncea as compared to B. napus in both fresh and mature siliques. Strong signals of shattering genes were observed in mature siliques in both species, while in fresh silique, the transcripts levels were low (Fig. 6). The correlation is completely noticeable in the evidence that shattering genes play a major role in shattering associated pathways by devoting to developmental pathways of lignification and valve margin associated transcriptional activity. Moreover, ALC gene expression was upregulated in fresh silique of B. napus while down regulation of ALC gene was observed in fresh silique of B. juncea. Similarly, higher expression of ALC genes was observed in mature silique of B. juncea compared to B. napus. PG gene downregulation was observed in fresh silique of B. napus while it expressed more in B. juncea. The expression of SHP1, SHP2, FUL and RPL were observed more in B. juncea in both fresh and mature siliques showed a difference expression patterns.

Fig. 6
figure 6

The expression levels of shattering genes in B. napus and B. juncea: Graph bar defining the difference and the correlation in the expression level among two tissues of B. napus and B. juncea. A1 A lower level of shattering genes expression in B. napus than B. juncea. A2 A higher level of shattering genes expression in B. juncea than B. napus in the given tissues. ALC gene expressed more in B. napus fresh silique. Lower expression of ALC gene was observed in fresh silique of B. juncea but highly expression was observed in mature silique

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