Genome-wide identification, phylogeny, and expression profiling analysis of shattering genes in rapeseed and mustard plants

Background Non-synchronized pods shattering in the Brassicaceae family bring upon huge yield losses around the world. The shattering process was validated to be controlled by eight genes in Arabidopsis, including SHP1, SHP2, FUL, IND, ALC, NAC, RPL, and PG. We performed genome-wide identification, characterization, and expression analysis of shattering genes in B.napus and B. juncea to gain understanding into this gene family and to explain their expression patterns in fresh and mature siliques. Results A comprehensive genome investigation of B.napus and B.juncea revealed 32 shattering genes, which were identified and categorized using protein motif structure, exon-intron organization, and phylogeny. The phylogenetic study revealed that these shattering genes contain little duplications, determined with a distinct chromosome number. Motifs of 32 shattering proteins were observed where motifs1 and 2 were found to be more conserved. A single motif was observed for other genes like Br-nS7, Br-nS9, Br-nS10, Br-jS21, Br-jS23, Br-jS24, Br-jS25, and Br-jS26. Synteny analysis was performed that validated a conserved pattern of blocks among these cultivars. RT-PCR based expressions profiles showed higher expression of shattering genes in B. juncea as compared to B.napus. SHP1, SHP2, and FUL gene were expressed more in mature silique. ALC gene was upregulated in fresh silique of B. napus but downregulation of ALC were observed in fresh silique of B. juncea. Conclusion This study authenticates the presence of shattering genes in the local cultivars of Brassica. It has been validated that the expression of shattering genes were more in B. juncea as compared to B.napus. The outcomes of this study contribute to the screening of more candidate genes for further investigation.

1 National Centre for Bioinformatics, Quaid-I-Azam University, Islamabad 45320, Pakistan Full list of author information is available at the end of the article Page 2 of 12 Afridi et al. Journal of Genetic Engineering and Biotechnology (2022) 20:124 species are natural hybrids of diploids and all species are interconnected [2]. Non-synchronous pod shattering remained a major problem of Brassica that results in yield loss. It also causes seed loss due to the dispersion of a silique following complicated physiological and biological mechanisms [3]. However, premature and unsynchronized pods shattering like the dehiscence results in a huge loss in crop yield [4]. Pod's shattering occurs when the adhesions among walls change into fragile and internal forces apply them to the moveable position [5]. Seed valves are responsible for the attachment and internal force creation that contributes to the necessary protection of the seed [6]. Seeds of B. juncea and B. napus are very important equally 14% of oil around the world is produced by these crops. Moreover, rapeseed is considered the third most important oilseed crop worldwide [7]. Distinct nutrients and biological molecules are reported to be involved in the evolution of shattering in canola [8].
Previous investigation over shattering revealed that shattering occurred because of molecular components excess production and enrichment in the valve margin and cellar portion around the pods in siliques. Lignin and cellulose play a key role in the hardening of pod walls, which lowers water content during the later development stages of rapeseed and Brassica species [9]. The shattering mechanism of B. napus and B. juncea are controlled by eight different genes, like SHATTERPROOF1 (SHP1), SHATTERPROOF2 (SHP2) FRUITFULL (FUL), INDEHISCENT (IND), ALCATRAZ (ALC), NAC, (NST1 and NST2) REPLUMLESS (RPL), and POLYGlACTOU-RANAZE [10]. For the development of shattering genes, distinct transcription factor binding sites are involved which are important both structurally and functionally [11]. Other genes like SHP1/2, FUL, IND, ALC, NAC, RPL, and PG of canola and Indian mustard were also reported [12]. In one study, a comparative analysis was performed to unveil the genomic maintenance for the evolutionary and functional correlation among shattering genes SHP1/2, FUL, IND, ALC, NAC, RPL, and PG [13] having functional and genetic conservation among them. The pattern of conservation in these shattering gene sequences was also found with comparative synteny approach by Krzywinski et al. [14].
The most desirable solution to the shattering problem of B. napus and B. juncea is to delay pod shattering by knocking out SHPS genes and stimulating the expression of FUL up to the susceptible crop is ready for harvesting. Therefore, before developing genome modified plants it is essential to study these genes elaborately in local plants B.napus and B. juncea. Therefore, this study was carried out to identify the orthologous of shattering genes in the local cultivars of B. napus and B. juncea and to study their expression pattern in fresh and mature siliques. This study further identified the syntenic and evolutionary relationship of shattering genes in the studied cultivars based on phylogenetic analysis with NJ algorithm.

Identification of shattering genes
BRAD database (http:// brass icadb. org/ brad/) was used to retrieve protein, genomic, CDS and cDNA sequences of the 8 shattering genes SHP1/2, FUL, ALC, NAC, IND, RPL, and PG and their orthologs in B.napus and B. juncea [15]. Other databases like NCBI (https:// www. ncbi. nlm. nih. gov/), TAIR (https:// www. arabi dopsis. org/), and Plants Ensembl (http:// plants. ensem bl. org/) were also consulted. A web tool from EMBL was used to identify different protein domains (http:// smart. embl. de/ smart/ set_ mode. cgi). The Basic local alignment search tool (BLAST) (htpp://www. ncbi. nlm. nih. gov/ BLAST/) was used to search the homology of the shattering genes in B.napus and B.juncea. ProtParam tool was used to study the primary structure of shattering genes (http:// expasy. org/ tools/ protp aram. html). The gene structure display server (GSDS) web tool was used to align the CDS sequences of shattering genes with genomic sequences to identify exons and introns [16].

Phylogenetic analysis of shattering proteins
B. napus and B. juncea shattering protein sequences were obtained from the BRAD database using reference sequences of shattering genes obtained from the TAIR database and the other plants protein sequences were obtained from the NCBI database and then aligned using the Clustal X program [17]. Using the Neighbor Joining (NJ) algorithm, a phylogenetic tree was constructed with MEGA 11 software [18]. The implication of nodes was calculated using a bootstrap study of 1000 replicates. For the surety of different domains that show the topology of NJ tree, pairwise gape deletion mode was used.

Analysis of conserved motifs in shattering proteins
MEME software (Multiple Em for Motif Elicitation, V4.9.0) was used to analyze MADS-box shattering genes protein sequences as described by Bailey et al. [19]. MEME search was run with the following parameters: (1) maximum number of motif identification = 10; (2) optimum motif width > 6 and < 200.

Chromosomal locations analysis
To find out identical genes, all shattering genes of B. napus and B. juncea were BLAST searched (htpp://www. ncbi. nlm. nih. gov/ BLAST/) against each other, with a query coverage and similarity percentage of candidate genes of more than 80% [20]. The Brassica database (http:// brass icadb. org/ brad/) was used to acquire positional information for all putative shattering genes along the 10 chromosomes of B. napus and B. juncea [15]. All genes were mapped along the 10 chromosomes, and the gene's location was observed.

Analysis of syntenic relationships
The comparative genomic synteny was performed to find the relationship among distinct shattering genes like SHP1/2, FUL, ALC, NAC, IND, RPL, and PG in B.napus and B. juncea using the circoletto program; genome visualization tool circoletto [14].

RNA extraction and cDNA synthesis
Total RNA from the fresh and mature siliques of B. napus and B. juncea was extracted using a Pure Link TM RNA Mini kit (Invitrogen). The RNAs were quantified by using BioSpec-nano Micro-volume UV-Vis Spectrophotometer (Shimadzu). The quality and integrity of RNA was checked on 1.5% agarose gel. cDNA was synthesized by using RevertAid TM reverse transcriptase enzyme (Fermentas TM Cat.No. K1621) following the manufacturer's guidelines.

Expression analysis of Shattering genes qRT-PCR
The expression pattern of shattering genes (SHP1/2, FUL, ALC, NAC, RPL, PG, and IND) was determined in fresh and mature silique of B. napus and B. juncea using comparative ΔCT method in real-time PCR (Applied Biosystems) with StepOnePlus software. For the execution of a relative expression, the Elongation factor (EF) was used as endogenous control. No template control (NTC) was also used as negative control in the assay. In total, 10 μl reaction volume, 5 μl Maxima SYBER Green (Thermo Fisher) genes specific primers (1 pmol of each), and 1 μl cDNA as a template were used. Real-time PCR conditions set were; denaturation at 94 °C for 10 min, the second stage followed by 40 cycles at 95 °C for 40 s, 58 °C for 32 s 72 °C for 32 seconds. Finally, a melt curve study was carried out at 52 °C to 95 °C. The statistical analysis of results was carried out by mean of relative fold expression of transcript ± standard deviation (SD). All the primers used in the qRT-PCR analysis listed in Table 1 were designed manually through the conserved region from the A and C subgenome of B. napus. The length of the amplified fragment ranged between 100 and 130 bp.

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:// brass icadb. 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).

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,     Fig. 1.

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,  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. 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. 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.

Syntenic relationship among shattering genes of B. napus and B. juncea
Comparative genomic synteny analysis was performed by circoletto tool (tools. bat. inspi re. org/ circo letto/) 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).

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.

Discussion
Brassicaceae is a large plant family consists of ~ 338 genera and 3700 species, important both economically and agriculturally [19]. In addition to this, plants of this family are grown like a weed in different parts of the world including North America, South America, and Austria [21]. Arabidopsis thaliana, a model plant from the family Brassicaceae was the first plant to be entirely sequenced [21]. Plants and vegetables from this family offer essential  [22]. In this study, SHP1, SHP2, FUL, IND, ALC, NAC, RPL, and PG when compared at the genomic level showed close similarity. Protein and nucleotide shows an important correlation at the sequence level. It has been showed that these genes are responsible in shattering and seed development of plants [23]. The phylogenetic analysis here showed that SHP1 as compared to other genes have fever dynamicity which is balanced in the connection of genomics but bear duplication. The duplicated genes determined with a distinct chromosome number in B. napus and B. juncea which recommended genomic flexibility as previously reported in Arabidopsis and B. rapa [24,25] shows similar results with our investigations. SHP2 shattering gene study uses a novel approach to phylogenetic analysis bears no duplication and triplication as previously reported in other Brassica species [26,27]. FUL is known for fruit development in different Brassica species. The Phylogenomics of FUL affords unusually different results than SHP1 and SHP2. The behavior observed more dynamics among the various species of Brassica family. FUL genes showing duplication and differential location in the genome of B. juncea and B. napus also previously described in B. rapa further strengthen our results [8].
In current research, we have study 32 shattering gene of B. juncea and B. napus which are more in number than the shattering genes reported for A. thaliana [28]. The syntenic analysis performed among B. napus and B. juncea shows the similar sequence feature and whole genome of both species go through triplication events since its divergence from Arabidopsis. The evolutionary and syntenic relationships among Arabidopsis and B. rapa is also supporting our results [29]. On the other hand, we observed the expression of shattering genes SHP1, SHP2, FUL, IND, ALC, NAC, RPL, and PG in B. napus and B. juncea like previously reported in Arabidopsis [30]. Our result also suggest that these genes are the reputed orthologous of Arabidopsis genes AGL1, AGL5, AGL8, AT5G67110, EDA33, At5g22380, BLH9, and At1g45015 might play the similar role, and they are expressed in both plants in fresh and mature siliques.
In previous studies, divergence in expression pattern was observed in shattering genes in B. napus. Wu et al. [31] determined the expression patterns and evolution of MADS-box TF family in B. napus. Becker and Theissen [32] reported that Shatterproof1/2 and genes which are members of MADS box family are engaged in controlling this pod shattering issue. SHP1 and SHP2 genes are involved in opening of silique in B. napus plants when the expression level is low [23,33,34].
The expression of these genes started from developed flower to mature silique with lower expression in the late stage of development of seed [31]. SHP1, SHP2, and FUL showed a relationship with IND, ALC that initiate acting to abrogate activity of DZ to forbid dehiscence at the time of seed formation follow indehiscence in the existence of multiple regulatory genes. The present analysis of all shattering genes showed different expression pattern in different tissues such as fresh and mature siliques of both plants as previously reported in Arabidopsis and B. rapa. These genes were expressed in both plant tissues, although in B. juncea they were slightly higher than in B. napus. These different expressions of shattering genes shows that they are important for cellular valve and margin evolution [24,35]. A similar study was conducted by Yasin et al. [36], Ahmad et al. [37], & Khan et al. [38] whose results agree with our results. They demonstrated higher expression of FUL gene in mature aerial part silique plant as compared to leaves and flowers of B. napus plants. Similarly, SHP1 and SHP2 transcripts were expressed in flower silique whereas; no expression was detected in the leaves. Our findings showed basic gene expression information about shattering cascade genes which can be useful for developing genome edited brassica plants which are resistant to shattering.

Conclusion
Conclusively, different orthologous of shattering genes are exists in the local cultivars of Brassica. After comparative phylogenetic study, molecular gene characteristics, motifs/domain identifications, and comparative expression study, it is validated that the sequences were conserved across B. napus, B. juncea as well as in Arabidopsis plant. The redundant expression was observed in fresh and mature siliques of both cultivars. The different expression patterns of shattering genes are also helpful to study the nature of both plants and their pathways related to transcription and regulation. Further analysis of shattering genes is required to uncover their functions involved in the regulation of different pathways.