Morphologically interspecific hybrid F1 and its colchicine induced amphidiploid were more similar to its parent 2 (OKP2). However, to identify the overall effect of polyploidization, different phenotypic features like plant height (Additional Fig. S1 A-D), leaf area, inflorescence (Additional Fig. S1 E), stem diameter (Additional Fig. S1 F-I), trichome density (Fig. S2), stomata etc. of amphidiploid, hybrid F1 and its parents (OBP1 and OKP2) were measured (Table 1). On comparing interspecific hybrid F1 with its parent plants and amphidiploid, F1 was found to be robust, rapidly growing, vigorous and tall (110.00±8.9). However, the leaf area of F1 (3.5± 0.18) was lesser than its amphidiploid (9.63 ± 0.75) and its parent plants. The leaf of interspecific hybrid F1 was long, medium broad and thin. Also, the inflorescence and stem were weak in interspecific hybrid F1 but the length of inflorescence of interspecific hybrid F1 was longer (nearly two fold) than amphidiploid and its parents. In contrast, amphidiploid was slow growing and medium tall (101.50 ± 9.30). The leaf of amphidiploid was oval shaped, broad, thick and the leaf area was greater than its parents. Besides these characteristics, trichome density was more in the hybrid F1 (nearly threefold higher than amphidiploid). Scanning electron microscopy revealed that size of trichome and stomata (nearly threefold greater than F1) was greater in amphidiploid (Fig. 1). Furthermore, the density of stomata was more in interspecific hybrid F1. However, oil yield per 100gm of fresh leaf was more in amphidiploid (0.48 ±0.02).
The results obtained from root-tip mitosis of the four target plants revealed the modal somatic chromosome number to be as: 2n (AA) = 48 for OBP1, 2n (BB) =76 for OKP2, n+n (A+B) = 62 for interspecific hybrid F1, and 2n+2n (AA+BB) =124 (Additional Fig. S3 A-D). This is in conformity that the F1 hybrid and the amphidiploids constitute the genuine genomic combination of the two progenitor parents employed in the present study.
Transcriptome Sequencing, Assembly and Annotation
Pair-end sequencing generated nearly 14,857,263 (OBP1), 13,017,783 (OKP2), 14,266,805 (interspecific hybrid F1), and 13,955,543 (amphidiploid) reads of 101bp average read length. After filtering and removing adapter sequences from raw reads, 14,431,491 (OBP1), 12,665,614 (OKP2), 13,866,646 (interspecific hybrid F1) and 13,479,705 (amphidiploid) high quality reads were acquired for further assembly (Table S1). High quality reads retained after filtering raw reads contained 1,437,320,230 (OBP1), 1,261,764,356 (OKP2), 1,382,322,028 (interspecific hybrid F1) and 1,334,646,724 (amphidiploid) high quality bases. High quality reads were used for merged assembly using Trinity assembler which generated total 243,647 numbers of transcripts of 148 Mbp in length. Total number of final transcripts obtained after the final assembly was 91,778 having final transcriptome length of 66 Mb with N50 value 942. BlastX search was carried out for 91,778 clustered transcripts against NRDB (Non redundant database) plant using e-value <=0.001. Out of the total only 78,928 transcripts got annotated while 12,850 remained un-annotated.
Comparative gene expression patterns between parents and progenies
The parent plants (OBP1 vs. OKP2) were investigated for their differential gene expression patterns (Fig. 2A). Further, the differentially expressed genes between F1 hybrids or amphidiploid compared to their parents were also analyzed (Fig. 2B). Out of 172 differentially expressed genes between interspecific hybrid F1 and parent 1 (OBP1), 60 showed up-regulation and 112 down-regulation. From 123 differential genes between amphidiploid and OBP1, 51 were up-regulated and 72 down-regulated. Similarly, 155 differentially expressed genes (47 Up-regulated and 108 down regulated) were obtained from the analysis of interspecific hybrid F1 and parent 2 (OKP2) and 215 (82 up-regulated and 133 down regulated) for amphidiploid and parent 2 (OKP2). In all comparisons, it was observed that the proportion of genes displaying the differential expression between interspecific hybrid F1 and amphidiploid and their parents was asymmetric (FDR<0.05; BH multiple correction test).
In order to identify the non-additively expressed genes, the expression levels of amphidiploid and hybrid F1 was compared with the mid parent values (MPVs) derived from the base mean values of the two parents assuming that one-third of the total transcription is from the genome from parent 1 (OBP1), one-third from parent 2 (OKP2) and one-third is from the interspecific hybrid F1. On the other hand, interspecific hybrid F1 was compared to the parent 1 (OBP1) and parent 2 (OKP2) assuming that the half of the total transcripts is from the genome of each parent (OBP1 and OKP2), respectively. The non-additively expressed genes between the F1 hybrid and MPV of parent1 and parent2 (OBP1 and OKP2) was found to be 38, of which 10 genes were up-regulated and 28 genes were down-regulated (Fig. 3A; FDR< 0.05). Likewise, of 786 non-additively expressed genes observed between the amphidiploid and the MPV of Parent1, Parent2 and F1, 395 genes showed up-regulation and 391 genes exhibited down-regulation (Fig. 3B; FDR <0.05). GO analysis of 10 up-regulated genes in F1 hybrid and MPV of OBP1 and OKP2 indicated enrichment of photosynthesis, terpene biosynthesis including sesquiterpenoid metabolism and lipid biosynthesis (Additional Fig. S4A; FDR< 0.05; Table S2) whereas, the 28 down regulated genes in F1 hybrid and MPV of OBP1 and OKP2 were mainly associated with lignan biosynthesis and metabolism, steroid metabolism (Additional Fig. S4B; FDR< 0.05; Table S3). In contrast, the 395 up-regulated genes in amphidiploid and the MPV of OBP1, OKP2 and F1 revealed the enrichment of developmental vegetative growth, regulation of leaf development and regulation of shoot apical meristem development (Additional Fig. S4C; FDR< 0.05; Table S4) while the 391 down regulated genes in the amphidiploid and MPV of OBP1, OKP2 and F1 were mainly enriched in flavonoid metabolism, starch catabolism, s-adenosylmethionine metabolism (Additional Fig. S4D; FDR< 0.05; Table S5).
Hybridization induced transcript expression
To understand the reasons for developmental changes between interspecific hybrid F1 and amphidiploid, pair-wise comparison between OBP1 vs. F1&Amphid2, OKP2 vs. F1&Amphid2, F1 vs. OBP1&OKP2 and Amphid2 vs. F1,OKP2&OBP1 were analyzed (Fig. 4A). 244 transcripts out of 38,040 common transcripts (obtained from pair-wise comparison between OBP1 vs. F1&Amphid2, OKP2 vs. F1&Amphid2, F1 vs. OBP1&OKP2 and Amphid2 vs. F1,OKP2&OBP) exhibited antagonistic expression pattern (log2 fold -1>=<1) in interspecific hybrid F1 and amphidiploid. Further analysis revealed similar expression pattern of these 244 transcripts in amphidiploid and parent 2 (OKP2). Among these 126 transcripts were up regulated in amphidiploid and parent 2 whereas these transcripts were down regulated in interspecific hybrid F1 (Fig. 5A; Table S6). In contrast to this, 118 transcripts were having higher expression in interspecific hybrid F1 in comparison to amphidiploid and parent 2 (Fig. 4C). These results indicated that the overall transcriptome of amphidiploid was more similar to its parent 2 (OKP2) and it matches to the morphological analysis which shows that amphidiploid plants were more similar to its parent 2 (OKP2). To identify the possible function of these transcripts GO enrichment analysis was conducted. This analysis revealed that these genes were mainly enriched to biological function such as shoot system development, flower development, cotyledon morphogenesis, embryonic morphogenesis, reproductive structure development, programmed cell death (Fig. 4D). It was also observed that many transcripts were reported as “PREDICTED” cellulose synthase-like protein G3 (proposed to synthesize non cellulosic polysaccharides that comprise plant cell walls)”, “1-deoxy-D-xylulose 5-phosphate synthase (catalyzes the first step of the MEP pathway)”, SWI/SNF complex component SNF12 homolog (activator of flower homeotic genes)”, oligopeptide transporter 3 (essential for early embryo development)”, casein kinase II subunit beta-like (involved in flowering-time regulation)”, AMP deaminase-like isoform X2 (essential for the transition from zygote to embryo)”, SNW/SKI-interacting protein (Splicing factor involved in post-transcriptional regulation of circadian clock and flowering time genes),” nuclear transcription factor Y subunit A-10 (positive Regulators of Photomorphogenesis)”, “Calmodulin-domain protein kinase 5 isoform 1 (involved in the many aspects of plant growth and development)”, “Epidermal patterning factor -like protein 4 (negative regulator of stomatal development)”, “WAT1-related protein At4g19185-like (involved in the secondary wall formation)”, “protein STRICTOSIDINE SYNTHASE-LIKE 11 (involved in anther development and pollen wall formation)” (Table S7). It was also noticed that many of the transcripts were predicted as “uncharacterized protein LOC105176273, hypothetical protein POPTR_0001s256302g, hypothetical protein MIMGU_mgv1a009003mg, hypothetical protein MIMGU_mgv1a005332mg, hypothetical protein JCGZ_24107, hypothetical protein M569_05704, uncharacterized protein LOC105177873, uncharacterized protein LOC105176091, uncharacterized protein LOC105165185, uncharacterized protein LOC105164617 isoform X1, etc.” and many of them were left un-annotated, perhaps due lack of its annotation in Ocimum. Further investigation of these transcripts could provide good candidates for understanding the role of genome doubling in the correction of phenotypic weakness induced by hybridization. Interestingly, these transcripts could be utilized for the identification of novel genes probably having their role in morphological and anatomical differences between parents and hybrid and the amphidiploid plants.
Comparison of gene expression between the F1 hybrid and amphidiploid
Between colchicine induced amphidiploid and F1 hybrids, 179 differentially expressed genes (DEGs) were identified including 132 up-regulated and 47 down-regulated genes when BH multiple test correction method was applied (Benjamini and Hochberg, 1995) having FDR < 0.05 (Additional Fig. S5A). These DEGs were mapped to reference canonical pathways in KEGG to find out their involvement in biological pathways and 44 out of 179 DEGs were assigned to 46 KEGG pathways (Additional Fig. S6). The largest cluster was of biosynthesis of secondary metabolites with 13 members and the second largest was of metabolic pathways with 9 members, indicating that many genes among these DEGs were involved in the biosynthesis of secondary metabolites. Thereafter, to investigate the probable function of these DEGs, GO enrichment analysis was performed. The results of GO enrichment analysis showed that they were mainly enriched in the secondary metabolic processes like sesquiterpenoid biosynthetic and metabolic process, isoprenoid biosynthetic process, jasmonic acid metabolic process in biological process category (Additional Fig. S5B) and were enriched in auxin:proton symporter activity, fatty-acyl-CoA reductase (alcohol-forming) activity, magnesium ion binding, lyase activity, cyclase activity etc (FDR < 0.05) (Additional Fig. S5C).
Detection of gene expression alterations in interspecific hybrid F1 and amphidiploid
Gene alteration events were calculated by the occurrence of new transcripts (lacking in parents) or by the lack of some transcripts (existing in parents) in interspecific hybrid F1 and amphidiploids. For this analysis, total transcripts of parent 1 (67,770), parent 2 (73,265), interspecific hybrid F1 (76,917) and the amphidiploid (76,563) were examined (Fig. 5). The result of this analysis illustrated that 5,766 common transcripts of parents and interspecific hybrid F1 were not detected in amphidiploid. In addition to this, about 6,432 transcripts present in interspecific hybrid F1 and amphidiploid were missing in parents. However, of these 6,432 transcripts, only 3,868 transcripts were common in interspecific hybrid F1 and amphidiploid. Therefore, total 8,330 transcripts of interspecific hybrid F1 were absent in amphidiploid. On the other hand, only two transcripts were found to be exclusive in amphidiploid with respect to interspecifc hybrid F1 and parents (OBP1 and OKP2). These alterations in gene expression may be because of gene silencing, activation or may be due to sequencing error, but here it was assumed that these transcripts were either suppressed or expressed in amphidiploid. Upon analyzing the annotations of these non-expressing 8,330 transcripts in amphidiploid, it was observed that these transcripts mainly included the genes which were involved in disease resistance, primary and secondary metabolism and cell cycle. It also included many transcription factors (“basic helix-loop-helix transcription factor”, “MYB/ MYB-related”, “MADS-box”, “APETALA”, “AP5/EREBP”, “WRKY” etc), cytochrome p450s and methyl-CpG-binding domain-containing proteins. Moreover, there were many transcripts which were predicted as “uncharacterized or unnamed protein” and many of them were left un-annotated also. In contrast, the 2 transcripts exclusive to amphidiploid were WNK lysine deficient protein kinase and geranylgeranyl transferase type-2 subunit beta 1-like proteins.
Chlorophyll content and DGE related to chlorophyll biosynthesis between F1 hybrid and amphidiploid
The result of chlorophyll estimation showed that the amount of Chla, Chlb and the total chlorophyll was 0.35 mg/g, 0.13 mg/g and 0.54 mg/g, respectively in interspecific hybrid F1 and 0.30 mg/g, 0.11 mg/g and 0.37 mg/g in amphidiploid. Here, it was observed that the amount of Chla, Chlb and the total chlorophyll contents were higher in the interspecific hybrid F1 than its amphidiploid (Fig.7A). To find the probable reason for this content change, 167 transcripts in interspecific hybrid F1 and 169 trancripts in amphidiploid related to 27 classic enzymes involved in chlorophyll metabolic pathway were analyzed (Table 2). Chlorophyll metabolic pathway in plants consists of ALA, Proto IX, heme and chlorophyll formation/degradation steps. In ALA, Proto IX, heme and chlorophyll biosynthesis steps, higher number of transcripts for enzymes like HemA, HemE, HemY, HemH, COX15, POR and CAO were detected while fewer transcripts for HemF, COX10, ChlD, ChlI, ChlM, ChlE, 4VCR and CLH were recorded. Single copy of enzyme ChlH and ChlG were identified from the trancriptome sequences of the interspecific hybrid F1 and amphidiploid. In the chlorophyll degradation steps, more than one transcript was identified for enzymes like NYC1, HCAR, PPH, PAO and RCCR. FPKM values and fold change values for 167 common transcripts in interspecific hybrid F1 and amphidiploid were averaged for the further analysis. Based on these identified transcripts, proposed chlorophyll metabolic pathway in interspecific hybrid F1 and amphidiploid was constructed (Fig.6).
The difference in the content change of Chla, Chlb and total chlorophyll were correlated with the average FPKM and the fold change values of the 27 classic enzymes involved in the chlorophyll metabolism. The trend of change in expression based on the average FPKM and fold change values (log2 fold change -0.1>=<0.1) of the main enzymes (HemA, HemL, HemC, HemE, HemF, HemY, HemH and COX10) involved in the Ala, Proto IX, Heme and the key enzymes (Chl, ChlI, ChlM, ChlE, 4VCR) of chlorophyll formation showed increased expression in interspecific F1 compared to amphidiploid. On the other hand, several enzymes (HCAR, PPH, PAO and RCCR) involved in the chlorophyll degradation showed increased expression in amphidiploid compared to interspecific hybrid F1. These results clearly demonstrated that the enzymes involved in chlorophyll biosynthesis (Ala, Proto IX, Heme and in Chlorophyll formation) were positively regulated in interspecific hybrid F1 but negatively regulated in amphidiploid. In contrast, the enzymes involved in chlorophyll degradation (NYC1, PPH, PAO and RCCR) were negatively regulated in interspecific F1 while positively regulated in the amphidiploid. These results correlates with the results obtained from the chlorophyll estimation.
Phenylpropanoid profiling and DEG related biosynthesis in F1 hybrids and amphidiploid
Phenylpropanoid biosynthetic pathway is the predominant pathway present in the different Ocimum spp. which produces different phenylpropenes, lignins and flavonoids. Therefore, to understand the effect of interspecific hybridization and whole genome duplication on phenylpropanoid biosynthesis, GC-MS profiling of essential oils of F1 and amphidiploid, total content change of lignin and flavonoid was analyzed (Fig. 7 B-D). The essential oil analysis showed that the amount of eugenol was 0.082 mg/g leaf in interspecific hybrid F1 and 0.063 mg/g leaf in amphidiploid whereas, the amount of methyleugenol was 0.087 mg/g leaf and 0.032 mg/g leaf in interspecific hybrid F1 and amphidiploid, respectively. In addition, the content of total flavonoid and total lignin was higher in the amphidiploid than interspecifc hybrid F1. In addition, the amount of flavonoid and lignin was 0.26 mg/g leaf and 0.63 mg/g leaf, respectively in interspecific hybrid F1 and 0.41 mg/g leaf and 1.03 mg/g leaf, respectively in amphidiploid. Thus, it was found that amount of eugenol and methyleugenol was higher in interspecific hybrid F1 but the amount of total flavonoid and total lignin was higher in amphidiploid. Several transcripts corresponding to 16 enzymes directly involved in the general phenylpropanoid biosynthesis pathway were identified and analyzed to address the question of differential biosynthesis of phenylpropanoids, lignin and flavonoids in the interspecific hybrid F1 and amphidiploid (Table 3). Among these enzymes, PAL, C4H and 4Cl are mandatory enzymes catalyzing the initial three steps of the phenylpropanoid pathway, while HCT, CCR, COMT, CCoAOMT and CAD are downstream enzymes directly involved in the biosynthesis of lignin in plants. These enzymes including PAL, C4H and 4CL belong to multigene family and hence, more than one transcript for these enzymes was identified. Similarly, for EGS and EOMT belonging to small gene families responsible for the production of eugenol and methyleugenol, respectively in Ocimum, only three transcripts for EGS and one transcript of EOMT were identified in the transcriptome. CHS is the first enzyme of flavonoid biosynthesis producing the first flavonoid naringenin chalcone with the involvement of 4-coumaroyl-CoA and three molecules of malonyl-CoA and CHI, the key enzyme of flavonoid biosynthesis pathway catalyses intramolecular cyclization of naringenin chalcone into naringenin. About 6 transcripts of CHS and 7 transcripts of CHI were identified in both interspecific hybrid F1 and amphidiploid. Reduction of dihydroflavanols at position 4 is catalyzed by enzyme belonging to DFR superfamily. About 14 and 15 transcripts of DFR were detected in interspecific F1 and amphidiploid, respectively. Likewise, 8 and 7 transcripts of F3’H and only 1 transcripts of F3’5’H and F3H were recorded in interspecific F1 and amphidiploid. About 4 transcripts of UFGT enzyme responsible for converting anthocyanidin to anthocyanin were found in the transcriptome sequences of interspecific F1 and amphidiploid. But the transcripts of CCoA-3H,F5H,CAAT and ANS/LDOX could not be annotated. The means of FPKM values and the values of differential gene expression of 184 common transcripts based on the above identified transcripts were used to correlate the trend of content change of phenylpropenes with its gene expression (Fig. 8).
The average of FPKM values and differential gene expression (log2 fold change -0.1>=<0.1) showed that the expression of four crucial genes (HCT, CCR, COMT and CAD) directly involved in the biosynthesis of lignin were up-regulated in amphidiploid but down-regulated in interspecific F1 hybrids. Conversely, expression of PAL, C4H, 4CL, EGS, EOMT gene were highest in the interspecific hybrid F1 while the expression of genes involved in flavonoid biosynthesis (CHS, CHI, F3’H, DFR and UFGT) were down-regulated in interspecific hybrid F1. To validate the gene expression profile of RNAseq Data, qPCR of seven genes involved in general phenylpropanoid biosynthesis was performed (Fig. 9). The result of qPCR correlates the decreased expression of COMT, CAD, CHS, DFR genes involved in lignin and flavonoid biosynthetic pathway and increased expression of PAL, 4CL, and EGS in interspecific hybrid F1 confirming the RNAseq data.
Identification of candidate DEGs involved in higher stomatal density in interspecific hybrid F1 and larger stomatal size in amphidiploid
Higher stomatal density in interspecific hybrid F1 and larger stomatal size in amphidiploid are the two peculiar characteristics which were observed through anatomical analyses in mature leaves. For this reason, 172 candidate DEGs (log2 fold change -0.1>=<0.1) putatively associated in the stomatal patterning and development were identified (Table S8). The means of FPKM values and DEGs of the identified genes showed that most of the identified genes including TOO MANY MOUTHS (TMM) which promotes cell fate progression in stomatal development, EPIDERMAL PATTERNING FACTOR 4 (EPF 4); a negative regulator of stomatal development, receptor like proteins such as CLAVATA1/ CLAVATA1-like, CLAVATA3/ CLAVATA3-like receptor kinases, ERECTA/ ERECTA-like, various type mitogen-activated protein kinase (MAPK) such as MAPK9/MAPK9-like, MAPK15/MAPK15-like, MAPK19/MAPK19-like, MAPK20/MAPK20-like, mitogen-activated protein kinase homolog MMK1-like, mitogen-activated protein kinase homolog NTF3, mitogen-activated protein kinase kinase kinase MAPKK2/MAPKK2-like, MAPKK5/MAPKK5-like, MAPKK6/MAPKK6-like were down regulated in interspecific hybrid F1 and up- regulated in amphidiploid. Besides this, MAP Kinase Kinase Kinase YODA (YDA) which act as a molecular switch in stomatal patterning and development were also negatively regulated in the interspecific hybrid F1 and positively regulated in amphidiploid. In contrast, EPIDERMAL PATTERNING FACTOR-like protein 9 (EPF9)/Stomagen and subtilisin-like protease SDD1, which act a positive regulator of stomatal density and patterning were up-regulated in interspecific hybrid F1. Additional ly, leucine-rich repeat receptor-like protein CLAVATA2, mitogen-activated protein kinase MAPK4/MAPK4-like, MAPK7/MAPK7-like, MAPK10/MAPK10-like, MAPK16/MAPK16-like, mitogen-activated protein kinase homolog NTF4-like, mitogen-activated protein kinase homolog MMK2-like, mitogen-activated protein kinase kinase kinase MAPKKK1/MAPKKK1-like, MAPKKK3/MAPKKK3-like etc were also up-regulated in interspecific hybrid F1. These results suggests that these differentially expressed genes (DEGs) in interspecific hybrid F1 and amphidiploid are involved in the higher stomatal density and larger stomatal size in interspecific hybrid F1 and amphidiploid, respectively.
Analysis of putative transcription factors involved in the altered phenotype of interspecific F1 and its improvement in amphidiploid
TFs play an important role in the growth and development of all organisms. In the present work, about 1,504 transcripts from interspecific hybrid F1 and 1,537 transcripts from amphidiploid were identified as TFs. These were categorized under 58 families as per plant transcription factor database PlantTFDB 4.0 (http://planttfdb.cbi.pku.edu.cn/) . Of these, MYB and MYB- related transcription factors were found to be most abundant TF family, followed by WRKY, AP2/ERF and NAC. Total 8 TFs were related to bZIP superfamily which is reported to be associated in various biological processes such as flower and vascular development, embryogenesis, organ differentiation, seed maturation. TFs which were annotated to have transcription factor activity but do not fall in any of the families as classified by plant transcription factor database PlantTFDB 4.0 were specified as ‘other’ (Fig.10). The differential gene expression (log2 fold change -0.1>=<0.1) and average FPKM values indicated highest number for MYB and MYB related super-family (35) followed by WRKY (33), NAC (17), MADS-BOX (14) and ethylene-responsive transcription factor (13) which are differentially expressed between interspecific hybrid F1 and amphidiploid. Also, a large number of TFs in WRKY (25) superfamily, MYB and MYB- related superfamily (22), NAC superfamily (12), MADS-BOX (7) and ethylene-responsive transcription factor (7) were down-regulated in interspecific hybrid F1 and up-regulated in amphidiploid. All the differentially expressed TFs were summarized according to their probable involvement in the floral development, leaf development, trichome development, seed development and xylem formation. Many other TFs putatively showing their role in different metabolic pathways such as chlorophyll biosynthesis/ catabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis and anthocyanin biosynthesis were also analyzed and listed (Table S9). In addition, many TFs involved in growth and developmental processes such as flowering time (MADS7, SOC1, FAR1-RELATED SEQUENCE 8 and 6 –like, CO-LIKE 9 etc), floral organ development (AP2, GAMYB, AGL9, SPL11, AGL8), cell expansion in leaf and shape (GRF1-interacting factor 2-like, GRF1-interacting factor 3, BEL1-like homeodomain protein 4), seed development (NAC025, bZIP11, DOF3.7-like) and xylem formation (NAC07, MYB48, MYB 86, GATA 12-like) were down-regulated in interspecific hybrid F1 and up-regulated in amphidiploid. Similarly, many TFs related to the repression of phenylpropanoids and regulation of flavonoids (NAC078, MYB3-like, MYB4) also showed down-regulation in interspecific hybrid F1. TFs involved conferring flowering time delay (RAP2-7), trichome development (MYB-like transcription factor ETC3, trihelix transcription factor GTL1, WRKY 44-like) were up-regulated in interspecific hybrid F1 and down-regulated in amphidiploid. Additional ly, the TFs having their contribution in chlorophyll degradation (NAC029, NAC100-like) and anthocyanin biosynthesis (MYB10) were negatively regulated in the interspecific hybrid F1 and positively regulated in amphidiploid. Thus, the expression of these TFs specifies their role in the altered phenotype of interspecific hybrid F1 and amphidiploid.