RNA-Sequencing based gene expression landscape of guava cv. Allahabad Safeda and comparative analysis to colored cultivars

doi:10.21203/rs.2.22915/v1

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Research article

RNA-Sequencing based gene expression landscape of guava cv. Allahabad Safeda and comparative analysis to colored cultivars

https://doi.org/10.21203/rs.2.22915/v1

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published 15 Jul, 2020

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Background

Guava ( Psidium guajava L.) is an important fruit crop of tropical and subtropical areas of the world. Genomics resources in guava are scanty. RNA-Seq based tissue specific expressed genomic information, de novo transcriptome assembly, functional annotation and differential expression among contrasting genotypes has potential to set the stage for the functional genomics for traits of commerce.

Results

Development of fruit from flower involves orchestration of myriad molecular switches. We did comparative transcriptome sequencing on leaf, flower and fruit tissues of cv. Allahabad Safeda to understand important genes and pathways controlling fruit development. Tissue specific RNA sequencing and de novo transcriptome assembly using Trinity pipeline provided us the first reference transcriptome for guava consisting of 84,206 genes comprising 279,792 total transcripts with N50 of 3,603 bp. Blast2GO assigned annotation to 116,629 transcripts and PFam based HMM profile annotated 140,061 transcripts with protein domains. Differential expression with EdgeR identified 3033 genes in Allahabad Safeda tissues and 68 genes among colored tissue comparisons. Mapping the differentially expressed transcripts over molecular pathways indicate significant hormonal changes during fruit development. Comparisons of red vs green peel in guava cv. Apple Colour, white pulp vs red pulp in Punjab pink and fruit maturation vs ripening in non-coloured Allahabad Safeda indicates up-regulation of ethylene biosynthesis accompanied to secondary metabolism like phenylpropanoid and monolignol pathways.

Conclusions

Benchmarking Universal Single-Copy Orthologs analysis of de novo transcriptome of guava with eudicots identified 93.7% complete BUSCO genes. In silico differential gene expression among tissue types of Allahabad Safeda and validation of candidate genes with qRT-PCR in contrasting colour genotypes promises the utility of this first guava transcriptome for its potential of tapping the genetic elements from germplasm collections for enhancing fruit traits.

Epigenetics & Genomics

Guava RNA-Seq

Fruit development and ripening

Allahabad Safeda

Punjab Pink

Apple Colour

Secondary metabolites

Candidate genes for fruit colour

Guava (Psidium guajava L.) fruit is a berry with edible pericarp tissue as flesh and has excellent antioxidant properties [1]. Guava is member of family Myrtaceae (possesses ~ 150 species) and has 2n = 22 chromosomes with a genome size of ~ 450 MB [2, 3]. Guava popularly known as ‘Apple of the Tropics’ is a native of tropical America from where it is distributed in all tropical and subtropical areas of the world [4, 5]. India, Mexico, Pakistan, Taiwan, Thailand, Colombia, Indonesia are major producers of guava and a small-scale plantation is done in Malaysia, Australia and South Africa [6].

Fruiting branches in guava bear three terminal flower buds and the central floral bud develop faster into fruit compared to other two lateral buds. In Northern India subtropics, there are two flowering seasons viz. April - May and August – September with peak anthesis time of flower bud between 5:00–7:30 AM. Guava flowers are hermaphrodite and carry 160–400 bilobed anthers and an ovary which is inferior, syncarpous with axile placentation and subulate terminal style [6]. Style being longer than filaments, self-pollination is less common and domestic honeybee (Apis mellifera) is the chief pollinator [7]. There are more than 400 guava cultivars grown around the world with variation in fruit pulp and peel colour. Fruit pulp colour ranges from white to deep pink and fruit skin turns green to yellow or red upon ripening and this character varies among cultivars and depends upon the season [7].

Guava is India’s fourth most important fruit crop after mango, banana, citrus and is popularly known as poor man’s apple because of low cultivation cost and high nutritive value. Guava possesses large quantities of vitamin C [6], is rich source of phenolic compounds [8] and carries secondary metabolites with medicinal properties [9, 10]. Guava intake induces resistance against infectious agents such as Staphyloccocus, scavenge cancer causing free radicals and helps in the structural protein, collagen synthesis which maintains integrity of blood vessels, skin, organs, and bones [11]. Guava fruit also contains reducing sugars, indigestible lignin fiber and carotenoids that increases as the fruit ripens [12] with major cell wall hydrolyzing enzymes like polygalacturonases, cellulases and starch hydrolyzing α-, β-amylases [13].

Coloured fruits are preferred by the consumer owing to higher nutraceutical properties. Colour in fruits and vegetables are controlled by secondary metabolism pathway genes mainly phenylalanine ammonia-lyase, chalcone synthase, dihydro- flavonol 4-reductase, flavanone 3-hydroxylase, UDP-glucose:flavonoid 3-O-glucosyltransferase, anthocyanidin synthase and MYB, bHLH and WD 40 domain transcription factors [14–19]. However there exists enormous gene sequence variation among species that generating consensus sequence-based markers and validation is labour intensive and non-targeted. Developing new coloured genotypes with desirable agronomic traits by hybridization without marker assisted selection for colour related genes based on sequence homology from other species is a time-consuming process. So, generating expressed genic sequence information at genome wide level is important to expedite gene cloning and tapping in colour trait controlling loci from agronomically less preferred coloured guava cultivars (owing to low yields and/or lesser shelf life). Tissue specific comparative gene expression within a genotype and comparison to contrasting genotypes by RNA-Seq is an alternate targeted approach in the absence of gold standard genome assembly.

To generate a global gene expression landscape in guava we generated RNA-Seq libraries from leaf, flower buds and fruit tissue of green skinned/white pulp table purpose guava cv. Allahabad Safeda (AS). In cultivar Apple colour (AC) fruit peel colour changes from green to apple colour (reddish) at fruit picking stage and peel becomes leathery within 3–5 days in winter season. Red pulp cv. Punjab Pink (PP) is commercially grown for red nectar and the colour develops during maturation process (immature fruits have white pulp) probably owing to the chromoplast development as found in other similar genotypes [20]. Comparative RNA-seq of leaf, flower and fruit at various developmental stages of AS, red vs green peel of AC and pink pulp of PP vs white pulp of AS in current study enhances our understanding of colour development in guava and identifying important colour controlling candidate genes. Most importantly this study provides the first de novo transcriptome of guava setting a stage for guava genomics at genome wide scale.

We have developed the first de novo reference transcriptome assembly of guava, performed gene annotations, compared different fruit development stages to understand molecular pathway in fruit ripening (Fig. 1 - methods & Fig. 2) and compared 3 different genotypes with variable coloration in pulp and fruit skin/peel to understand the fruit colour development pathway in guava. AS is the widely grown table purpose guava genotype of India and has green foliage. Figure 1A shows that the flower buds at all the growth stages of AS are green in color, with white coloured flower petals. The immature and mature fruits both have white pulp and green skin. During ripening fruit skin turns yellow within 3 days after harvesting and stays yellow thereafter. PP has darker green foliage (Fig. 1B) and flower buds compared to AS. Although pulp colour in PP is white in immature fruit but turns pink in mature fruit (Fig. 1B). AC has green foliage (Fig. 1C), green flower buds, white flowers and white pulp of immature and mature fruits but the skin of fruit changes its colour from green to crimson red (apple color) at maturation within 3–5 days in winter season (Fig. 1C). We have compared the RNA-Seq (methods) of Allahabad Safeda LSt, MFb and MFr and fruit growth stages ImF, 0DF, 3DF, 7DF to understand the landscape of molecular changes in fruit development of guava. To identify inducible genes resulting into apple color development in colored genotypes, we compared red vs green skin of AC and 0DF of AS vs PP.

RNA-Seq data generation, de novo transcriptome assembly and annotation

The pair end libraries from different tissue types of AS, AC and PP were sequenced and 137.3, 20.24 and 20 million raw reads of 100 bp each were generated, respectively (Table 1). The low quality sequences were filtered at quality score ≥ 30 and 120 million high quality reads of AS belonging to 13 libraries were used for generating de novo reference transcriptome using Trinity assembler [21, 22]. A total of 2,79,792 transcript belonging to 84,206 components/genes with N₅₀ of 3603 bp were obtained (Table 2). Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis [23] with eudicots identified 93.7% (1987/2121) complete BUSCO genes, 4.5% (95) fragmented orthologs and 1.8% (39) orthologs were missing ( Figure S1). Blast search against the nr protein database identified homologs for 219,924 transcripts. Protein family search identified 140,061 protein family domains. Gene ontology assessment with Blast2GO assigned gene ontology terms to 116,629 transcripts (Table 2; Data S1), where biological process consists of 87,954 transcripts, cellular components consist of 82,820 and molecular function consist of 96,308 transcripts (Fig. 3, Figure S2).

Table 1

Description of RNA-Seq paired-end data through Illumina high-throughput sequencing
Genotype	Tissue type	Raw reads (Millions)			Total Reads (Millions)
		Replicate 1	Replicate 2	Replicate 3
Allahabad Safeda	LSt	11.92	13.93	11.57	137.3
	MFb	9.05	8.47	9.60
	MFr	8.95	10.88	8.86
	ImF	12.0
	0DF	10.34
	3DF	10.23
	7DF	11.50
Apple Colour	Green Peel	10.35			20.24
	Red Peel	9.89			20.24
Punjab Pink	ImF	9.66			20.0
	0DF	10.34			20.0

Table 2

Allahabad Safeda transcriptome assembly statistics
Transcriptome Assembly
Contigs/Transcripts	279,792
Components/Genes	84,206
% GC content	43.08
Contig N50	3,603
Assembly length (MB)	647.4
Functional annotation
Transcripts with homologs	219,924
Match with predicted protein	9,958
Match with hypothetical protein	7,790
Protein Family annotation
Transcripts with Pfam domains	140,061
Gene Ontology Annotation
Transcripts with assigned GO terms	116,629
Biological Processes	87,954
Cellular Component	82,820
Molecular Function	96,308

Differential Expression analysis of Leaf, Flower and Fruit of Allahabad Safeda

In AS, 2,777 transcripts representing 2,139 genes were found differentially expressed in mixed fruit vs mixed flower buds, mixed fruit vs leaf & shoot tip and mixed flower bud vs leaf & shoot tip (Data S2). Clustering analysis shows a high correlation among the replicated samples > 0.96 for LSt, > 0.93 for MFb and > 0.97 for MFr (Table S1; Fig. 4A).

We identified 2,125 differentially expressed transcripts (DETs) in MFr compared to LSt, with 971 being up and 1,154 down regulated. In MFr and MFb comparison, 1445 DETs were found, of which 719 were up-regulated and 726 down regulated. A least number of 660 DETs were identified between MFb and LSt, with 447 up and 213 down-regulated (Data S2). Only 33 transcripts among the DETs were common among the three tissues types (Fig. 4B). In order to identify genes involved in fruit development, top 20 up-regulated transcripts were selected from MFr comparison to LSt and/ or MFb and 7 genes were found common with > 10 Log2FC. Interestingly, putting together transcripts of these two comparisons none of the gene was found down regulated (Table S2).

The most up-regulated gene (comp27411_c1) represented by six transcripts Hydroxycinnamoyl CoA shikimate (quinate hydroxycinnamoyltransferase, HCT) belonging to BAHD family of acyl-CoA-dependent acyl-transferases controls lignin [24, 25] and cutin biosynthesis [26]. Cinnamyl alcohol dehydrogenase (CAD) important for lignin biosynthesis [27, 28], expansins involved in cell wall loosening [29], ABC transporter encoding ATP dependent channels [30], Palmitoyl transferase involved in fatty acid oxidation [31], 1-aminocyclopropane-1-carboxylate oxidase (ACO) an ethylene biosynthesis gene [32], Subtilisin-like protease with a role in plant-pathogen interactions [33], 9-cis-epoxycarotenoid dioxygenase (NCED) a major ABA biosynthesis gene [34, 35] and Rbcx, a Rubisco assembly chaperon [36] are the top protein families represented by up-regulated transcripts in guava fruit (Table S2).

Metabolic Pathway Analysis of Fruit Tissue in Comparison to Leaf and Flower

The metabolic and regulatory pathway analysis of fruit, the major sink in comparison to strongest source, the leaf was performed with MAPMAN software [37] (http://mapman.gabipd.org) with all DETs at FDR < 0.001. A total of 2125 transcripts were found significantly regulated in fruit compared to leaf (Fig. 5 and Data S2). General metabolism shows that transcripts involved in light reactions, C3 cycle, photosynthesis, tetrapyrole pathway (controlling chlorophyll biosynthesis), starch synthesis, amino acid biosynthesis except phenylalanine (input for secondary metabolism), lipid degradation, Raffinose biosynthesis, cell wall associated leucin rich repeat and arabinogalactan-proteins are down-regulated in fruit. Importantly sucrose biosynthesis, gluconeogenesis, conversion of starch to reducing sugars like glucose and fructose, wax biosynthesis, phenylalanine generation, glycolipid synthesis (for generating mono and di galactosyl diacylglycerol for food reserve storage in seeds), cellulose synthesis, trehalose biosynthesis, mitochondrial electron transport chain, cell wall degradation pectate lyases (PeLs) and PGs are up-regulated in fruit. Discreet furcation of these pathways in fruit tissue are in general concordance with its biological role of alluring birds for seed dispersal [16]. However, pectin esterases involved in plant cell wall modification and subsequent breakdown and long chain fatty acid biosynthesis genes catalyzing the cutin synthesis exhibit a mixed response (Fig. 5A). Regulation overview (Fig. 5B) with MAPMAN shows that most of the transcripts mapping to ABA, ethylene, cytokinin, gibberellin and salicylic acid signal transduction pathways were up-regulated whereas Jasmonate, Auxin, and Brassinosteroid were down-regulated with few transcripts showing up-regulation.

In ethylene pathway the ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate synthase (ACS), ACC oxidase 3, Ethylene receptor 2 (ETR2) homolog of Arabidopsis, ethylene response factor ERF-1, basic helix-loop-helix (bHLH) TF and pyridoxine biosynthesis gene PDX1.2 were found up-regulated. ABA biosynthesis and signaling factors including NCED and ABA binding factor 4 (ABF4), B3 domain containing high-level expression of sugar-inducible gene 2 (HSI2), highly ABA-induced 1 (HAI1), hypostatin resistance 1 (HYR1), a UDP glycosyltransferase (UGT) and GRAM domain family protein were highly up-regulated, only ABA-responsive TB2/DP1 (HVA22 family protein) showed down-regulation. Auxin leucine-rich repeats (LRR), F-box TIR receptor, TCP family, ARF and AUX/IAA transcription factors were found down regulated indicating the auxin signaling down-regulation in guava fruit development. Interestingly, Brassinosteroid insensitive (BRI) encoding receptor kinase is up-regulated indicating overall up-regulation of BR signal transduction and responses.

We identified forty transcription factor families with multiple transcripts belonging to MYB, MADS, HB, WRKY, ARF, bHLH, AP/EREBP, bZIP, NAC, AUX/IAA, B3, Jumonji, Polycomb. These families showed both up and down regulation, indicating their importance in modulation of fruit development. Sucrose cytosolic invertase 2 (CINV2), responsible for conversion of sucrose to monosaccharides like fructose and glucose showed up-regulation and is in line with increase in sucrose catabolism in developing fruits.

Cellular response (Fig. 5C) depicts down regulation of transcripts belonging to biotic stress and is in line with fruits being more prone to pathogen and insect damage in comparison to leaves. Phytoene synthase (PSY) and lycopene beta cyclase (lcy-b) responsible for accumulation of α and β- carotene shows over-expression indicating up-regulation of carotenoid biosynthesis pathway. Ubiquitin and autophagy dependent degradation pathways (Fig. 5D) shows up-regulation of 44 transcripts, indicating increased protein turnover process. Near similar results were obtained in a comparison of fruit vs flower transcripts (Data S2).

Up-regulation Of Secondary Metabolites During Fruit Ripening

We compared RNA-Seq at different fruit maturity and ripening stages in AS. Comparison of mature fruit 0DF to ImF identified 220 differentially regulated transcripts, with 75 showing up-regulation and 145 showing down regulation (Data S3). However, at ripening 3DF vs 0DF 366 transcripts were differentially regulated with 232 up-regulated and 144 down-regulated (Data S4). Interestingly, during over-ripening 7DF vs 3DF only 11 transcripts showed differential regulation with only one down regulated (Data S5). The major up-regulated genes in 0DF vs ImF (Data S3; Figure S3) include Alpha-Expansin, cellulose synthase, phospho-enol-pyruvate carboxylase kinase, β-amylase, PSY, CAD and COMT family of lignin biosynthesis genes and other o-methyl transferases. However, flavonoid pathway genes other than lignin biosynthesis, pectin methyesterases, light reactions, calvin cycle and photorespiration were down-regulated.

In 3D vs 0D (Data S4, Figure S4 A) there is upregulation of transcripts for cellulose synthase, expansins, increased fatty acid synthesis and elongation, PSY, phenylalanine biosynthesis genes arogenate dehydratase, flavonoid biosynthesis related transcripts like UGT - Hypostatin Resistance 1 (HYR1), Flavonoid 3',5'-hydroxylase 2 (F3′5′H), Phenylalanine ammonia-lyase 3 (PAL 3) and 4-coumarate–CoA ligase 2 (4CL2). All the transcripts belonging to ABA, brassinosteroid, ethylene, cytokinin, and salicylic acid were up-regulated, while AUX and TCP transcripts related to auxin and gibberelic acid Insensitive (GAI) were down-regulated (Data S4, Figure S4 B). Transcripts corresponding to TF families of WRKY, AP2, bHLH, PHOR1 (ubiquitin ligase activity), MYB and C2C2.CO like were up-regulated. Ubiquitin and autophagy dependent protein turnover pathway were found up-regulated (Data S4, Figure S4 C).

Apple color in fruit skin is derived from up-regulation of secondary metabolism

Apple colour skin of AC develops in a short time period of ~ 3–5 days during winter season during fruit maturation. Comparison of FPKM value of transcript belonging to red vs green skin, identified only 52 DETs indicating very specific pathways involved in fruit colour development. Interestingly, all the transcripts of phenylpropanoid and lignin pathway showed up-regulation indicating the colour development in the skin of guava is result of over expression of phenypropanoid and lignin biosynthesis pathway (Figure S5; Data S6). Comparison of PP mature 0DF with AS 0DF identified 19 DETs with 9 up-regulated and 10 down-regulated (Data S7). However, only omega-hydroxypalmitate O-feruloyl transferase-like indicated a footprint of secondary metabolism pathway.

Fruit colour development in coloured guava genotypes is concomitant with ripening process

We hypothesized that pink to reddish fruit skin or pulp colour development may share the fruit ripening related genes as the colour development in apple colour skin or pulp is concomitant with ripening process. The cluster analysis of DETs in AS ImF vs 0DF, 3DF vs 0DF, 7DF vs 3DF, PP ImF vs 0DF, and AC_RP vs AC_GP was carried out. AS mature fruit, PP mature fruit, AC green peel and AC red peel cluster in single group of mature fruit stages. AS 3DF and AS 7DF made another group with mostly similar expressions and 11 DETs. Third cluster consisted of AS ImF and PP ImF, again a result of similar fruit stage (Figure S6; Data S8).

qRT-PCR for two well-known candidates for coloration in fruits and vegetables PAL and PSY 2 was carried out to see the reproducibility of differential FPKM expression values. Expression of PAL in AS fruit was maximum at 3DF green to yellow peel colour turning stage (~ 9.5X compared to ImF_AS; Fig. 6A). However, expression in 0DF_ PP (pink pulp) is ~ 3X higher compared to ImF_AS. Interestingly, expression in AC_RP was found the highest (~ 1.5 X compared to 3DFr_AS). These results indicated that the expression of PAL increases with ripening, but is also genotype dependent and might have contribution towards red coloration in peel of apple colour genotypes. However, PSY 2 expression (Fig. 6B) shows a general trend of increase in expression with maturity and ripening in all the three genotypes. These results also indicated that observations recorded in our comparative transcriptomic data are in line with qRT-PCR analysis.

Candidate Genes For Colour Development In Guava

To identify the genes involved in colour development, GO enrichment of DETs was performed (Figure S7). For the potential candidates FPKM values were compared in AC_RP vs AC_GP and 0DF PP vs 0DF AS for all the tissue types. The FPKM value showed higher expression of genes at colour turning stages (Table 3). Interestingly, the FPKM comparative analysis indicated reticulin o-methyltransferase (responsible for converting alkaloid reticulin to laudanine) as top candidate gene. The moderate expression of transcripts was present in green and maturing fruits of AS. However, huge increase in expression of reticulin o-methyltransferase in red peel of AC and mature PP fruit suggested increased levels of such alkaloids in coloured fruit tissues. Maximum FPKM expression value of glycerol-3-phosphate acyltransferase 5 (GPAT 5), peamaclein, CTP synthase-like, chloroplastic monodehydroascorbate (MDA), probable 2-oxoglutarate-dependent dioxygenase AOP1 (2OG-AOP1) and methionine synthase (MS) corresponding transcripts in red peel of guava suggested more candidate genes for red coloration in peel of guava. Higher expression of transcripts for Secoisolariciresinol dehydrogenase (SDH), BEL1-like homeodomain 1(BLH1) in PP indicates additional candidates for red colour in pulp of guava.

Three transcripts (R, S)-reticuline 7-O-methyltransferase-like (comp25759_c1_Seq1, comp25759_c1_Seq5 & comp25759_c1_Seq11) showed high homology, so common primers were designed for qRT-PCR. Results show that expression of (R, S)-reticuline 7-O-methyltransferase Like (RML) transcripts increases to 100X at 0DF_AS compared to ImF_AS set to unity (Fig. 6C). However, expression did not change in 3DF_AS compared to 0DF_AS and reduced to 20X in 7DF_AS. Interestingly, expression in ImF_PP is ~ 3X compared to ImF_AS and increases to ~ 300 X in 0DF_PP. Expression in AC_GP is ~ 21X but show an increase to ~ 300X in AC_RP, almost to similar levels as in 0DF_PP suggesting RML, a candidate for coloration in both guava peel and flesh. Surprisingly, expression of RML_ comp25759_c1_seq4 (Fig. 6D) stayed at low level in AS and PP at all the stages, however expression is ~ 30X in AC_GP and reaches to 80X in AC_RP. In AC this result indicated genotype specificity of RML members. High expression of RML family members emphasizes their role in color development in guava. High expression of G3PAT, SDH and AOP1 in AC_RP indicates the additional candidates for colour development in guava (Fig. 6). In general, qRT-PCR for all these candidates supported our transcriptome based FPKM results.

Table 3

FPKM value of top differentially regulated transcripts of red peel vs green peel of Apple Colour (AC) and Punjab Pink (PP) mature red fruit vs Allahabad Safeda (AS) mature fruit in development stages of AS, PP and AC
Transcript ID	Description	Genotypes
		Allahabad Safeda						Apple Colour		Punjab Pink
		LSt Avg ^$(S.E)	MFb Avg ^$(S.E)	ImF	0DF	3DF	7DF	GP	RP	ImF	0DF
AC peel Red vs Green up-regulated
comp25759_c1_seq1	(R,S)-reticuline 7-O-methyltransferase-like	0.5 (0.3)	0.3 (0.3)	2.3	79.7	13.9	21.7	2.2	828.6	0.0	402.8
comp25759_c1_seq4	(R,S)-reticuline 7-O-methyltransferase-like	0.0 (0.0)	10.5 (2.2)	0.0	12.3	3.7	3.2	1.5	183.3	0.0	88.8
comp25759_c1_seq5	(R,S)-reticuline 7-O-methyltransferase-like	0.2 (0.2)	2.5 (0.5)	0.0	66.1	16.5	24.6	5.2	382.1	0.0	220.4
comp25759_c1_seq11	(R,S)-reticuline 7-O-methyltransferase-like	0.1 (0.1)	0.4 (0.2)	0.0	22.7	19.7	1.6	2.4	111.6	0.0	63.9
comp14631_c0_seq1	glycerol-3-phosphate acyltransferase 5	25.6 (0.8)	1.2 (0.1)	0.3	0.6	1.0	8.4	0.6	34.2	0.4	0.0
comp12564_c0_seq1	peamaclein	71.8 (9.2)	57.8 (0.8)	2.6	18.3	34.5	46.9	4.3	130.3	1.8	18.4
comp22486_c0_seq10	CTP synthase-like	0.4 (0.4)	0.0 (0.0)	0.0	3.1	6.3	0.0	0.7	18.0	0.0	0.0
comp26385_c2_seq73	monodehydroascorbate chloroplastic	6.4 (1.4)	3.5 (2.8)	3.2	18.9	1.0	26.9	5.5	69.4	1.2	28.6
comp26017_c0_seq1	probable 2-oxoglutarate-dependent dioxygenase AOP1	38.3 (1.6)	97.4 (4.0)	3.3	1.9	8.7	57.9	11.8	150.5	2.4	2.64
comp23125_c0_seq2	methionine synthase	87.7 (3.8)	168.3 (4.0)	227.5	128.0	382.3	43.6	60.0	723.8	98.4	298.5
PP 0DF vs AS 0DF Up-regulated
comp28595_c0_seq1	Secoisolariciresinol dehydrogenase	67.8 (3.1)	54.9 (2.1)	6.2	1.3	21.4	42.1	302.9	1106.1	1.3	66.8
comp17046_c0_seq2	BEL1-like homeodomain 1	0.2 (0.2)	1.4 (1.0)	0.0	2.8	16.3	13.1	11.7	27.1	26.3	46.2
comp22451_c2_seq1	1-aminocyclopropane-1-carboxylate oxidase 1-like	0.2 (0.2)	5.8 (1.6)	0.0	43.1	190.4	103.7	35.5	10.5	0.0	551.5
comp27248_c1_seq24	PREDICTED: uncharacterized protein LOC104449412	2.6 (1.4)	3.5 (1.8)	5.0	4.1	6.9	0.0	15.9	6.8	2.5	51.4
$ For S.E. n = 3

Fruit ripening and maturation involves ABA, Ethylene and secondary metabolite up-regulation

Climacteric fruits show increased rate of respiration and ethylene biosynthesis which in turn triggers the activity of enzymes like PGs, PeLs, pectate methylesterases (Pme). Process of ripening is accelerated by the conversion of complex polysaccharides into simple sugars leading to increased sugar to acid ratio concomitant with textural and colour changes. Conversion of l-aminocyclopropane-l-carboxylic (ACC) acid from S-adenosylmethionine (SAM) is catalyzed by ACC synthase and is the rate limiting step in ethylene biosynthesis [38]. Expression value comparison in AS, PP and AC showed differential increase in transcripts corresponding to ACO 1, ACO 2, ACS and ETR2 during fruit maturation (Data S9). ABA biosynthesis and signaling is also found up-regulated during fruit maturation and ripening [39]. We observe increased expression of ABA biosynthesis gene NCED, receptor Pyrabactin resistance 1 like 9 (PYL9) and TF ABA insensitive 5 (ABI5) during fruit ripening in AS, PP and AC (Data S9). Ethylene and ABA are stress hormones and up-regulates defense system of plants against pathogens by stimulating phenylpropanoid pathway, pathogenesis-related proteins and inducing systemic resistance [40]. Expression of pathogenesis related PR-4 is high in fruit tissues with the highest expression in red peel and PR-10 in green peel.

Guava is rich in secondary metabolites. Expression of genes controlling secondary metabolites like isoflavone reductase (IFR) is maximum in immature fruits, flavanone 3-hydroxylase (F3H) is high in leaf, flower and immature fruits and flavonoid 3, 5 -hydroxylase (F3'5'H) 2-like in leaf and flower tissue. Interestingly, expression of PAL, isoflavone 2 -hydroxylase-like and flavonol synthase F3H -like are high during fruit maturation with maximum expression in ripe guava (Data S9). Hydrolysis of starch by β-amylase generates maltose leading to sweet flavor in ripened fruits [41]. Expression of β-amylase increased at 0DF and 3DF while reduced at over-ripe stage 7DF. Also, the expression is much higher in AC peel and 0DF of PP (Data S9). High expression of β-amylase underscores the sweet-smelling nature of guava at fruit ripening in general and specifically higher in AC and PP cultivars.

Synthesis of lignin monomers involve the phenylpropanoid pathway initiated by PAL and followed by Cinnamate 4-hydroxylase (C4H), 4CL, HCT, Caffeoyl CoA 3-O-methyltransferase (CCoAOMT), Cinnamoyl CoA reductase (CCR), Caffeic acid 3-O-methyltransferase (COMT) and CAD genes. Several CAD family members generally show up-regulation in the fruit flesh and respond to ABA, ethylene and various biotic and abiotic stresses as observed in melon [27]. However, expression of COMT gene is directly associated with increase in lignin content and is maximum in immature AS fruit. HCT and CAD genes control the crucial step in suberin [24, 25] and cutin biosynthesis [26]. HCT and CAD genes are most up-regulated transcripts (Table S2) in MFr vs LSt. Their expression increases during maturation and was the highest at 3DF followed by reduction at over-ripe 7DF stage indicating lignin, suberin and cutin synthesis during maturation and ripening. PGs expression increases during ripening. We also identified the highest expression of transcript corresponding to PGs at 7DF in AS, and high expression in red peel of AC compared to green peel and 0DF compared to ImF in PP (Data S9). Results of comparative transcriptome analysis in this manuscript are in concordance with protein and metabolic studies reported in different fruit species supporting that this first transcriptome of guava will set the stage for functional genomics in guava.

(R,S)-reticuline 7-O-methyltransferases is a potential candidate for colour development in guava

Transcript FPKM value and qRT-PCR (Table 3; Fig. 6) of RML, GPAT 5, Peamaclein, CTP synthase-like, chloroplastic MDA, 2OG-AOP1 and MS genes show that expression level of these are maximum in AC fruit skin begging the question if increased expression of these genes is responsible for coloured guava skin. RMLs are involved in tetra hydrobenzyl isoquinoline alkaloid production in poppy at the step of reticuline to laudenine conversion [42]. Up-regulation of 4 isomeric transcripts of RML in peel of guava suggests that red guava fruits may serve as a good source for extracting alkaloids like reticulin and laudenin rather than a much-regulated source like poppy. Although the reticulin and laudenin are extracted from poppy for medicinal purposes as an anti-diaaheral, anti-dysentric, anticough, the use of guava fruit having similar properties have been described since ancient time. BLAST search of four RML transcripts identified its homologs in eucalyptus, grape and cork oak tree with 74–86% identity. Expression of all the four RML transcripts is the second highest in PP 0DF- mature fruit (red pulp) after AC_RP - red peel (Table 3).

GPAT 5 is required for synthesis of suberin in Arabidopsis root and seed coat [43]. Increased expression of HCT and GPAT 5 indicates high suberisation occurring in fruit coat in a short duration accompanying the ripening process. Peamaclein originally identified in peach fruit, is a recently identified gibberellin induced fruit allergen conserved among various fruits [44]. Higher expression of peamaclein transcripts in red peel (Table 3) might be an indicator of high fruit allergen prevalence in red peel. CTP synthase-like is an enzyme associated with cytosine synthesis. Increased CTP cellular level in yeast has been associated with increased phospholipid synthesis via the Kennedy pathway[45]. This suggests increased levels of phospholipids during reddening of fruit skin.

MDA can be reduced to ascorbate through reactions by MDA reductase (MDAR) in chloroplast and help relieving the oxidative stress by reactive oxygen species (ROS) evolving from the photosynthesis reactions [46]. Overexpression of MDAR in tomato has been associated with stress enhanced tolerance to temperature and methyl viologen-mediated oxidative stresses [46]. High expression of chloroplastic MDA transcript indicates the release of ROS in the fruit peel during colour change. 2OG-AOP1 is involved in glucosinolate synthesis in Arabidopsis. Interestingly, 2OG is also the second largest enzyme family in plants involved in hormone and flavonoid biosynthesis [47]. Increased expression of 2OG-AOP1 is convincingly in the line of increased expression of flavonoid biosynthesis related genes during colour changing in AC.

Methionine not only acts as a building block for protein synthesis but also serve as immediate precursor of S-adenosylmethionine, a major methyl-group donor in transmethylation reactions and as an intermediate for biosynthesis of polyamines and ethylene [48]. Up-regulation of methionine synthase, an enzyme catalyzing the last step of methionine biosynthesis supports the increased demand of ethylene biosynthesis during fruit ripening in general and highest expression in red skin (Table 3; Fig. 6H).

SDH converts (2)-secoisolariciresinol into (2)-matairesinol in Forsythia intermedia, a precursor for the biosynthesis of antiviral and anticancer agent, podophyllotoxin [49, 50]. Although, SDH is identified in a comparison of AS 0DF to PP 0DF but the expression was the highest in AC red skin with > 3-fold expression increase compared to green skin (Table 3; Fig. 6G). Presence of RML and SDH high expression in red guavas indicated colored guava being a rich source of rare secondary metabolites. BLH1 have been implicated in leaf and ovule development in Arabidopsis [51] and overexpression of apple BEL1-Like MDH1 in Arabidopsis [52] leads to reduced fertility and other pleiotropic effects, however no implication in colour development are reported. RNAi of SlBEL1- like 11 in tomato increased the chlorophyll content [53] and delayed fruit ripening. Increased expression of BEL1 in PP pulp might have role in chromoplast development. Comp27248_c1_seq24 is another highly expressed transcript in PP 0DF that does not have any other functional evidence but shows 90% identity with Eucalyptus grandis uncharacterized LOC104449412.

Up-regulation of DETs in red skin of guava cv. Apple Colour implicates cross talk among myriad dynamic processes

Up-regulation of Mannan endo-1,4-beta-mannosidase (β-mannanase) indicates utilization of mannans in producing mannose oligosaccharides from lignocellulosic polysacchrarides [54]. Kinesis light chains (KLCs) are involved in microtubule mediated organelle transport in eukaryotes [55]. Up-regulation of KLC corresponding transcripts indicate high metabolic activity in skin colour change in guava. EG45-like domain proteins belong to plant natriuretic peptides (PNPs), a class of systemically mobile molecules distantly related to expansins with implication in regulating water and ions homeostasis and participation in plant immune response [56], so are in line with increased demand in leathery red skin. (-)-germacrene D synthase is involved in biosynthesis of a sesquiterpene germacrene D generally obtained from cubebe oil [57]. Golgi α-mannosidase II is a key glycosyl hydrolase in the N-linked glycosylation pathway [58], indicating N-glycosylation role in reddening of skin. E3-ubiquitin ligases catalyse final step of ubiquitin conjugation demanding tight regulation to ensure accurate substrate ubiquitylation and finally degradation via 26 S proteasomal pathway marking an important checkpoint for protein turnover [59]. Up-regulation of 2 transcripts belonging to E3 ubiquitin- ligase SHPRH in apple colour skin development indicated high turnover of specific proteins. Omega-6 fatty acid destaurases or Fatty Acid Destuarse 2 (FAD2) desaturates oleic acid to linoleic acid (18:2) for enhancing stress tolerance in endopasmic reticulum in response to abiotic stresses [60]. Omega-6 fatty acid endoplasmic reticulum isozyme 2-like up-regulation in red and leathery skin might be an indicator of better protection to biotic and abiotic stresses. Arabinogalactan peptides (AGPs) in wheat are encoded by grain softness protein genes [61]. Wheat-AGPs derived down-regulation of GSPs in RNAi lines increases the grain hardness and decreases viscosity of aqueous extracts. Such extension like activities in the reddening skin might be attributes of AGPs as Arabinogalactan peptide 13-like is up-regulated. Knock-down of FAB1A/B, 1-phosphatidylinositol-3-phosphate 5-kinase in Arabidopsis causes defect in the membrane recycling by auxin transporters [62]. FAB1 also plays an important role in endosomes maturation for mediating cortical microtubule association of endosomes [63]. Increased expression of 1-phosphatidylinositol-3-phosphate 5-kinase/FAB1D during colour change is suggestive of increased auxin transport. Hemicellulose 4-O-methyl glucuronoxylan is a major component present in the secondary cell walls of eudicots. Arabidopsis GXMT catalyzes 4-O-methylation of the glucuronic acid substituents in hemicellulose [28]. Up-regulation of GXMT 1-like indicates hemicellulose synthesis at colour change stage.

Phenylpropanoid Pathway In Coloured Guava

Anthocyanin biosynthesis genes like F3H [64], Isoflavone-2′-hydroxylase [65], Anthocyanidin 3-O-glucosyltransferase 2-like / UGT [66], 4-Coumarate:CoA ligase and PAL are up-regulated in red skin compared to green skin (Data S6). However, expression of multiple transcripts corresponding to these genes are also up-regulated during fruit ripening in AS and PP, pointing the shared phenomenon between fruit ripening and color accumulation. However, doubled expression of PAL in AC red skin compared to AS 3DF (Data S9, Fig. 6A) compels the idea that increased anthocyanin accumulation in red skin might be the resultant of PAL accumulation. PAL is the check point between primary and secondary metabolism and activity increases in response to biotic and abiotic stresses. PAL in combination with other phenylpropanoid enzymes like 4-Coumarate-CoA ligase, Chalcone synthase, Cinnamic acid 4-hydroxylase, F3H, Flavonol synthase, Stilbene synthase, Isofavone synthase, Resveratrol synthase etc. has been used in different hosts, like Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida and Streptomyces spp. to synthesize a wide range of phenylpropanoid-derived compounds like flavanones, naringenins, kaempferol, quercetin, stilbenes and many more [67]. Preliminary work of oral or subcutaneous administration of PAL to Phenyl Ketone Uric patients leading to substantial reduction of plasma L-Phe levels has been reported [67]. Increased expression of PAL in red peel indicates a potential new source of this enzyme in guava. Expression increase in PSY and ACO (Data S9; Fig. 6) during ripening in AS, PP and AC supports high correlation in colour development and maturation in guava but might need interaction with specific factors like RML / GPAT 5 / peamaclein / CTP synthase / MDA chloroplastic / 2OG-AOP1 / SDH / BLH1.

Tissue type and genotypic comparative transcriptome analysis of guava reported here corresponds to the metabolic changes expected and observed in guava and other fruit crops. The transcriptome assembly generated in this study will set the stage for functional genomics in guava, the second most important fruit crop of Northern India. Importantly, identification of candidate genes for red colour will aid in generating EST-SSR/SNP markers and help in increasing the density of existing linkage maps proving a boon for marker assisted breeding in guava.

Original source of the plant materials

Punjab Pink is a hybrid between Portugal x L-49 = F1 x Apple colour and was developed by Punjab Agricultural University, Ludhiana and released in year 2009. CISH-G5/Lalima is an apple colour selection of Central Institute of Sub-tropical Horticulture, Lucknow, Uttar Pradesh, India. Allahabad Safeda is an open pollinated seedling selection from Uttar Pradesh, India. The three cultivars were clonally propagated and raised in the mother block of Regional Fruit Research Station - PAU, Bahadurgarh, Patiala, Punjab, India. The 10 years old clonal propagated trees growing in fruit research orchards of PAU, Ludhiana, India have been used in current study.

Source of Fruits or Plant material and tissue sample collection

In the present study we used Psidium guajava L. local cultivars Allahabad Safeda (AS), Apple Colour (AC: CISH-G5) and Punjab Pink (PP) 8 years old trees growing in Orchards of PAU, Ludhiana, India. Allahabad Safeda is the most prominent cultivar grown throughout India for table purpose. AS fruits are green skinned at immature and harvesting stage (mature fruit, 0DF), starts turning yellow and skin turns completely yellow within 3 days after picking (ripe fruit, 3D) and becomes over-ripe thereafter. Foliage and flower buds of AS, PP and AC are all green however pulp of PP turns to deep pink on maturity (0DF), whereas immature fruit pulp is white. Fruits of AC turn their skin colour from green (-5DF) to reddish (0DF) in winter season (Fig. 1).

In AS, we collected actively growing immature young leaves, fully expanded mature leaves, actively growing shoot tips, flower buds at six different developmental stages from immature to mature (1 day before anthesis) and 0D opened flower, 80DPA (immature fruit) with seeds and without seed (ImF), fully mature ready to harvest fruit tissue with seeds and without seeds (0DF), fruit tissue 3 days after harvesting without seeds (3DF) and fruit tissue 7 days after harvesting without seeds (7DF) in three replicates. All the tissues were flash frozen in liquid N₂ and stored in -80 °C before proceeding to RNA extraction. PP immature fruit (80 DPA) without seed (ImF_PP), mature fruit without seed (0DF_PP) and AC green peel (AC_GP, -5D fruit) and red peel (AC_RP, 0D fruit) were also harvested, similarly.

Rna Extraction

Total RNA was extracted from all the tissues using Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) followed by on-column DNase I digestion (Sigma-Aldrich) for removing DNA contamination. RNA integrity was analysed on 1.2% agarose denaturing gel as described before [68]. High quality RNA from different tissues was pooled in equimolar ratios to reduce the number of libraries to be sequenced. Briefly, total RNA extracted independently from immature young leaves, fully expanded mature leaves and actively growing shoot tips was pooled in equal amount to make one RNA sample Leaf shoot tip (LSt), 6 flower bud stages and opened flower sample- Mixed flower bud (MFb), 80 DPA immature fruit (ImF), 0D fruit (0DF), 3D ripe fruit (3DF) and 7D overripe fruit (7DF) sample- Mixed fruit (MFr). All the fruit stage samples used to pool MFr were a slice of guava fruit containing peel, pulp and seed altogether. However, independent fruit samples ImF, 0DF, 3DF and, 7DF were also sampled with skin but without seed. So, in total 7 samples representing 7 tissue types of AS were LSt, MFb, MFr, ImF, 0DF, 3DF and 7DF. For PP and AC we extracted 2 RNA samples each ImF_PP & 0DF_PP and AC_GP & AC_RP, respectively.

Library Preparation, Rna Sequencing And Reference Assembly Generation

Bioanalyzer (Agilent) was used to asses RNA quality before preparing RNA-Seq libraries. Libraries were constructed with ribo-zero treated RNA from 3 biological replicates of AS LSt, MFb, MFr and a single sample for AS ImF, 0DF, 3DF, 7DF, ImF_PP, 0DF_PP, AC_GP & AC_RP. RNA had RIN value ≥ 8.3 and TruSeq Stranded RNA Library Prep kit (Illumina) was used with an insert size of ~ 300 bp. The paired end (PE) libraries were sequenced on Illumina HiSeq2500. 100 bp high quality PE reads were generated. All the libraries were run in a single lane to avoid any discrepancies while calculating differential expression (FPKM). Read quality was assessed using FASTQC toolkit [69, 70]. Adapter and low quality sequences were trimmed at minimum PHRED quality score 30 using Trimmomatic read filtering tool [71]. de novo RNA-seq assembly was generated by pooling AS libraries of LSt, MFb, MFr, ImF, 0DF, 3DF, 7DF with Trinity transcriptome assembler [21] as no reference assembly for guava is available. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis was performed using BUSCO pipeline with eudicot model to estimate the completeness of transcriptome assembly.

Functional Annotation of de novo Assembled Allahabad Safeda Transcriptome

The assembled transcripts were annotated on the basis of corresponding homologs identified from BLASTX [72] program with search against NCBI protein “nr” database at e-value of 1e^− 3. Gene ontology (GO) terms associated with transcripts were determined using BLAST2GO program [73]. GO enrichment was done among the DETs in specific comparisons using GOseq [74]. KEGG annotations were also assigned using Blast2GO KEGG mapping. Transdecoder tool in Trinity package was used to identify longest open reading frame (ORF) and protein families were assigned by searching against the Pfam database using pfamscan [75]. Datafile with transcript ID description, GO annotation and enzyme name are provided in Data S1.

Measuring Gene Expression And Identification Of Differentially Expressed Genes/transcripts

The trimmed reads were mapped to reference transcriptome and abundance of transcripts was measured in FPKM values using RSEM estimation tool, for each sample [76]. Trimmed mean of M-values (TMM) normalization for libraries was performed with Trinity. The expected count values were used for determining the differential expression with edgeR [77] on biological replicated data. Default dispersion value of 0.1 was used to calculate the differential expression for samples with no replicates. Transcripts exhibiting ≥ 4-fold change (Log2FC) in expression and < 0.001 false discovery rate (FDR) were considered significant DETs among different tissues, developmental stages and /or genotypes.

Pathway Analysis

DETs in tissues/genotypes were put together in a fasta file and pathway annotation were determined using online Mercator analysis tools (http://mapman.gabipd.org) [78]. DETs were binned into functional categories by Mercator. MAPMAN software [37] was used for graphical representation of metabolic and signaling pathways. The results of metablolic pathway analysis described in manuscript can be reproduced by installing MAPMAN and uploading the data set in suppl. files on a computer following authors guidelines described in original manuscript [37].

Qrt-pcr Analysis

Two micro grams of RNA was reverse transcribed by reverse transcriptase (Maxima First Strand cDNA Synthesis Kit for RT-qPCR containing oligo(dT)18 and random hexamer primers; Thermo-Scientific, Surrey, UK), in a 25 µl reaction further diluted to 100 µl with nuclease free water. Diluted cDNA template (2 µL) was used for a 15 µl PCR reaction. Gene-specific primers (Table S3) for Phenylalanine Ammonia-Lyase (comp27773_c0_seq1), Phytoene synthase (comp27725_c0_seq1), (R,S)-reticuline 7-O-methyltransferase-like (comp25759_c1_seq1, comp25759_c1_seq5, comp25759_c1_seq11), (R,S)-reticuline 7-O-methyltransferase-like (comp25759_c1_seq4), Probable 2-oxoglutarate-dependent dioxygenase AOP1 (comp26017_c0_seq1), Glycerol-3-phosphate acyltransferase 5 (comp14631_c0_seq1), Secoisolariciresinol dehydrogenase (comp28595_c0_seq1), Methionine synthase (comp23125_c0_seq1), PREDICTED: uncharacterized protein LOC104449412 (comp27248_c0_seq1) and 1-Aminocyclopropane-1-carboxylate oxidase 1-like (comp27730_c0_seq1) were used to amplify cDNAs. Histone 3 (comp27670_c0_seq1) was used as internal control. qRT-PCR was performed using PowerUp™ SYBR™ Green Master Mix on a LightCycler® 480 Instrument. Fold change was calculated as described before [79]. Oligonucleotide primers (Table S3) were designed using Primer3 design (http://frodo.wi.mit.edu/).

AC:Apple Colour/CISH-G5/Lalima; AC_GP:Apple Colour green peel; AC_RP:Apple Colour red peel; AS:Allahabad Safeda; BUSCO:Benchmarking Universal Single-Copy Orthologs; DETs:Differentially expressed transcripts; FPKM:Fragments Per Kilobase Million; ImF:Immature fruit; ImF_PP:Immature fruit of Punjab Pink; LSt:Leaf and shoot tip; MFb:Mixed Flower buds tissue; MFr:Mixed stage Fruit tissue; PP:Punjab Pink; 0DF_PP:Mature Fruit of Punjab Pink; 0DF:Mature ready to harvest fruit; 3DF:3 days after harvesting fruit; 7DF:7 days after harvesting fruit.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

Trinity generated transcripts of length > 200bp submitted to NCBI Transcriptome Shotgun Assembly (TSA) has accession no. GGPP00000000. RNA-seq data of cv. Allahabad Safeda is submitted under Bioproject PRJNA472130 with 13 biosamples (SAMN09227265 - 77). Raw reads are submitted under Short Reads Archive (SRA) database for LSt (SRR7186630, SRR7186631, SRR7186633), MFb (SRR7186632, SRR7186634, SRR7186635), MFr (SRR7186629, SRR7186636, SRR7186637), ImF (SRR7186628), 0DF (SRR7186640), 3DF (SRR7186639) and 7DF (SRR7186638). Reads for Punjab Pink (PP ImF - SRR7471728 & PP 0DF - SRR7471727) and Apple Colour – CISH G5 (AC_GP - SRR7471740 & AC_RP - SRR7471739) can also be found at NCBI - SRA.

Competing interest

Authors declare no financial and non-financial competing interest.

Funding

Experiments were financially supported by initial grant to Amandeep Mittal from self-financing scheme of Punjab Agricultural University, Ludhiana and Department of Biotechnology Grant no BT/PR24373/AGIII/103/1012/2018.

Author Contribution

AM, KS and MISG conceived the idea; AM, KS, ISY, NKA and MISG planned experiments; MISG, NKA and RSB maintained and provided the guava genotypes; AM, NKA and RSB collected the plant tissue; MM and AM optimized the RNA extraction method from various tissue types & prepared RNA for sequencing; ISY and AM analyzed the RNA-Seq data and did bioinformatics analysis; AM, PC and MM planned and conducted qRT-PCR for validating the assembly and colour responsive candidate genes; WE and PK supported Bioinformatics data analysis; AM, ISY and PC wrote the paper. All the coauthors read the manuscript and approved it.

Acknowledgements

Authors are thankful to the subDIC center (BTISNET, DBT) PAU for providing computational facility. Authors thank Dr. M.R. Dinesh, Director-Indian Institute of Horticultural Research, Bengaluru, and Dr. Binay Panda, Ganit Labs, Bengaluru for useful discussions and Mr. Bhupinder Singh & Mr. Manish Jindal for their technical support.

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Additional Files

Additional file 1: TableS1. Correlation matrix values among Leaf Shoot tip (LSt), Mixed Flower bud (MFb) and Mixed Fruit (MFr) tissue samples and the replicates.
Additional file 2: TableS2. Expression of top 20 co-up regulated transcripts in Allahabad Safeda fruit tissue compared to leaf and flower with FDR <0.001.
Additional file 3: TableS3. Primer Sequences for candidate genes/internal control for qRT-PCR analysis.
Additional file 4: FigureS1. Summary of conserved orthologous genes (BUSCO) in the assembled guava transcriptome.
Additional file 5: FigureS2. Blast2GO distribution of assembled transcripts into A) Biological processes B) Cellular component C) Molecular function.
Additional file 6: FigureS3. Metabolic overview with MAPMAN analysis of differentially expressed transcripts of mature fruit (0DF) vs Immature fruit (ImF) of cv. Allahabad Safeda. Up- and Down- regulated DETs are represented with blue and red squares, respectively with log2 transformed values.
Additional file 7: FigureS4. MAPMAN analysis of differentially expressed transcripts of ripe fruit (3DF) vs mature fruit (0DF) of cv. Allahabad Safeda A) Metabolic Overview B) part of regulation overview C) proteasome and autophagy. Up- and Down- regulated DETs are represented with blue and red squares, respectively with log2 transformed values.
Additional file 8: FigureS5. MAPMAN analysis of differentially expressed transcripts of apple colour skin vs green skin of Apple Colour – CISH-G5, shows up-regulation of the secondary metabolism pathway. Up- and Down- regulated DETs are represented with blue and red squares, respectively with log2 transformed values.
Additional file 9: FigureS6. Cluster analysis of differentially expressed transcripts among fruit stages of Allahabad Safeda (AS), Apple Colour (AC) and Punjab Pink (PP) immature fruit (IMf), mature fruit (MF), 3 days after harvesting (3DF), 7 days after harvesting (7DF), green peel and red peel.
Additional file 10: FigureS7. Gene Ontology enrichments between A) red and green peel of Apple Colour B) mature fruit of Punjab Pink and Allahabad Safeda.
Additional file 11: Guava_Reference_Transcriptome_Data files.

Data S1. Functional Annotation of Allahabad Safeda Transcriptome Assembly.

Data S2. Differential expression among different tissue types, mixed flower buds vs leaf & shoot tip (MFb vs LSt), mixed fruit stages vs leaf & shoot tip (MFr vs LSt) and mixed fruit stages vs mixed flower buds (MFr vs MFb) of Allahabad Safeda.

Data S3. Differential expression between mature but unripened fruit (0DF) vs immature fruit (ImF) of Allahabad Safeda.

Data S4. Differential expression between 3 days ripe fruit (3DF) vs mature unripened fruit (0DF) of Allahabad Safeda.

Data S5. Differential expression between 7 days over-ripe fruit (7DF) vs 3 days ripe fruit (3DF) of Allahabad Safeda.

Data S6. Differential expression between red peel vs green peel of Apple Colour.

Data S7. Differential expression between Punjab Pink mature fruit (PP_0DF) vs Allahabad Safeda mature fruit (AS_0DF).

Data S8. FPKM values of DETs among fruit and peel stages of 3 genotypes.

Data S9. Expression of genes involved important pathways for fruit maturation, ripening and pathogenesis in different tissues of 3 guava genotypes.

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RNA-Sequencing based gene expression landscape of guava cv. Allahabad Safeda and comparative analysis to colored cultivars

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Journal Publication

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Abstract

Figures

Background

Results

RNA-Seq data generation, de novo transcriptome assembly and annotation

Differential Expression analysis of Leaf, Flower and Fruit of Allahabad Safeda

Metabolic Pathway Analysis of Fruit Tissue in Comparison to Leaf and Flower

Up-regulation Of Secondary Metabolites During Fruit Ripening

Apple color in fruit skin is derived from up-regulation of secondary metabolism

Fruit colour development in coloured guava genotypes is concomitant with ripening process

Candidate Genes For Colour Development In Guava

Discussion

Fruit ripening and maturation involves ABA, Ethylene and secondary metabolite up-regulation

(R,S)-reticuline 7-O-methyltransferases is a potential candidate for colour development in guava

Up-regulation of DETs in red skin of guava cv. Apple Colour implicates cross talk among myriad dynamic processes

Phenylpropanoid Pathway In Coloured Guava

Conclusions

Methods

Original source of the plant materials

Source of Fruits or Plant material and tissue sample collection

Rna Extraction

Library Preparation, Rna Sequencing And Reference Assembly Generation

Functional Annotation of de novo Assembled Allahabad Safeda Transcriptome

Measuring Gene Expression And Identification Of Differentially Expressed Genes/transcripts

Pathway Analysis

Qrt-pcr Analysis

Abbreviations

Declarations

References

Supplementary Files Legend

Supplementary Files

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Journal Publication

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