Characterization of the Rosa roxbunghii Tratt transcriptome and … · Page 2 of 27 15 Abstract 16 Rosa roxbunghii Tratt belongs to the Rosaceae family, and the fruit is flavorful,

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1 Characterization of the Rosa roxbunghii Tratt transcriptome and

2 analysis of MYB genes

3

4 Xiaolong Huang 1,2¶, Huiqing Yan1*¶, Lisheng Zhai1,2, Zhengting Yang1,2, Yin Yi2*

5

6 1Key Laboratory of Plant Physiology and Development Regulation, school of life

7 science, Guizhou Normal University, Guiyang, China

8 2Karst Key Laboratory of biodiversity conservation in Southwest China, National

9 Forestry Administration, Guiyang, China

10

11 *Corresponding author

12 E-mail: [email protected] and [email protected]

13

14 ¶X Huang and H Yan contributed equally to this work.

.CC-BY 4.0 International licenseacertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was notthis version posted August 15, 2018. ; https://doi.org/10.1101/392720doi: bioRxiv preprint

https://doi.org/10.1101/392720http://creativecommons.org/licenses/by/4.0/

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15 Abstract

16 Rosa roxbunghii Tratt belongs to the Rosaceae family, and the fruit is flavorful,

17 economic, and highly nutritious, providing health benefits. MYB proteins play key roles

18 in R. roxbunghii’ fruit development and quality. However, the available genomic and

19 transcriptomic information are extremely deficient. Here, a normalized cDNA library was

20 constructed using five tissues, stem, leaf, flower, young fruit, and mature fruit, with three

21 repetitions, and sequenced using the Illumina HiSeq 2500 platform. De novo assembly

22 was performed, and 470.66 million clean reads were obtained. In total, 63,727 unigenes,

23 with an average GC content of 42.08%, were determined and 59,358 were annotated. In

24 addition, 9,354 unigenes were assigned the Gene Ontology category, and 20,202

25 unigenes were assigned to 25 Eukaryotic Ortholog Groups. Additionally, 19,507

26 unigenes were classified into 140 pathways of the Kyoto Encyclopedia of Genes and

27 Genomes database. Using the transcriptome, 18 candidate MYB genes that were

28 significantly expressed in mature fruit, compared with other tissues, were obtained.

29 Among them, 10 R2R3 MYB and 1 R1 MYB were identified. The expression levels of 12

30 MYB genes randomly selected for qRT-PCR analysis were consistent with the RNA-seq

31 results. A total of 37,545 microsatellites were detected, with an average EST-–SSR

32 frequency of 0.59 (37,545/63,727). This transcriptome data will be valuable for

33 identifying genes of interest and studying their expression and evolution.

34 Key words: Rosa roxbunghii Tratt, transcriptome, MYB transcription factor, EST-SSR

35




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36 Introduction

37 Rose roxburghii Tratt, a fruit crop and deciduous horticultural shrub, belongs to the

38 Rosaceae family and is mainly distributed in southwestern China. It has several

39 nutritional and functional components, including polysaccharides, flavonoids,

40 troterpenes, and superoxide dismutase activity [1, 2]. Current pharmacological research

41 indicates that it could be used for treatments of arosclerosis and senescence-retardation.

42 In addition, its fruits have radio protective, anti-tumor, antimutagenic, and genoprotective

43 activities [3-5]. As a traditional Chinese medicine, it can be further processed into fruit

44 juice, preserves, and fruit wine [6]. MYB proteins play key roles in fruit development and

45 quality [7, 8]. However, genes of the R. roxburghii MYB superfamily have not been

46 comprehensively identified and evaluated. Consequently, we located and characterized R.

47 roxburghii MYB genes.

48 Among the different transcription factor families, MYBs form the largest and most

49 functionally diverse superfamily, and they are involved in regulating cell activities and

50 plant development. The N-terminus of a MYB domain is composed of adjacent tandem

51 repeats [8]. The repeat encodes 50–53 amino acid residues and contains helices forming a

52 helix-turn-helix domain that interact with the major grooves of specific DNA sequences

53 [9]. MYB superfamily members can be classified into several subfamilies, including R1

54 MYB, R2R3 MYB, R1R2R3 MYB, 4R MYB, and atypical MYB-like proteins [10].

55 Many MYB superfamily proteins and their functions have been determined in different

56 species. Due to the conserved domains, the R2R3 MYB type is predominant in plants




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57 [11]. MYBs are involved in regulating plant growth, development, and stress resistance,

58 including the anthocyanin biosynthetic pathway, trichome initiation and development,

59 flavonoid or phenylpropanoid metabolism, secondary wall biosynthesis, sugar signaling

60 and responses to abiotic or biotic stress [12, 13].

61 Genomic information is not presently available for R. roxburghii in an online

62 database. Thus, high-throughput Illumina sequencing could be performed to determine

63 new transcripts, gene expression levels, and an accurate transcriptome profile [14]. Now

64 it becomes a powerful methodology for species that lack reference genome information.

65 Assembled unigenes with different database annotations can be assayed to evaluate

66 genetic characteristics and metabolic pathways. Recently, fruits of R roxburghii at three

67 different developmental stages were collected and analyzed using Illumina sequencing

68 technology. A previous study generated 106,590 unigenes using de novo assembly. Using

69 a BLAST algorithm-based search, 9,301 and 2,393 unigenes were categorized into Gene

70 Ontology (GO) and Clusters of Orthologous Group (COG) categories, respectively.

71 Additionally, 7,480 unigenes were assigned to 124 pathways in the Kyoto Encyclopedia

72 of Gene and Genome (KEGG) pathway database. Among the unigenes, 9,131 potential

73 EST–SSR (simple sequence repeat) loci were identified [15].

74 To better understand the profiles of different tissues in R. roxburghii, leaf, stem,

75 flower, young fruit and mature fruit were collected. In this study, the Illumina platform

76 was used to construct a cDNA library using 15 mixed tissues to obtain transcriptome

77 information. MYBs, which are significantly expressed in mature fruit, were identified.




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78 For verification, randomly selected MYB genes were analyzed by qRT-PCR. EST–SSRs

79 were subjected to a genetic diversity analysis of R. roxburghii. The obtained information

80 provides a valuble resource for functional gene analyses, in particular of MYB genes.

81 Materials and methods

82 Biological materials

83 Wild Rose roxburghii were planted in Guizhou Normal University, Guizhou, China.

84 Different tissues were collected including leaf, stem, flower, young fruit (50 days after

85 flowering, YF) and mature fruit (120 days after flowering, MF). Each tissue was in three

86 independent experiments. These materials were immediately frozen in liquid nitrogen, then

87 mechanically grounded into fine powder and stored at −80 °C for further experiment.

88 RNA extraction and sequencing

89 A total of 1 g of frozen tissues from leaf, stem, flower, young fruit and mature fruit were

90 weighed. RNA was isolated using the Trizol method (Takara, Japan), flowing the

91 manufacturer’s guidelines. RNA quality was determined with Nanodrop (Wilmington,

92 USA) and Agilent 2100 (Santa Clara, USA). Fifteen libraries were created; three for each

93 of the individual tissues or growth stage. Samples of mRNA were selected and randomly

94 fragmented. Using these fragments as templates, cDNA was transformed and purified. The

95 adapters were connected to distinguish different sequencing samples. Strand specific

96 cDNA libraries were constructed for sequencing through Illumina HiSeq 2500 (Illumina,

97 San Diego, USA).

98 De novo assembly and functional annotation




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99 The clean reads were selected from raw data with filtering out adaptor-only reads, reads

100 with more than 5% N bases unknown, and low-quality reads (reads containing more than

101 50% bases with Q-value≤10). Trinity assembly program was used to obtain data. To

102 determine the function of the unigenes, BLASTx alignment with an E-value cut off of 10−5

103 was performed with different database, including KOG (Eukaryotic Ortholog Groups,

104 http://www.ncbi.nlm.nih.gov/KOG/) [16], Nr (NCBI non-redundant protein database,

105 http://www.ncbi.nlm.nih.gov/) , KEGG (Kyoto Encyclopedia of Genes and Genomes,

106 http://www.genome.jp/kegg/) [17], Gene Ontology (GO, http://www.geneontology.org/)

107 and Swiss-Prot (http://www.expasy.ch/sprot) [18, 19]. The best-aligning results were used

108 to identify sequence direction of the unigenes. If different databases conflicted, the results

109 were prioritized in the following order: Nr, Swiss-Prot, KEGG, COG and GO [20]. When

110 transcripts did not align to any of the databases, EST Scan (http://myhits.isb-sib.ch/cgi-

111 bin/estscan) was conducted to decide its sequence direction.

112 Nucleic acid sequences from strawberry (Fragaria X ananassa), apple (Malus x

113 domestica) and cherry (Pruns. avium) were aligned by blast of Uni-prot database used for

114 Rose roxburghii. This was done in order to compare the four species based on exactly the

115 same search parameters and database type.

116 Identification and conserved motif analysis of Rosa roxbunghii superfamily

117 All genes related to MYBs were selected and clustered based on Nr annotation and made

118 descriptions. The expression level of all allergen MYB genes in various tissues were

119 showed. Conserved motifs shared by MYB proteins, significantly expressed in mature fruit




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120 using a threshold value of absolute log2 FC (fold change) ≥1 with FDR (False discovery

121 rate) ≤ 0.01, were analyzed using Multiple Em for Motif Elicitation (MEME V 5.0.1

122 http://meme.nbcr.net/meme/cgi-bin/meme.cgi) online tool through uploading the amino

123 acid sequences of the MYB superfamily members. The following parameter settings were

124 used: 1R-MYB proteins, having one R; R2R3-MYB proteins, having two Rs; R1R2R3-

125 MYB proteins, having three Rs; 4R-MYB proteins, having four Rs. Others belong to

126 atypical MYB families were determined [21].

127 Quantitative real-time PCR (qRT-PCR) assays

128 Twelve cDNAs encoding MYB transcriptional factor, all of which have potential roles in

129 regulation plant development, were selected for qRT-PCR validation. Primers were

130 designed (S1 Table) with primer premier 6. Total RNAs were isolated from leaf, stem,

131 flower, young fruit (YF) and mature fruit (MF) using the Trizol, followed by purification

132 with an RNA purification kit (Takara, Japan). Real tine RT-PCR was performed on a Roche

133 LightCycler480 machine using SYBR green I, with β-actin as an endogenous control.

134 Amplification was performed for 95°C for 2 min, 40 cycles with 95°C for 15 s, annealed

135 at 58°C for 30 s, and 72°C for 30 s. The expression levels relative to the control were

136 estimated by calculating △△Ct and subsequently analyzed using 2−△△Ct method.

137 Microsatellite detection

138 The program MISA (http://pgrc.ipksgatersleben.de/misa/) was used to detect

139 microsatellite repeat motifs for each unigene in order to understand distributions of

140 microsatellites (also known as SSRs) and to develop new markers in the transcriptome of




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141 Rose roxburghii. The number of core repeat motifs in mononucleotide, di-nucleotides, tri-

142 nucleotide tetra-nucleotide, penta-nucleotide and hexa- nucleotides was counted.

143 Results

144 Illumina sequencing and sequence assembly

145 To identify more genes, RNA-seq of different tissues, leaf, stem, flower, young fruit and

146 mature fruit, was performed with an Illumina HiSeq 2500 (Fig 1A–F). After trimming

147 and quality filtration of the raw data, each tissue was represented by an average of 31.38

148 million reads. A total of 470.66 million reads were obtained for all five tissues with three

149 repetitions. Total reads per biological condition are showed in S2 Table. For each sample,

150 86.21% of the reads could be mapped uniquely. A total of 212,534 transcripts were

151 obtained by assembling clean reads using the Trinity program, with an average GC

152 content of 42.08%, an average length of 1,077 bp and an N50 length of 2,085 bp.

153 Transcripts were further clustered and assembled into 63,727 unigenes. The unigenes

154 were all longer than 200 bp, with average and N50 lengths of 995 bp and 1,895 bp,

155 respectively (Table 1). Of the 63,727 unigenes, 78.03% (49,727) were longer than 600 bp

156 and 56.07% (35,732) were longer than 1 kb (Fig 2). In addition, most unigenes (60,901)

157 had a length of less than 5,200 bp (95.57%) (S3 Table and Table 1).

158 Functional annotation of unigenes

159 To obtain sequence information, GO, KEGG, Nr, Swiss-prot, and Eukaryotic

160 Orthologous Groups (KOG) databases were used to compare 59,358 (93.14%) of the

161 63,727 unigenes using a BLASTX-based algorithm (S4 Table). Swiss-prot and Nr




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162 contained the most homologous unigenes, at 55,118 and 55,151, respectively. In total,

163 3,284 unigenes were annotated to all database (Fig 3A). Comparative approaches

164 effectively found differences and similarities among different species. Nucleic acid

165 sequences from Rose species, including strawberry, apple, and cherry, were aligned using

166 a BLAST algorithm-based search of the Uniprot database. We found that strawberry was

167 the closest model species, followed by cherry and then apple, with the numbers of

168 unigenes similar to those of R. roxburghii being 30,577 (47.98%), 20,105 (31.55%), and

169 17,677 (27.74%), respectively (Fig 3B).

170 There are three GO categories: biological process, cellular component, and

171 molecular function (S5 Table). Category “biological process” consisted of 20 functional

172 groups, with the major groups, metabolic process (56.56%) and cellular process

173 (54.02%), having the same and higher numbers of annotations, respectively. For the

174 category “cellular part”, 16 groups were predicted. Cell, cell part, and organelle were the

175 three major groups. For “molecular function”, binding (49.02%) and catalytic activity

176 (46.01%) were the dominant groups, followed by structural molecule activity (14.89%)

177 (Fig 4).

178 A total of 20,202 unigenes were identified using the KOG database (S6. Fig) and

179 annotated to 25 functional categories. General function prediction (46.37%) was the

180 largest group, followed by signal transduction mechanisms (24.26%), posttranslational

181 modification, protein turnover, and chaperones (23.49%), and translation, ribosomal

182 structure, and biogenesis (19.80%). The numbers of unigenes assigned to transcription




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183 (11.81%), carbohydrate transport and metabolism (11.70%), energy production and

184 conversion (11.67%), and intracellular trafficking, secretion, and vesicular transport

185 (11.29%) were almost the same. In addition, lipid transport and metabolism represented

186 10.29%. However, there were still 1,939 unigenes with unknown functions. KOG

187 classifications revealed potential biological functions and provided an insight into the

188 chemical reactions involved in the molecular processes in R. roxbunghii.

189 A total of 19,507 annotated unigenes were assayed to determine the biological

190 pathways represented in R. roxbunghii. Briefly, these unigenes matched 140 KEGG

191 pathways, as summarized in S7 Table. Translation in genetic information processing

192 (3,005) was the dominant pathway, followed by carbohydrate metabolism (1,988),

193 folding, sorting and degradation (1,375), energy metabolism (1,285), transport and

194 catabolism (1,184), amino acid metabolism (1,161), and lipid metabolism (901) (Fig 4).

195 KEGG pathways can provide new insights into R. roxbunghii biology and contribute to

196 the prediction of the higher-level complexity of cellular processes and organismal

197 behavior based on this information.

198 Genes involved with MYB transcriptional factors in five different tissues

199 Among the functional database annotations, 163 MYB proteins were detected.

200 Descriptions of the MYBs are listed in S8. Fig and S9 Table. MYBs in R. roxburghii

201 were similar to the 61 members in Fragaria vesca. MYBs regulate secondary

202 metabolism, gene expression and are involved in environmental stress responses. The

203 expression levels of 159 putative MYB genes in five tissues are shown clustered. Various




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204 MYBs were expressed in different tissues. The black boxes represented MYBs that were

205 significantly expressed in mature fruit.

206 To investigate features of homologous domains and each repeat MYB domain that

207 was significantly expressed in mature fruit compared with the other four tissues, an

208 online MEME was used to search for conserved motifs shared by these proteins by

209 uploading the amino acid sequences. In total, 18 candidate MYB genes were analyzed

210 using the transcriptome. Among them, 10 R2R3 MYB, and 1 R1 MYB were identified,

211 while the remaining 7 MYB genes belonged to atypical MYB families (Fig.5). Sequences

212 of the different conserved motifs are shown in S10. Fig.

213 Verification of RNA-seq results by qRT-PCR

214 Real-time RT-PCR was conducted to validate the identification of MYB genes by RNA-

215 seq in five different tissues. With β-actin as the internal control, 12 genes related to MYB

216 transcription factors were randomly selected. Validation results showed that the change

217 trends of the 12 genes were nearly consistent with the gene expression patterns identified

218 by RNA-seq (Fig 6). These conclusions highlight the fidelity and accuracy of the RNA-

219 seq analysis in the present study.

220 Microsatellite analysis and SSR distribution

221 Using MISA software, 63,727 unigenes with a total length of 109,644,660 bp were

222 screened for microsatellite determination. A total of 37,545 potential EST–SSRs were

223 identified. The average distribution of the SSRs was calculated to be 1:2,920 bp

224 (37,545/109,644,660), and the average frequency of an EST–SSR was 0.59




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225 (37,545/63,727). In total, 20,321 unigenes contained one kind of SSR (54.12%), and

226 8,757 contained more than one SSR (23.32%; S11 Table). In total, 5,275 (14.05%) SSRs

227 were present in a compound formation. Transcriptome types of SSRs, from single

228 nucleotide to hexa-nucleotide, were abundant.

229 Among the identified 37,545 SSRs, repeats having mononucleotide motifs were the

230 most abundant (19,589, 52.17%), followed by di-nucleotides (11.504, 30.64%) (Table 2).

231 The most abundant motif was A or T (18,855, 50.22%), followed by AG or CT (8,055,

232 21.45%) (Table 2). Among SSRs with tri-, tetra-, and penta-nucleotides, the most

233 abundant types were AAG/CTT (2,189, 5.83%), AAAT/ATTT (141, 0.38%), and

234 AAAAT/ATTTT (19, 0.05%), respectively. The hexa-motifs AAGGAG/CCTTCT and

235 ACCTCC/AGGTGG, were the most abundant types and were equally present (7, 0.02%)

236 (Table 2). The repeat positions of the SSR types were analyzed and ranged from 5 to 121.

237 Most SSR types were repeated more than 15 times, at 19.01% (7,136), while those

238 repeated 10 times were 17.10% (6,422) (Table 3). Except mononucleotides, the repeat

239 numbers for most SSRs ranged from 5 to 12 (9,612, 75.9%), with only small percent

240 being repeated more than 15 times (1,177, 9.3%).

241 Discussion

242 Illumina mRNA sequencing technology is considered as an effective approach to assess

243 transcriptional expression in different tissues. However, studies of the R. roxburghii

244 transcriptome are limited. A previous study resulted in 53 million reads using fruit at

245 three developmental stages [15]. Genomic information for R. roxburghii using next-




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246 generation sequencing technologies has also been obtained. In total, 30.29 Gb of

247 sequence data was identified by HiSeq 2500 sequencing [5], covering ~60.60% of the R.

248 roxburghii genome. However, whole-genome sequencing was not performed, and only

249 genes related to the biosynthesis of ascorbic acid were the focus. The limited available

250 genomic information for R. roxburghii has constrained genetic studies. To obtain

251 sequences of as many expressed genes as possible, 15 mixed tissues were used for library

252 construction and 470,657,040 clean reads were obtained, which was greater than

253 previously reported. In addition, 9,354 unigenes were assigned to GO categories,

254 compared with 9,301 unigenes in a previous study. Moreover, 19,507 unigenes were

255 assigned to 140 KEGG pathways compared with the previous 7,480 unigenes assigned to

256 124 pathways. The greater amount of data increased the coverage depth and accuracy,

257 and a large number of genes involved in different metabolic pathways can now be

258 identified [22].

259 Because 15 tissues were applied to construct the cDNA libraries, a large portion of

260 the whole transcriptome was available for annotation. In fact, 93.14% of the R roxburghii

261 unigenes were annotated through BLAST algorithm-based searches against five

262 databases. A similarity analysis in the Nr database revealed that unigenes were more

263 homologous to cDNA sequences of strawberry than the two Rosaceae species apple and

264 cherry. This indicated that R. roxburghii has a closer evolutionary relationship with

265 strawberry. The genomic resources of strawberry have been available on line

266 (http://bioinformatics.towson.edu/strawberry/). Strawberry has a short life cycle (3.5 to 4




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267 months) and a facile transformation system, while R. roxburghii is a deciduous

268 horticultural shrub with a long-life cycle. However, it is still feasible to study R.

269 roxburghii gene functions using strawberry as a model system, as was previously done

270 for fruit crops in Rosaceae, including apple, peach, and cherry [23].

271 Based on MYB expression patterns, 18 MYB genes were significantly expressed in

272 mature fruit compared with in other tissues. In total, 10 R2R3 MYBs were identified, and

273 this was the dominant MYB type. MYBs presented similar patterns and conserved motifs,

274 suggesting that their conserved features play similar roles in group-specific functions. As

275 part of a myb-bHLH-WD40 complex, they are involved in plant secondary metabolism

276 and responses to abiotic and biological stresses [24, 25]. Several R2R3 MYBs were

277 analyzed for their possible involvement in anthocyanin biosynthesis and accumulation,

278 and fructan exohydrolase regulation when subjected to different stress and hormone

279 treatments [26, 27]. MYBs were implicated in sugar signaling, fruit-skin coloration, and

280 phenylpropanoid metabolism [28-30]. Most R2R3 MYBs were implicated in fruit

281 development and should be studied to improve fruit quality and stress resistance. The

282 results of this study will aid functional studies of interesting MYB genes involved in R.

283 roxburghii fruit development.

284 Using qRT-PCR, the accuracy of RNA-seq was validated. Both assays were limited

285 to the transcriptional level. Although 15 tissues were used to construct cDNA libraries,

286 the whole R. roxburghii transcriptome was still not completely covered because some

287 transcripts, not expressed in these tissues, may be missed.




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288 A total of 37,545 microsatellites with different repeat types were detected from

289 63,727 unigenes, indicating that each unigene, on average, contained 0.59 SSR. SSR

290 locus density was 1:2,920 bp, compared with 1:4.00 kb, in a previous study. Various

291 criteria and parameters for SSR detection, as well as the diversity of genomic structures

292 and compositions can influence SSR density [31]. The differences found here might

293 reflect genome size [32], which implied that we obtained more abundant data. Errors in

294 sequencing and assembly mistakes that resulted in mononucleotide SSRs were relatively

295 low [33]. Except mononucleotides, the most common SSR motif was dinucleotide repeats

296 in the transcriptome, which was similar to previously reported results. Here, AC/GT was

297 the most common type. In conclusion, detected EST–SSRs (37,545) were more

298 associated with functional genes and could provide valuable genetic and genomic

299 analyses. These EST–SSRs will be useful for genetic linkage mapping construction,

300 comparative mapping and MAS breeding.

301 Taken together, a deep RNA-seq analysis was conducted on five tissues, and a total

302 of 469.5 million reads were generated. In total, 63,727 unigenes were obtained using

303 Trinity assembly, in which nearly 90% (59,358) were successfully annotated. The data

304 provided comprehensive coverage of the R. roxburghii transcriptome and allowed us to

305 identify the MYBs significantly expressed in mature fruit. Future studies are necessary to

306 elucidate the MYBs involved in fruit quality and development. Finally, EST–SSRs were

307 identified and different SSR repeat numbers isolated from the transcriptome were

308 analyzed. This study provides a valuable resource for bioengineering and biological




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309 studies of R. roxburghii.

310 Conclusions

311 In present study, a deep RNA-seq analysis was conducted on five tissues, and a total of

312 469.5 million reads were generated. In total, 63,727 unigenes were obtained using Trinity

313 assembly, in which nearly 90% (59,358) were successfully annotated. The data provided

314 comprehensive coverage of the R. roxburghii transcriptome and allowed us to identify the

315 MYBs significantly expressed in mature fruit. Future studies are necessary to elucidate the

316 MYBs involved in fruit quality and development. Finally, EST–SSRs were identified and

317 different SSR repeat numbers isolated from the transcriptome were analyzed. This study

318 provides a valuable resource for bioengineering and biological studies of R. roxburghii.

319

320

321 Acknowledgements

322 This work was supported by grants from the National Natural Science Foundation of

323 China (Grant No. 31660554, 31600214 and 31660046), Science Foundation of Guizhou

324 Provinces (Qiankehe J zi (2015) 2117).

325

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456 Supporting information

457 S1 Table. Primers for real-time PCR.

458 S2 Table. Summary of read mapping in leaf, flower, stem, young fruit and mature

459 fruit with three repetitions.

460 S3 Table. Analysis of size distribution of unigene for Rosa roxbunghii Tratt.

461 S4 Table. The number of unigenes annotated according to the NCBI non-redundant

462 (Nr), Swiss-Prot, KOG, Gene Ontology (GO) and KEGG database in Rosa

463 roxbunghii Tratt.

464 S5 Table. The number of unigenes annotated with GO and classified with three

465 categories.

466 S6 Fig. Histogram of Eukaryotic Ortholog Groups (KOG) classification of assembled

467 unigenes.

468 S7 Table. The number of unigenes involved in KEGG pathways.

469 S8 Fig. Heat map diagram of the expression levels of MYB transcriptional factor in

470 five different tissues. The black box represented MYBs which significantly

471 expressed in mature fruit compared with other four tissues.

472 S9 Table. Analysis of genes related with MYB transcription factor in five different

473 tissues of Rosa roxbunghii Tratt.

474 S10 Fig. The different color represented conserved motifs.

475 S11 Table. Summary of different type EST-SSRs identified from the transcriptome.

476




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477 Figures and tables

478 Fig 1. Rosa roxbunghii plant and different tissues collected in this study. (A) The whole

479 plant, Bar = 50 cm; (B) Leaf and stem, Bar = 0.5 cm; (C) Flower bud, Bar = 1mm;

480 (D) Flower, Bar =1 cm; (E) Young fruit (50 days after flowering, DAF), Bar = 1

481 cm; (F) Mature fruit (120 DAF), Bar =1 cm.

482 Fig 2. Length distribution of unigenes assembly for Rosa roxbunghii Tratt.

483 Fig 3. Overview of functional annotation for unigenes of Rosa roxbunghii (A) Venn

484 diagram of the number of unigenes annotated by BLASTx against five different

485 databases. The number in the circles represented the number of unigenes

486 annotated by single or multiple databases; (B) Homology to other model Rosa

487 species, including strawberry (Fragaria X ananassa), apple (Malus domestica)

488 and cherry (Pruns avium).

489 Fig 4. (A) Histogram of Gene Ontology (GO) classification of assembled unigenes. Blue

490 indicated biological process, green indicated cellular process, and red

491 represented molecular function. (B) Pathway assignment based on KEGG

492 analysis. The bottom x-axis indicated the number of unigenes in a specific

493 category. The y-axis indicates the clustered function groups, the right y-axis

494 indicates the specific category of genes in the main category.

495 Fig 5. Motif distribution of MYB proteins which significantly expressed in mature fruits.

496 Various colors represented different conserved motifs.

497 Fig 6. Comparison of the relative expression levels of twelve randomly selected MYB




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498 genes by RNA-Seq and RT-PCR.

499 Table 1. Summary of the trinity assembly for Rosa roxbunghii.

500 Table 2. Summary of EST-SSRs identified from the transcriptome of Rosa roxbunghii.

501 Table 3. Summary of different repeat times for SSRs isolated from the transcriptome of

502 Rosa roxbunghii Tratt.

503

504 Table 1. Summary of the trinity assembly for Rosa roxbunghii Tratt.

Length range (bp) Transcript Unigene

Total Number 212,534 63,727

Total length 305,558,508 85,677,327

N50 length 2,085 1,895

Median Length 1,077 995

505

506

507

508

509

510

511

512




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513 Table 2. Summary of EST-SSRs identified from the transcriptome of Rosa roxbunghii

514 Tratt.

SSR Type Number Percentage Dominat motif

Number of

dominant

motif

Percentage of

dominant motif

in all EST-SSR

Mononucleotide 19,589 52.17% A/T 18,855 50.22%

Dinonucleotide 11,504 30.64% AG/CT 8,055 21.45%

Trinucleotide 5,879 15.66% AAG/CTT 2,189 5.83%

Tetranucleotide 373 0.99% AAAT/ATTT 141 0.38%

Pentanucleotide 93 0.25% AAGAG/CTCTT 19 0.05%

AAGGAG/CCTTCT 7 0.02%Hexnucleotide 107 0.28%

ACCTCC/AGGTGG 7 0.02%

515

516




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517 Table 3. Summary of different repeat times for SSRs isolated from the transcriptome of Rosa roxbunghii Tratt.

518

SSR Type 5 6 7 8 9 10 11 12 13 14 15 >15 Total

Mononucleotide 0 0 0 0 0 5448 2864 2019 1380 1165 905 5808 19589

Dinonucleotide 0 2441 1765 1368 1158 883 985 858 216 259 263 1308 11504

Trinucleotide 2965 1385 727 461 91 86 69 40 20 8 7 20 5879

Tetranucleotide 242 95 15 7 4 5 0 3 2 0 0 0 373

Pentanucleotide 67 17 8 1 0 0 0 0 0 0 0 0 93

Hexnucleotide 74 22 7 1 1 0 1 1 0 0 0 0 107

Total 3348 3960 2522 1838 1254 6422 3919 2921 1618 1432 1175 7136 37545

Percentage 8.92% 10.55% 6.72% 4.90% 3.34% 17.10% 10.44% 7.78% 4.31% 3.81% 3.13% 19.01% 100%

519




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Characterization of the Rosa roxbunghii Tratt transcriptome and … · Page 2 of 27 15 Abstract 16 Rosa roxbunghii Tratt belongs to the Rosaceae family, and the fruit is flavorful,