Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line

GENE-40818; No. of pages: 9; 4C: Gene xxx (2015) xxx–xxx

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Research paper

Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line Ning Zhang, Shasha Wang, Xiangfen Zhang, Zhongdong Dong, Feng Chen ⁎, Dangqun Cui ⁎ Agronomy College, Collaborative Innovation Center of Henan Grain Crops, National Key Laboratory of Wheat and Corn Crop, Henan Agricultural University, Zhengzhou 450002, China

a r t i c l e

i n f o

Article history: Received 19 April 2015 Received in revised form 26 August 2015 Accepted 1 September 2015 Available online xxxx Keywords: RNA-seq Transcriptome Wheat EMS mutant line qRT-PCR

a b s t r a c t Roche 454 next-generation sequencing was applied to obtain extensive information about the transcriptomes of the bread wheat cultivar Yunong 201 and its EMS mutant line Yunong 3114. Totals of 1.43 million and 1.44 million raw reads were generated, 14,432, 17,845 and 27,867 isotigs were constructed using the reads in Yunong 201, Yunong 3114 and their combination, respectively. Moreover, 29,042, 34,722, and 48,486 unigenes were generated in Yunong 201, Yunong 3114, and combined cultivars, respectively. A total of 50,382 and 59,891 unigenes from the Yunong 201 and Yunong 3114 were mapped on different chromosomes. Of all unigenes, 1363 DEGs were identified in Yunong 201 and Yunong 3114. qRT-PCR analysis confirmed the expression profiles of 40 candidate unigenes possibly related to abiotic stresses. The expression patterns of four annotated DEGs were also verified in the two wheat cultivars under abiotic stresses. This study provided useful information for further analysis of wheat functional genomics. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Wheat (Triticum aestivum L.) is an important cereal crop alongside maize and rice; more than 600 million tons of wheat are harvested annually (Shewry, 2009). A total of 704, 723, and 883 million tons of wheat, rice, and maize were produced in 2011, respectively (http:// faostat.fao.org/). These data show that wheat yield increases more slowly than other crop yields. The genome sizes of the three cereal crops are also remarkable; for instance, the genome of rice is approximately 400 Mbp (Goff et al., 2002; Yu et al., 2002), and that of the ancient allotetraploid maize is approximately 2.3 Gbp (Schnable et al., 2009). By contrast, the genome size of wheat is 16 Gbp. Such a large and complex genome complicates research on wheat genome; the depth and breadth of such a research are inferior to those of rice, maize, barley, and other crops. Modern biotechnologies have been applied to improve wheat yield, nutritional content, and salinity, drought, and biotic tolerance (Tester and Langridge, 2010). However, information on the genome sequences and transcriptome of wheat remain insufficient.

Abbreviations: EMS, ethyl methanesulfonate; NGS, next-generation sequencing; qRTPCR, quantitative real-time PCR; EST, expressed sequence tag; DEG, differentially expressed gene; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; FDR, false discovery rate; ddH2O, double distilled H2O; RLK, receptor-like protein kinase; RLK, receptor-like protein kinase; TLP, thaumatin-like protein; TF, transcription factor; Hsp, heat-shock protein; RWC, relative water content. ⁎ Corresponding authors at: Agronomy College, Henan Agricultural University, Wenhua Road 95, Zhengzhou 450002, PR China. E-mail addresses: [email protected] (N. Zhang), [email protected] (S. Wang), [email protected] (X. Zhang), [email protected] (Z. Dong), [email protected] (F. Chen), [email protected] (D. Cui).

The next-generation sequencing (NGS) has been successfully applied in wheat and its closely related species for several years; other extensive applications and studies were conducted in the past 2 years. For instance, the first homolog-specific sequence assembly of wheat transcriptome provides a reference transcriptome for future wheat studies based on Roche 454 and Illumina GAIIx (Schreiber et al., 2012). A high-throughput RNA sequencing has been performed using Illumina NGS to characterize the transcriptome of Wangshuibai during Fg infection (Xiao et al., 2013). RNA sequencing (RNA-seq) methods have also been applied to generate the transcriptome profiles of the wheat cultivar Chinese Spring in response to 10 d of phosphate (Pi) starvation and to elucidate the molecular mechanisms associated with such conditions (Oono et al., 2013). RNA-seq can accurately measure the transcript levels of Pina, Pinb, and each of the four Pinb-2 variants in developing wheat seeds, whereas Northern blots cannot accurately quantify Pinb-2 transcript levels because of cross-hybridization (Giroux et al., 2013). Interactions between stem rust and wheat were studied using NGS of rust genomes and transcriptomes of infected wheat tissues; RNA-seq expression profiling demonstrated race- and hostspecific responses in different combinations of stem rust and wheat genotypes (Akhunov, 2013).Nowadays, ethyl methanesulfonate(EMS) mutation has also reached a mature stage, in which damages in plants are reduced and abundant plant mutations are generated by controlling the usage of EMS. EMS mutants have been employed as basic materials in extensive studies. Until now, RNA-seq and EMS mutants were rarely combined in wheat transcriptome studies. The Chinese winter wheat cultivar Yunong 201 we used in this study, developed by Agronomy College of Henan Agricultural University, was released as a high-quality noodle

http://dx.doi.org/10.1016/j.gene.2015.09.002 0378-1119/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

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N. Zhang et al. / Gene xxx (2015) xxx–xxx

wheat cultivar by Henan province in 2006. An elite M2 line was screened from the EMS mutagenized population encompassing 2000 lines because of its different plant architecture, large kernel size, and high grain weight. This line was self-crossed thrice into the M5 line Yunong 3114.Compared with Yunong 201, Yunong 3114 showed relatively larger kernel size, higher thousand grain weight and higher yield per plot. Yunong 201 and Yunong 3114 also showed obvious difference on tolerance to abiotic stresses (e.g. drought, coldness, dry-hot wind). Therefore, comparison of transcriptomes of Yunong 201 and Yunong 3114 could provide valuable information for further dissection of molecular and genetics basis of the phenotypes related to abiotic stresses as well as yield and quality in bread wheat. In the study, Roche 454 sequencing was applied in bread wheat Yunong 201 and Yunong 3114 to generate their transcriptomes. The gene profiles of Yunong 201 and Yunong 3114 were obtained by de novo sequencing. Differentially expressed genes (DEGs) related to the growth, stimuli, and photosynthesis of Yunong 201 and Yunong 3114 were comprehensively analyzed. Subsequently, the expression patterns of four annotated DEGs were verified in the two varieties under abiotic stresses. This study provided important information for future understanding the transcriptome of hexaploid wheat.

polygenetic analysis with CD-HIT version 4.0 (Huang et al., 2010) at a sequence identity threshold of 99%. The trimmed reads without redundancy were analyzed by comparing with the NT library. Assembly and quantification were conducted by 454 software Newbler and RPKM algorithm. Trimmed reads assembled using Newbler 2.5 were called contigs; isotigs were formed by the assembly of one or more contigs. Three groups comprising Yunong 201, Yunong 3114, and their combination were finally generated. 2.4. Sequence annotation Isotigs + singlets sequences were subjected to polygenetic analysis by using CD-HIT version 4.0 (Huang et al., 2010) with an identity of 95%. Chromosome mapping using ssahaSNP software and comparing with known genome segments were also performed. The BLAST program (Camacho et al., 2009) (a searched threshold of 1e–10) was used to search for unigenes in EST libraries of 10 species related to NCBI (http://www.ncbi.nlm.nih.gov/) and DFCI (ftp://occams.dfci. harvard.edu/pub/bio/tgi/data). Nucleic acid and protein were separately annotated by comparing the unigenes with those in the NT library (E-value b 1e–5) and the NR library (BLASTX; E-value b 1e–5; similarity of protein N 30%).

2. Materials and methods 2.5. GO and KEGG orthology 2.1. Plant materials The Chinese winter wheat cultivar Yunong 201 (released No. Yushenmai 2006006) and its EMS-derivative Yunong 3114 as mentioned above were planted and grown at the Zhengzhou Scientific Research and Education Center of Henan Agricultural University (N34.9°, E113.6°) during 2011 to 2012 cropping seasons at nonstressed conditions. The leaves and stems at the three-leaf stages were collected to generate the cDNA libraries of Yunong 201 and Yunong 3114.

The gene ontology (GO) project is a major bioinformatics initiative that aims to standardize the representation of gene and gene product attributes across species and databases (http://www.geneontology. org/). We identified the annotated unigene sequences for the possible functions by using this method. We mapped sequences to the reference authoritative pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) to determine the active biological pathways in annotated unigene sequences. KEGG is a database used to understand the high-level functions and utilities of a biological system.

2.2. RNA extraction, library construction, and Roche 454 sequencing 2.6. Analysis of gene expression Full-length cDNA libraries were sequenced using a GS FLX sequencer (Roche) in accordance with the standard single read shotgun 454 sequencing protocol with titanium chemistry (Roche). The total RNAs of Yunong 201 and Yunong 3114 were extracted using Invitrogen Trizol Reagent. The two 454 libraries of Yunong 201 and Yunong 3114 were constructed using a GS FLX titanium general library preparation kit. The quantity and quality of the total RNA were analyzed via spectrophotometry (Ultrospec™ 2100 pro UV/Visible spectrophotometer) and gel electrophoresis. mRNA was then isolated from the total RNA by using oligo (dT)-attached magnetic beads. cDNA was synthesized using a random Roche primer and via PCR amplification. The quality of the doublestranded cDNA was checked by running on a 2% 1 × TAE agarose gel with 0.1 mg/mL ethidium bromide (carcinogen) for approximately 30 min. cDNA concentration was determined using a Bioanalyzer 7500 kit. cDNA samples sheared by ultrasonication should be N80 ng/μL (total N 1 μg) and should range from 100 bp to 800 bp, which is the appropriate fragment size range for 454 sequencing. RL adaptors or RL MID adaptors were ligated to the fragmented cDNA, and the small fragments were removed. A TBS 380 fluorometer (Turner Biosystems) was used to quantify the cDNA library. The quality of the cDNA library was assessed, and the average fragment length ranged between 600 and 1200 bp with a low size cut-off b10% of 500 bp. 2.3. Pretreatment of data A total of 5 Gb raw data were generated from each cultivar. Raw reads were trimmed using Newbler 2.5 (Margulies et al., 2005). Lowquality bases and vector sequences were filtered; reads less than 50 bp were also removed. The trimmed reads were then subjected to

The total unigenes of the combined Yunong 201 and Yunong 3114 were divided into three sections to analyze the expression difference in unigenes: unique unigenes in Yunong 201 and Yunong 3114, and common unigenes in the first two groups. False discovery rate (FDR) statistical test was conducted in accordance with the RPKM value of the common unigenes in Yunong 201 and Yunong 3114. At FDR b 0.05, DEGs were present; at FDR N 0.05, no differences were present between the expression levels of common unigenes. 2.7. Validation of DEGs by quantitative real-time PCR (qRT-PCR) Based on the functional annotation of unigenes, the 44 DEG we selected are possibly related to stresses, and corresponding specific primers were designed by software Primer 3.0. We verified the expression profiles of 40 wheat candidate unigenes that were chosen from the 1363 DEGs. 2.8. Stress treatments and validation of DEGs by qRT-PCR Seeds of Yunong 201 and Yunong 3114 were immersed sterilized with 0.01% (w/v) H2O2 for 0.5 h and then thoroughly washed with distilled water. Sterilized seeds were grown in glass dishes (9 cm diameter) with double distilled H2O (ddH2O). Seedlings were maintained in illuminated incubator at 25/15 °C day/night temperatures under 16/8 h light/dark photoperiod and 250 μmol m−2 s−1 light intensity. Twoweek old seedlings with similar heights were used to analyze the effects of different abiotic stresses. In order to investigate abiotic stress of the Yunong 201 and Yunong 3114 plants, ten seedlings for each cultivar

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

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were treated with 20% PEG 6000 (drought treatment) for 0 h, 12 h, 24 h and 48 h, and 400 mM NaCl (salt treatment) for 0 h, 1 h, 2 h and 3 h were used to measure the salt resistance of two wheat cultivars. Three replicates were performed. Relative water content (RWC) was examined to evaluate drought resistance of the two cultivars according to method of Tavakol (Tavakol and Pakniyat, 2007). Some seedlings were transferred to an illuminated incubator at 4 °C or 42 °C for the low- or hightemperature stress treatments. Some seedlings were exposed to 200 mM NaCl (salt treatment) and 20% PEG 6000 (drought treatment) by immersing the roots, respectively. The shoots of the two wheat cultivars were collected at 0, 1, 2, 6, 12, and 24 h after the different treatments and then stored at − 80 °C. Total RNA was extracted using the total RNA kit (TaKaRa, Dalian, China). Two-Step PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa) was used for the RT reactions. The temperature program was adjusted as follows: 2 min at 42 °C, 15 min at 37 °C, 5 s at 85 °C, and then 4 °C. For each candidate, three biological replicates were performed. qRT-PCR was conducted using a Bio-Rad IQ5 Real-Time PCR Detection System. Each reaction included 20 μL of products from the diluted RT reactions, 0.4 μL of each primer (forward and reverse), 10 μL of Go Taq® qPCR Master Mix (Perfect Real Time; Promega), and 7.2 μL of nuclease-free water. The reactions were incubated in a 96well plate at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. All reactions were run in triplicates for each sample. The β-actin gene (AB181991) served as the endogenous control (Chen et al., 2012).

3. Results 3.1. Phenotype analysis of the Yunong 201 and Yunong 3114 To evaluate the tolerance levels of the Yunong 201 and Yunong 3114, four treatments of drought (PEG), cold, heat and salt were performed at the three-leaf stages of the Yunong 201 and Yunong 3114, respectively. Under the cold and heat treatments, phenotype of the Yunong 201 and Yunong 3114 did not show significant difference (data not listed) even though seedlings of Yunong 3114 showed slightly stronger tolerance to low and high temperatures than that of Yunong 201. However, under the drought treatments, leaf relative water content (RWC) of Yunong 201 decreased more quickly than that of Yunong 3114 after 24 h and the RWC of Yunong 3114 still remained above 75% at 72 h (Fig. 1a). Under the salt treatment, the seedlings of Yunong 3114 showed better tolerance than that of Yunong 201 during 1–2 h (Fig. 1b).

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3.2. Overview of the transcriptome dataset in Yunong 201 and Yunong 3114 High-throughput RNA sequencing was performed with Roche 454 NGS to obtain a comprehensive view of the transcriptomes of Yunong 201 and its EMS mutant line Yunong 3114. A total of 1.43 million reads with an average length of 440 bp and 1.44 million reads with an average length of 384 bp were generated from Yunong 201 and Yunong 3114, respectively. Furthermore, 1.41 million good reads with an average length of 446 bp and 1.31 million good reads with an average length of 393 bp were obtained in Yunong 201 and Yunong 3114, respectively. These reads were also used for the succeeding analysis after low-quality bases, vector sequences, and reads with less than 50 bp were trimmed and filtered in SeqClean 86–64, Newbler 2.5. A summary of the trimmed reads is presented in Table 1.

3.3. Annotation and assembly of transcriptome data Blast results indicated that 1.37 million reads of Yunnong 201 and 1.26 million reads of Yunong 3114 sequences could be annotated in the NT database of the NCBI website with the expected hit value be−10, which corresponds to annotation percentages of 97.6% and 95.8%, respectively. Further analysis of the annotation results showed that the percentages of high-quality hits were 48.6%, 22.0%, and 13.0% for Yunong 201 compared with those for T. aestivum, Taeniatherum caput-medusae, and Hordeum vulgare subsp. Vulgare. Similar results (43.0%, 19.1%, and 14.0%, respectively) were observed in Yunong 3114. These results suggested that we obtained high-quality sequencing data. CD-HIT version 4.0 was used to remove duplicate sequences with a criterion of 99% identity. The results of polygenetic analysis indicated that 281,294 and 304,075 sequences were obtained from Yunong 201 and Yunong 3114, respectively. Newbler 2.5 was used to assemble the reads from Yunong 201 and Yunong 3114. The first step in transcriptome assembly involves constructing a contig graph in the Newbler project. However, several contigs were possibly obtained from each transcript because of the splice variants (and other sequence particularities) that formed a small contig graph. The transcriptome projects showed numerous subgraphs called isogroups, each of which potentially represented one gene. The contigs in the subgraphs of each isogroup were traversed by Newbler to generate transcript variants called isotigs. Sequence variants between reads obtained from different homologous chromosomes formed two or more almost identical contigs for a region of the transcript. As a result, the number of isotigs inflated, and several isotigs were almost identical.

Fig. 1. Comparison of stress resistance of the Yunong 201 and Yunong 3114. a. The relative water content of wheat leaves during treatment of the drought stress (20% PEG 6000). b. Phenotypic changes of wheat seedlings during treatment of 400 mM NaCl for 0 h, 1 h, 2 h, 3 h.

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

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Table 1 Summary of sequencing and assembly of the transcriptome.

Yunong 201 Yunong 3114 Combination

Number of total reads trimmed

Isotig number

Isotig Length (bp)

Isotig N50 (bp)

Isotig longest (bp)

Isotig average depth

Singlet number

1407516 1311103 2718619

14432 17845 27867

7979664 9731845 15558942

585 580 592

6326 9581 8431

19.59 17.25 20.93

33015 33323 60465

Assembly results obtained using Newbler 2.5 showed that 14,432, 17,845, and 27,867 isotigs were constructed from the reads of Yunong 201, Yunong 3114, and their combination, respectively. The N50 lengths of the isotigs were 585, 580, and 592 bp in Yunong 201, Yunong 3114, and their combination, respectively. The resulting N50 length of the isotigs indicated the good quality of our assembled results. The numbers of the singlets were 33,015, 33,323, and 60,465 for Yunong 201, Yunong 3114, and their combination, respectively (Table 1). 3.4. Functional annotation Complementary approaches were employed to annotate the assembled sequences. Isotig + singlet sequences merged from the three groups were subjected to polygenetic analysis using CD-HIT version 4.0 to remove duplicate sequences. The sequences were then clustered into one group with a criterion of 95% identity. The longest sequence was used as a representative to merge the depth of each type (the depth meter of a singlet for 1). These representatives constituted the libraries of unigenes. Yunong 201, Yunong 3114, and their combination had 29,042, 34,722, and 48,486 unigenes, respectively (Table 3). Unigenes from the Yunong 201 and Yunong 3114 exhibiting distinct characteristics in the expressed sequence tag (EST) sequence were selected with a criterion of E-value b1e–10, and then were mapped in the wheat chromosome, including three genome databases IWGSP1, (ftp://ftp.ensemblgenomes.org/pub/release-26/plants/fasta/triticum_ aestivum/dna/) wheat Agenome (http://gigadb.org/dataset/100050), and wheat D genome (http://gigadb.org/dataset/100054). A total of 50,382, 45,256, 48,156 unigenes from Yunong 201 and 59,891, 53,163, 57,116 unigenes from Yunong 3114 were mapped on different chromosomes, A and D genomes, respectively (Additional file 7 and Table 4). Of them, 3317 and 3848 unigenes from the Yunong 201 and Yunong 3114 were mapped on the chromosome 3B, respectively. Among the unigenes on other chromosomes, the number of unigenes mapped on 5BL and 5DL were relatively higher (Additional file 7 and Table 4). Considering that no reference genome for wheat is available, we also collected wheat mitochondrial and chloroplast genome sequences, cloned 3B chromosome sequences, assembled 5A chromosome sequences, and assembled wheat genome sequences published by other researchers. We obtained 5,633,795 sequences with a total size of approximately 4 G. Unigenes from the combined Yunong 201 and Yunong 3114 were blasted with the obtained genomic sequences Table 2 Comparison of unigenes from the combination of Yunong 201 and Yunong 3114 with 11 species from EST libraries.

Library name

Sequence number

Matched unigene number

Matched sequence number

Arabidopsis Brachypodium distachyon Glycine max Gossypium Gossypium raimondii Hordeumvulgare Oryza sativa Physcomitrella patens Secale cereale Sorghum bicolor Triticum aestivum

1532821 29260 55990 114394 24891 82981 202142 63781 5587 46025 128088

2307 11523 2432 2650 559 25831 14082 1367 6580 10086 33603

777 7816 959 780 303 16104 8115 259 2710 5247 22255

with a criterion of E-value b1e–10. The results revealed that 42,759 unigenes matched the genome fragments, accounting for 88.2% of the total unigenes. Unigenes from the combined Yunong 201 and Yunong 3114 were also compared with the EST libraries of 11 species (Additional file 1), namely, Arabidopsis, Brachypodium distachyo, Glycine max, Gossypium sp., Gossypium raimondii, H. vulgare, Oryza sativa, Physcomitrella patens, Secale cereale, Sorghum bicolor, and T. aestivum, from NCBI/DFCI (ftp://occams.dfci.harvard.edu/pub/bio/tgi/data) with a criterion of E-value b1e–10. The results of the sequence alignments are listed in Table 2. The largest number of matched unigenes corresponded to the wheat EST library containing 33,603 unigenes. The nucleic acids and proteins of the unigenes in the three groups were annotated. We then compared the results with those in the NT library (E-value b1e–5). A total of 19,359, 22,747, and 30,820 unigenes were annotated in Yunong 201, Yunong 3114, and their combination, which corresponded to 294, 312, and 428 species, respectively, including barley, wheat, rice, and maize (Table 3). Among the total annotations, 17,666 (39%) unigenes from the combined Yunong 201 and Yunong 3114 did not exhibit similarities. This result indicated that new genes were possibly generated in our transcriptome data. The matched unigenes (61%) containing homologous genes could be used as candidates for future studies. We also compared the unigene sequences of the three groups with those in the public NR and SwissProt databases by using BlastX (E-value b1e–5; similarity of protein N30%). The results showed that 16,331 (56.2%), 19,389 (55.8%), and 25,747 (53.1%) unigene sequences matched the known protein sequences. Gene ontology (GO) annotation of the unigenes in the three groups was performed. The GO terms were categorized into molecular functions, biological processes, and cellular components. A total of 25,610 (88.2%), 26,885 (77.4%), and 38,237 (78.9%) unigenes in Yunong 201, Yunong 3114, and their combination, respectively, were assigned in the secondary category in accordance with the GO assignments (Additional file 2). Approximately 9646 (37.7%), 10,579 (39.3%), and 14,425 (37.7%) unigenes were annotated in the molecular function category; 7657 (29.9%), 8001 (29.8%), and 11,275 (29.5%) in the biological process category (Additional file 2); and 8305 (32.4%), 8380 (30.9%), and 12,527 (32.8%) in the cellular component category. Among the molecular function categories, the most highly represented categories were binding and catalytic activities (91.0%; Fig. 2). Among the biological processes, the largest proportion was assigned to the secondary level GO terms of metabolic and cellular processes (Fig. 2). For the cell component category, almost all of the unigene sequences in the three groups were annotated in the cell subcategory; 35% of these sequences were annotated in the cell and cell part categories in the three unigene groups; and 22% of the sequences were assigned in the organelle category of Yunong 201 and the combined Yunong 201 and Yunong 3114. The percentage of the organelle category of Yunong 3114 was 20% because the organelles, membrane-enclosed lumen, extracellular region, and macromolecular complex only comprised 7% of Yunong 201 and combined Yunong 201 and Yunong 3114; 9% was observed in Yunong 3114 (Fig. 2). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was employed to characterize the intricate biological behavior of unigenes involved in functional classification and pathway mapping. Overall, 5396, 5979, and 8159 unigene sequences were assigned to 285, 288, and 287 KEGG pathways in Yunong 201, Yunong 3114, and their combination, respectively (Additional file 3). The largest

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

N. Zhang et al. / Gene xxx (2015) xxx–xxx Table 3 Annotations of nucleotide and protein from three groups of unigenes. Library name

Unigene number

Matched unigene number in NT

Matched unigene number in NR

Yunong 201 Yunong 3114 Combination

29042 34722 48486

19359 22747 30820

16331 19389 25747

categories of 5856, 5769, and 8557 unigene sequences of the three groups with specific enzyme commission numbers were assigned to metabolism (Table 5). This classification included carbohydrate metabolism, amino acid metabolism, energy metabolism, biosynthesis of other secondary metabolites, nucleotide metabolism, lipid metabolism, energy metabolism, glycan biosynthesis and metabolism, xenobiotic biodegradation and metabolism, terpenoid and polyketide metabolism, and cofactor and vitamin metabolism. Approximately 1125, 1450, and 1732 unigene sequences were classified in genetic information processing. Many of these sequences are involved in transcription, translation, folding, sorting and degradation, replication, and repair. Cellular processes, including transport and catabolism, cell growth and death, cell motility, and cell communication, accounted for 654, 738, and 965 of the KEGG-annotated sequences. Furthermore, environmental information processing, including signal transduction and membrane transport, was represented by 362, 425, and 564 KEGG-annotated sequences.

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Table 5 KEGG biochemical mappings for wheat. Number of genes KEGG pathway

Yunong 201 Yunong 3114 Assembly

Metabolism Carbohydrate metabolism Amino acid metabolism Energy metabolism Biosynthesis of other secondary metabolites Nucleotide metabolism Lipid metabolism Metabolism of other amino acids glycan Glycan biosynthesis and metabolism Xenobiotics biodegradation and metabolism Metabolism of terpenoids and tolyketides Metabolism of cofactors and vitamins Genetic information processing Transcription Translation Folding, sorting, and degradation Replication and repair Environmental information processing The signal transduction Membrane transport Cellular processes Transport and catabolism Cell growth and death Cell motility Cell communication Total

5856 2082 524 1897 187 176 298 152 80 175 176 162 1125 162 477 370 116 362 334 28 654 284 200 43 127 7997

5769 1951 588 1615 226 223 362 185 103 182 139 195 1450 227 602 478 143 425 401 24 738 324 245 49 120 8382

8557 3042 706 2841 299 275 412 208 142 236 159 237 1732 258 707 573 194 564 525 39 965 399 318 70 178 11,818

3.5. Differential gene expression Differential gene expression was analyzed by identifying the difference in expression levels between samples. For a normal experimental design/processing, the ratio of expression between samples (fold change) and statistically significant P-value/false discovery rate (FDR) are generally used. FDR is the proportion of misjudgment derived by rejecting a null hypothesis test. Fold change directly provides the difference in copies between two samples; however, fold change was only applicable in the gene expression analysis of two samples. Statistically significant P-value/FDR method is applicable not only in the gene expression analysis of two samples but also in that of one sample.

Table 4 Number of unigenes from the Yunong 201 and Yunong 3114 mapped on the different wheat chromosomes, A and D genomes. Number of mapped Chromosome unigenes from Chromosome/Genome Yunong 201/3114 1AS 1AL 1BS 1BL 1DS 1DL 2AS 2AL 2BS 2BL 2DS 2DL 3AS 3AL 3B 3DS 3DL 4AS 4AL 4BS 4BL 4DS 4DL

675/837 1844/2154 320/356 1308/1596 736/953 1747/2020 1235/1395 1681/2022 1219/1522 1852/2067 993/1110 1522/1824 885/997 1290/1639 3317/3848 673/813 1299/1628 966/1218 1439/1670 1115/1364 1127/1295 874/999 1410/1503

Number of mapped unigenes from Yunong 201/3114

5AS 639/750 5AL 1538/1853 5BS 682/804 5BL 2255/2670 5DS 761/867 5DL 2111/2560 6AS 963/1135 6AL 1040/1343 6BS 843/994 6BL 1028/1294 6DS 856/939 6DL 1263/1590 7AS 1004/1258 7AL 987/1251 7BS 824/923 7BL 1074/1244 7DS 1569/1860 7DL 1416/1723 Total 50,382/59,891 A genome (T. urartu) 45,256/53,163 D genome (Ae. tauschii) 48,156/57,116

The FDR results could be the priority selection criterion when determining DEGs. In this study, DEGs were analyzed using fold change in accordance with the unigene expression abundance value (RPKM) from the combined Yunong 201 and Yunong 3114. DEGs were observed between samples when the difference in expression quantities was more than twice the original expression value. This condition indicated that the fold change was transformed from log2 N 1 or b −1. Two groups of the binomial distribution statistical test method were utilized to calculate significant P-values and FDR. Significant P-values were calculated by subjecting the two groups to a binomial distribution statistical test method; FDR was calculated using the Benjamini and Hochberg method. We recommend using the FDR result as a reference when searching for DEGs. The difference in unigene expression between Yunong 201 and Yunong 3114 was considered when the total reads were N20 and FDR was b 0.05. A total of 1363 differentially expressed unigenes containing 1212 annotated genes were detected in the public NR-based FDR method (Additional file 4). Upregulated genes were those with significantly higher expression levels in Yunong 3114 than in Yunong 201. These genes were those encoding for pathogenesis-related (PR) protein, chitinase, plasma membrane intrinsic protein heat-shock protein (Hsp), aquaporin PIP1, auxin-induced protein X10A-like, MYB-related protein, NAC transcription factor (TF), Histone H3, histone H2B, and ubiquitinlike protein 5. Downregulated genes were those with significantly higher expression levels in Yunong 201 than in Yunong 3114. These genes were those encoding for photosystem I antenna protein, photosystem II CP43 chlorophyll apoprotein, chlorophyll a-b binding protein, photosystem II protein H, LHCI-680, extracellular invertase, and peroxidase. In this study, 1126 unigenes were upregulated and 237 unigenes were downregulated. Specificity was considered significant for the unique unigenes in Yunong 201 and Yunong 3114 when the total_reads were N 10 and FDR was b0.05. Approximately 54 and 213 unique unigenes in Yunong 201 and Yunong 3114 were identified, respectively. These unique unigenes of Yunong 201 and Yunong 3114 matched 23 and 99 different proteins in the public NR, respectively (Additional file 5). More unique unigenes were identified in Yunong 3114 than in

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

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Table 6 Four annotated DEGs related to stress responses. Gene-ID

Length(nt)

201-RPKMa

3114-RPKMa

FDR

Annotation

all_isotig10337 all_isotig22065 all_isotig07518 all_isotig07897

580 388 664 651

302.56 9.16 21.40 60.02

544.42 100.25 72.37 124.19

2.61E-11 2.34E-09 4.11E-05 6.63E-04

MYB-related protein [Aegilopsspeltoides] CRT/DRE binding factor 9[Triticummonococcum] TPA: NAC transcription factor[Hordeumvulgaresubsp. vulgare] cold acclimation protein COR413-TM1[Triticumaestivum]

a

201 and 3114 indicate Yunong 201 and Yunong 3114, respectively.

Yunong 201. The matched unique unigenes of Yunong 201 mainly contained categories of ribulose bisphosphate carboxylase, cell wallassociated hydrolase, and two-component response regulator-like PRR95-like molecule. Unique unigenes in Yunong 3114 mainly involved sec-23-like transport protein, histone H2B, 2-like isoform 1, 10-like ubiquitin carboxyl-terminal hydrolase, carbonyl reductase, LHCI-680, photosystem I antenna protein, cinnamyl alcohol dehydrogenase, UVH6-like DNA repair helicase, translation initiation factor 4G, and lipoxygenase 3. To further validate the reliability of the DEGs, 44 candidates were which possibly related to stresses were chosen from the differentially expressed unigenes to examine their expression levels via reverse transcriptase Real-Time PCR. cDNAs were generated from seedlings at the three-leaf stages of Yunong 201 and Yunong 3114, and specific primers for quantitative real-time PCR (qRT-PCR) were designed to amplify the unique cDNA fragments. The results showed that 40 out of the 44 primer sets were specific and were further used in qRT-PCR analysis (Additional file 8). Furthermore, 27 of the 40 candidate unigenes (70%) showed similar differences in expression levels to the results analyzed by 454 sequencing between Yunong 201 and Yunong 3114. That is, 21 of these unigenes were upregulated and the other 6 were downregulated (Fig. 3).

3.6. qRT-PCR analysis of four selected DEGs in response to abiotic stresses Due to the different stress resistance for the Yunong 201 and Yunong 3114, four DEGs (Additional file 6; Table 6) among the annotated genes were predicted to be intimately associated with stress response (cold, heat, drought, and salt) and were selected to examine their expression pattern in two wheat samples (Fig. 4).

3.6.1. Cold treatment At 2 h of cold stress, all_isotig22065 increased by more than 400-fold in Yunong 3114 but only by 275-fold in Yunong 201. Four genes (all_isotig10337, all_isotig22065, all_isotig07518, and all_isotig07897) were upregulated in Yunong 3114, whereas two genes (all_isotig10337 and all_isotig07518) initially upregulated but later downregulated in Yunong 201. Under cold stress (0-24 h), all_isotig07897 was downregulated in Yunong 201. 3.6.2. Heat treatment In response to heat treatment, four genes (all_isotig10337, all_isotig22065, all_isotig07518, and all_isotig07897) were downregulated in Yunong 201, and all_isotig10337 was downregulated in Yunong 3114. The remaining three genes (all_isotig22065, all_isotig07518, and all_isotig07897) initially increased and then decreased in Yunong 3114. 3.6.3. Drought treatment Three genes (all_isotig22065, all_isotig07518, and all_isotig07897) were upregulated in Yunong 3114 (0 to 24 h), whereas two genes (all_isotig07518 and all_isotig07897) were upregulated before 2 and 6 h of drought stress in Yunong 201 and then downregulated. Only the all_isotig22065 gene was upregulated at the later stage of drought stress in Yunong 201. 3.6.4. Salt treatment The all_isotig10337 gene was downregulated in both Yunong 201and Yunong 3114. The all_isotig22065 gene was also downregulated in Yunong 201. Two genes (all_isotig07518 and all_isotig07897)

Fig. 2. Functional classification of contig sequences based on GO categorization. Note: x-axis represents unigenes that were classified in particular components and terms. y-axis indicates the number of unigenes in a category as a percent of the total number of unigenes.

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

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the two wheat samples under abiotic stresses. These results possibly revealed that Yunong 201 and Yunong 3114 differed in their adaptation mechanisms under abiotic stresses. Our data not only provided useful information on wheat transcriptome by presenting new resources that could be used in future studies on wheat functional genomics, but also laid the foundation for the further study of Yunong 3114 withthe relative superior phenotypes. 4.1. Functional characterization

Fig. 3. Expression patterns of differentially expressed genes in the Yunong 201 and Yunong 3114. Top five up-regulated unigenes: 1–5 represent all_isotig05062, all_isotig09359, all_isotig22065, all_isotig07518, and all_isotig10049, respectively; Top five downregulated unigenes: 6–10 represent all_isotig07041, all_isotig07510, all_isotig03109, all_isotig21806, all_isotig01111, respectively.

were upregulated in Yunong3114 from 0 to 24 h under salt stress. However, the expression levelsof all_isotig07518 and all_isotig07897 were upregulated at 1and 2 h after the treatment in Yunong 201, respectively. 4. Discussion RNA-Seq was used to obtain a large amount of transcriptome information of the bread wheat cultivar Yunong 201 and its EMS mutant line Yunong 3114. Functional characterization of the unigene sequences were performed through GO annotation and KEGG pathway. qRT-PCR analysis confirmed the expression profiles of 40 candidate unigenes, and the expression patterns of four annotated DEGs were verified in

We obtained large amounts of transcriptome information of the bread wheat cultivar Yunong 201 and its EMS mutant line Yunong 3114 by Roche 454 NGS. Approximately 29,042, 34,722, and 48,486 unigenes were identified in the three groups, respectively. The unigenes in Yunong 201, Yunong 3114, and their combination were subjected to GO annotation. The GO terms included several categories, including molecular functions, biological processes, and cellular components. The largest percentages of unigenes among the three groups were classified in the molecular function category. The major categories of molecular function were binding and catalytic activities (91.0%); this result is similar to that reported in other studies on wheat and corn (Alexandrov et al., 2009; Kikuchi et al., 2003; Manickavelu et al., 2012; Zhang et al., 2004). In the three groups, biological, metabolic, and cellular processes exhibit important functions; cell and cell part categories accounted for 70% of the cell component category. No significant difference was observed in the proportion of the genes in the three GO term categories between Yunong 201 and Yunong 3114. This result suggested that a similar distribution of unigenes in the three functional categories could be observed. We also performed KEGG pathway mapping that categorized gene functions in terms of the biochemical pathways of the unigene sequences. All of the unigenes were mainly involved in the categories of metabolism, genetic information processing, cellular processes, and environmental information processing. Numerous

Fig. 4. qRT-PCR analysis of four DEGs genes in the Yunong 201 (201) and Yunong 3114 (3114) in response to different abiotic stresses. Four (a, b, c, and d) abiotic stresses: cold, heat, drought, and salt; Each color represents a range of relative expression. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

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unigenes with different functions were potential candidates for further studies on wheat genetics and breeding. Due to the large differences on plant architecture, kernel size, thousand grain weight and higher yield per plot as well as processing quality of the Yunong 201 and Yunong 3114, comparison of transcriptomes of Yunong 201 and Yunong 3114 would provide valuable information for further dissection of molecular and genetics basis of some wheat phenotypes related to yield and quality in bread wheat. 4.2. Differential gene expression In the present study, DEGs related to growth, development, stress, and photosynthesis were identified by comparing the expressed genes of Yunong 201 and Yunong 3114. The following representatives were obtained: PR protein, chitinase, plasma membrane intrinsic protein, Hsp, aquaporin PIP1, MYB-related protein, NAC TF, and UVH6-like DNA repair helicase. Economic traits such as growth and yield are of particular interest to researchers of cereal crops (e.g., wheat, rice, and maize). Such traits are either directly or indirectly affected by genes. The sequence and annotation information from the BLAST, GO, and KEGG annotations of DEGs provided valuable sources for molecular studies that determine these economical traits. We also identified DEGs that encode for different groups of growth factors associated with cell growth and target gene expression. These genes included those that encoded small upregulated Ras-related GTP-binding protein, which is involved in the control of a diverse set of important cellular functions, including growth, differentiation, cytoskeletal organization, intracellular vesicle transport, and secretion (Hall, 1990). As a part of the largest plant-specific TF families, the NAC TF has an important function in plant development processes, response to biotic and abiotic cues, and hormone signaling (Singh et al., 2013). Similar to the TF, MYB proteins form one of the largest TF families and exhibit a crucial function in development and stress response in plants (Du et al., 2013). CRT/DRE binding factor 9 was also upregulated; dehydration-responsive-element-binding protein/Crepeat binding factors belong to the APETALA2 family of TFs that bind to DRE/CRT cis-element and regulate the expression of stressresponsive genes (Akhtar et al., 2012). Phosphatase serine/threonineprotein phosphatase PP1-like (PP1) was upregulated; PP1 is one of two major functional groups of phosphatases that regulate many signaling pathways; function in the negative feedback of different kinases to regulate the phosphorylation of diverse substrates and developmental processes; and participate in diverse cellular processes, such as cell cycle progression, protein synthesis, carbohydrate metabolism, transcription, and neuronal signaling (Dai et al., 2013; Fischer, 1999; Wera and Hemmings, 1995). The coordinated development of multicellular plants requires a regulated cell division and expansion; cell surface receptors, known as receptor-like protein kinases (RLKs), have emerged as potential key regulators. RLKs also function in diverse biological processes, such as self-incompatibility and disease resistance (Lease et al., 2001). Among the DEGs, three types of RLKs were upregulated. In this study, AFC2-like serine/threonine-protein kinase was upregulated. Several protein kinases are important regulators of cell proliferation, survival, and cell death (Reyland, 2009). The identification of stress-related DEGs may be of interest to wheat researchers because of the increasing environmental pressures and devastating effects of diseases. KEGG and GO analyses could be performed to identify DEGs that are potentially involved in response to environmental pressures and stimuli. These DEGs include those that encode for PR protein, chitinase, Hsp, aquaporin PIP1, cinnamyl alcohol dehydrogenase, cell wall-associated hydrolase, and subtilisin-chymotrypsin inhibitor 2. Most plants are immobile; therefore, physiological processes that rapidly respond to changing environmental conditions are essential for plant survival. In contrast to other organisms, plants may need a more sophisticated tuning of water balance. Among other conditions,

such water balance is indicated by aquaporin PIPs in plants (Kaldenhoff and Fischer, 2006); these substances are membrane channels that facilitate water movement across cell membranes (Péret et al., 2012) and can be divided into two phylogenetic groups: PIP1 and PIP2. The PIP1 isoforms are tightly conserved, sharing N 90% amino acid sequence identity (Fraysse et al., 2005). The expression levels of aquaporin PIP1, which could be related to the osmoregulation and metabolite transport of aquaporin PIP1, were higher in Yunong 3114 than in Yunong 201. Heat shock proteins (Hsps)/chaperones are responsible for protein folding, assembly, translocation, and degradation in many normal cellular processes; Hsps also stabilize proteins and membranes as well as assist in protein refolding under stressful conditions. Moreover, Hsps protect plants from stress by reestablishing normal protein conformation and cellular homeostasis (Wang et al., 2004). Hsps were upregulated in Yunong 3114, conferring strong protection to proteins against degradation. PR protein and chitinase are upregulated genes. PR proteins are “proteins encoded by the host plant but induced only in pathological or related situations,” in which the latter refers to non-pathogenic situations (Reddy, 2013). Chitinases are highly characterized classes of PR proteins because these enzymes degrade chitin, a structural component of fungal cell wall (Punja, 2001). The disease resistance of mutant Yunong 3114 significantly increased. This result is similar to that in a recent report, indicating that the expression of a rice chitinase gene in transgenic banana provides resistance to black leaf streak disease (Kovács et al., 2013). We also observed the upregulation of thaumatin-like protein (TLP), an intensely sweet PR protein isolated from the fruits of the West African rain forest shrub Thaumatococcus daniellii. TLP can be classified into three groups: (i) those produced in response to pathogen infection; (ii) those produced in response to osmotic stress (or osmotins); and (iii) antifungal proteins present in cereal seeds. TLPs are generally resistant to proteases and pH- or heat-induced denaturation. Lignin is a phenolic heteropolymer in secondary cell walls and participates in plant development and defense against pathogens; lignin is also upregulated. The biosynthesis of monolignols, the main component of lignin, involves many enzymes; cinnamyl alcohol dehydrogenase is a key enzyme in lignin biosynthesis and catalyzes the final step in monolignol synthesis (Barakat et al., 2009), which was induced in mutant Yunong 3114. These results indicated a stronger defense capability of mutant Yunong 3114 than Yunong 201. UVH6-like DNA repair helicase was triggered in mutant Yunong 3114. This enzyme repairs DNA to prevent errors in DNA replication. Ubiquitin-like protein 5 was also upregulated, suggesting its possible function in degrading error codes of proteins in mutant Yunong 201. However, the expression levels of photosystem I antenna protein, photosystem II CP43, chlorophyll apoprotein, chlorophyll a-b binding protein, photosystem II protein H, and LHCI-680 decreased in Yunong 3114. These substances are related to a decrease in the photosynthetic ability of the mutant Yunong 201. 4.3. Gene expression analysis in abiotic stresses Abiotic stresses trigger a wide range of plant responses from the alteration of gene expression and cellular metabolism to changes in plant growth and development and crop yields (Barakat et al., 2009). To survive under abiotic stresses, plants have evolved complex signaling pathways and induced stress-related genes (Barakat et al., 2009). In the present study, the public information in NCBI was used as a basis to predict that four of the identified DEGs were intimately associated with abiotic stress. The expression patterns of these DEGs were compared between Yunong 201 and Yunong 3114. The selected DEGs were TFs and stress-related genes. The four DEGs were upregulated in Yunong 3114 under cold stress; by contrast, one of them was downregulated in Yunong 201, and two were upregulated before 1 h cold stress. Under heat stress, the four DEGs were downregulated in Yunong 201

Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002

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during the heat stress (0 to 24 h), whereas three of them were upregulated at the early stage of heat stress in Yunong 3114. Overall, the expression levels of the four DEGs were higher in Yunong 3114 than in Yunong 201. This result might be ascribed to the relatively stronger adaptability to abiotic stress of Yunong 3114. Therefore, the DEGs we selected in this study would be useful for further uncovering the genes related to abiotic stress in bread wheat. 5. Conclusions In this study, high-throughput RNA-Seq was applied to obtain transcriptomes of bread wheat cultivar Yunong 201 and its EMS derivative Yunong 3114. Genes were detected, annotated, and functionally characterized; differentially expressed genes were analyzed. A total of 50,382 and 59,891 unigenes from the Yunong 201 and Yunong 3114 were mapped on different chromosomes in bread wheat. The annotated genes could be used as potential candidates. qRT-PCR analysis confirmed the expression patterns of 40 candidate genes. Additionally, the expression patterns of four annotated DEGs were illustrated in the two cultivars and those DEGs could be useful for further examining genes related to abiotic stress in bread wheat. Our results provided information of wheat transcriptome by using new resources and benefits the future studies on wheat functional genomics. Availability of supporting data: The authors confirm that all data underlying the findings are fully available without restriction. The data sets supporting the results of this article are available in the NCBI SRA repository, [http://www.ncbi.nlm.nih.gov/sra/?term=SRR1685717]. TSA repository accession number: GBZP00000000. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.gene.2015.09.002. Competing interests The authors declare that they have no competing interests. Authors' contribution FC and DC designed the project. NZ and FC performed RNA-seq experiments and wrote the paper. NZ, XZ, SW and ZD performed the computational analyses. Acknowledgments This project was funded by the 973 projects (2014CB138105 and 2014CB160303), National Natural Science Foundation (31370031) and Program for New Century Excellent Talents in University of China (NCET-13-0776). References Akhtar, M., Jaiswal, A., Taj, G., Jaiswal, J., Qureshi, M., Singh, N., 2012. DREB1/CBF transcription factors: their structure, function and role in abiotic stress tolerance in plants. J. Genet. 91, 385–395. Akhunov, E., 2013. Genomic Architecture Of Rust-Wheat Interaction: Implications For Breeding Disease-Resistant Crops. Plant and Animal Genome XXI Conference. Plant and Animal Genome. Alexandrov, N.N., Brover, V.V., Freidin, S., Troukhan, M.E., Tatarinova, T.V., Zhang, H., Swaller, T.J., Lu, Y.-P., Bouck, J., Flavell, R.B., 2009. Insights into corn genes derived from large-scale cDNA sequencing. Plant Mol. Biol. 69, 179–194. Barakat, A., Bagniewska-Zadworna, A., Choi, A., Plakkat, U., DiLoreto, D., Yellanki, P., Carlson, J., 2009. The cinnamyl alcohol dehydrogenase gene family in populus: phylogeny, organization, and expression. BMC Plant Biol. 9, 26. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T., 2009. BLAST+: architecture and applications. BMC Bioinf. 10, 421. Chen, F., Zhang, F.-Y., Xia, X.-C., Dong, Z.-D., Cui, D.-Q., 2012. Distribution of puroindoline alleles in bread wheat cultivars of the Yellow and Huai valley of China and discovery of a novel puroindoline a allele without PINA protein. Mol. Breed. 29, 371–378.

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Please cite this article as: Zhang, N., et al., Transcriptome analysis of the Chinese bread wheat cultivar Yunong 201 and its ethyl methanesulfonate mutant line, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.09.002