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

Highlights

• Of all unigenes, 1363 DEGs were identified in Yunong 201 and Yunong 3114.

• The response ability to drought and salt stress of the two cultivars were investigated.

• qRT-PCR analysis of four selected annotated DEGs in response to abiotic stresses.

Abstract

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.

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.

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 (P_i) 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 host-specific 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 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.