Development of a 44 K custom oligo microarray using 454 pyrosequencing data for large-scale gene expression analysis of Camellia sinensis

Abstract

Tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important woody crop with a currently unsequenced full genome, which limits the ability to perform functional genome studies. Next-generation Sequencing (NGS) technologies, such as RNA sequencing (RNA-seq), provide a powerful approach for quantifying the transcriptome. In this study, RNA-seq was conducted using the Roche 454 platform in tea new shoots, generating 612,014 high-quality reads with an average sequence length of 343 base pairs. After the de novo assembly, 60,479 unigenes consisting of 32,384 contigs and 28,095 singletons were detected, including 10,141 novel genes which showed no homology with reported Camellia sinensis sequences. To investigate the large-scale gene expression in different tea plant clones, a custom oligonucleotide microarray was developed using sequences from the RNA-seq study to design the probes because a commercial array is unavailable for the tea plant. A 4×44K 60-mer oligo microarray consisting of 43,833 probes covering 42,440 unigene sequences and 1417 positive and negative Agilent control sequences was designed and used to analyze the expression profiles of four tea plant clones. The microarray data demonstrated that the dataset can be used for typical analyses, including principal component analysis (PCA) and differential gene expression analysis. The PCA showed that the four genotypes were differentiated from each other with good biological repeatability. To investigate the mechanism of higher amino acid content in ‘Zhongcha 108’, ‘Zhonghuang 1’ and ‘Zhonghuang 2’, genes involved in amino acid metabolism were analyzed and significant differential expression of a number of these genes was seen in the three cultivars compared to ‘Longjing 43’. Twenty differentially expressed genes were validated by qRT-PCR, and the qRT-PCR results displayed a similar tendency in variation as the microarray results. We have developed a tea plant oligonucleotide microarray that can be used to analyze gene expression in the shoots of different tea plant clones without prior sequence knowledge. The combination of RNA-seq and custom microarray design was shown to be a powerful method for identifying differential expression genes among different tea cultivars.