Rna-seq Analysis Of Zanthoxylum Bungeanum Toidentify Putative Genes Involved In Prickle Differentiation

Huajiao (Zanthoxylum bungeanumMaxim.) is a common medicinal plant, and the driedfruit follicles are used as an important culinary spice in China. As the increase of demand forthe fruit, picking becomes a big challenge. Due to its sharp prickle outgrowth of the epidermis,it usually injure the farmers in harvesting, as a result picking cost doubled. Finding out theessential genes in prickle differentiation will help us breeding none-prickle huajiao. However,the lack of extensive Z. bungeanum cDNA libraries and large expressed sequence tag databaselimited the efficiency of breeding. Fortunately, the Next Generation Sequencing provides anapproach to obtain massive transcriptome sequences rapidly with acceptable cost. Two typetissues, node and internode of stem tip, were sequenced by Illumina HiseqTM2500platform.The main results were as follows:1. We totally obtained10.23G short reads from the two type tissues and these readswere de novo assembled by Trinity software to obtain45,057non-redundant Unigenes with anaverage length of610base pairs. The total length is32.5M and N50is846base pairs.2. For validation and annotation of the assembled Unigenes, sequence similarity searchwas conducted in the Swiss-Prot, NR, Pfam, KEGG, COG and GO databases, and the resultsindicated that49.82%,80.34%,40.23%,78.83%,43.34%and70.26%Unigenes, using acut-off p-value of105, showed significant similarity to these databases, respectively.3. Of the45,057Unigenes,3,315Unigenes were identified to contain3,814SSRs,422of the Unigenes contained two or more SSRs.4. Comparison between assembled Unigenes of two samples indicated that411Unigenes were significant difference (p-value<0.05, fold change<2), including276up-regulated,135down-regulated genes.5. GO and Pathway enrichment analysis identifies significantly enriched metabolicpathways or signal transduction pathways in DEGs comparing with the whole transcriptomebackground. With the threshold of p-value <0.05,133GO terms and23pathways wereobtained which including Flavonoid biosynthesis, Pyruvate metabolism, Citrate cycle andGlycolysis/Gluconeogenesis, etc. 6. FifteenUnigenes were selected for qRT-PCR analysis using the same RNA sample asfor deep sequencing, including7differentially expression genes (EXLB1, YAB1, Unknown319,YAB2, YAB4, TMM, and AS1), two ARFs (ARF5, ARF23), WRK18and5candidate referencegenes (ACT7, TUBB8, TUBA3, APT1and UBI3). The qRT-PCR results confirmed the dataobtained from deep sequencing analysis and showed similar trends in up regulated Unigenes.In order to catch the essential genes in prickle differentiation, the sampling plan shouldbe designed very carefully, since the transcriptome of a cell is changing all the time. Samplesof two type tiny stem tissues were collected carefully from the shoot apical meristem with asingle tree. We mixed the node of stem where prickle differentiation, and the inner node ofstem is took as control sample. Finally, we obtained massive transcriptome and annotationdata. In addition, lots of SSRs were scanning from the data. SSR technology has wideapplications in vegetable breeding, including genetic mapping, DNA fingerprinting andgenetic diversity studying of germplasms.411different expressed genes were screened bycalculating RPKM value, and10of them which mainly including ARFs and YABBYs genefamily were selected, since they may related to the polar cell division. These10candidategenes were verified through qRT-PCR. Abundance of transcripts are match with the RNA-Seqresults. Massive data yield in this study provide a wealth of information which helps us tounderstand the molecular mechanism of prickle differentiation.