Sequencing analysis of pollen transcriptome of 'Yali' and its spontaneous self-compatible mutant 'Jinzhui'

Abstract:【Objective】‘Jinzhui' (JZ) is a spontaneous self-compatible mutant of‘Yali' (Pyrus bretschneideri Rehd., YL) with a typical S-RNase-based gametophytic self-incompatibility (GSI) . The phenotypic changes of the pollen-part mutation (PPM) ‘Jinzhui'might be due to a natural mutation in the pollen-S gene. However, the molecular mechanisms behind these phenotypic changes remain unclear. To understand the possible mechanisms in response to SI, a comparative transcriptomic analysis with pollenes of YL and JZ was performed in order to provide valuable information for analyzing the candidate self-incompatibility associated genes of P. bretschneideri Rehd.【Methods】Pollen samples of YL and JZ were collected as experimental materials and high-throughput next generation sequencing technology RNA-seq was used to conduct sequencing. Sequence comparison with designated reference genome was performed to obtain mapped data. From the comparison of transcriptomic data of YL pollenes and JZ pollenes, differentially expressed genes (DEGs) were identified and the regulated models were analyzed. To better understand the distribution of gene functions at the macro level, the GO function classi-fication of the DEGs were analyzed using the WEGO online tool. To further investigate the influence of the DEGs on pathways, statistical pathway enrichment analysis of DEGs was performed based on KEGG database. To identify differentially expressed genes associated with self-incompatibility (SI) in P. bretschneideri Rehd., the expression levels of SI related DEGs were measured based on the fragments per kb per million of the mapped reads (FPKM) value.【Results】Through transcriptome sequencing data analysis, totally 44 778 208 and 44 995 100 clean reads were generated in the YL and JZ libraries after removing adaptor, ambiguous and low-quality reads, and the GC contents were 47.5% and47.7% respectively. The Q30 contents of two samples were both over 95%, indicating the high quality of transcriptome sequencing and the high accuracy of the data. Of these high-quality reads, 66.35% and66.08% were aligned to reference genome or gene sequences, respectively, and for unique alignment position the sequence alignment efficiency between reads of both samples and reference genome and genes was over 55.02%. For these aligned genes in YL and JZ, FPKM method was used to get the standard measure. The significance of gene expression differences of YL and JZ was determined using the threshold of FDR≤ 0.001 and |l|log2 Ratio|≥|≥ 1. Totals of 136 differentially expressed genes (DEGs) were obtained between samples YL and JZ. Specifically, the expression levels of 76 genes were up-regulated and 60 genes were down-regulated in sample YL compared with sample JZ. For these up-and downregulated genes, GO and KEGG analysis were performed. When the DEGs matched to the GO terms, a total of 68 of these DEGs were associated with 33 subcategories belonging to 3 categories, biological process, cellular component and molecular function. Among the biological process category, “metabolic process”and“cellular process”were the main functional groups, which were followed by“single organism process”and“response to stimulus”. In terms of cellular component, “cell part”and“cell”were the most highly represented subcategories. For the molecular function category, “catalytic activity”and“binding”were the two main groups. To further investigate biological behavior, the DEGs were assigned to the biochemical pathways described in the KEGG database. A total of 86 DEGs were assigned to the 49 KEGG pathways, including metabolic pathways, biosynthesis of secondary metabolites, plant-pathogen interaction, signal transduction, ubiquitin-mediated proteolysis, and RNA degradation. To identify differentially expressed genes associated with SI in YL and JZ, the expression levels of 41 DEGs were measured based on the fragments per kb per million of the mapped reads (FPKM) value. Several notable genes were potentially involved in SI responses, such as those involved in pollen tube growth, RNA degradation and stress resistance, polyubiquitin, ubiquitin conjugating enzyme complex member, vesicle-mediated transporter were identified. Some defense-related genes were upregulated in JZ pollen sample, which might function not only in defense but also in response to recognition process. Notably, a gene ubiquitin-conjugating enzyme E2 variant 1 D-like (103966960) was found for over 40 times difference of the gene expression. Further study is necessary on those genes that might be associated with SI.【Conclusion】The results of transcriptome analysis suggested that multiple genes might be associated with SI in P. bretschneideri Rehd. We hypothesized that the gene, ubiquitin-conjugating enzyme E2 variant 1 D-like, might be crucial for self-recognition. However, further studies are required to fully understand the role of the candidate gene in SI. Our study provides a pool of SI-related genes in P. bretschneideri Rehd. and offers a valuable resource for elucidating the mechanisms of SI in Pyrus.