RNA-Seq Analysis of Compatible and Incompatible Styles of Pyrus
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bioRxiv preprint doi: https://doi.org/10.1101/344044; this version posted June 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of 2 pollination 3 Running title: RNA-seq of Pyrus 4 5 Yaqin Guan 6 College of Horticulture, Qingdao agricultural university, China, Qingdao City, 7 [email protected] 8 Kun Li 9 College of Horticulture, Qingdao agricultural university, China, Qingdao City, 10 [email protected] 11 Yongzhang Wang 12 College of Horticulture, Qingdao agricultural university, China, Qingdao City, 13 [email protected] 14 Chunhui Ma 15 College of Horticulture, Qingdao agricultural university, China, Qingdao City, 16 [email protected] 17 Corresponding author 18 Haiyong Qu* 19 College of Horticulture, Qingdao agricultural university, No. 700 Changcheng road, 20 Chengyang, Qingdao City, Shandong province, China, [email protected]. Phone 21 number: 86-0532-86080740, Postcode: 266109 22 1 bioRxiv preprint doi: https://doi.org/10.1101/344044; this version posted June 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 Abstract: In Rosaceae, incompatible pollen can penetrate into the style during the 24 gametophytic self-incompatibility response. It is therefore considered a stylar event rather 25 than a stigmatic event. In this study, we explored the differences in gene expression between 26 compatibility and incompatibility in the early stage of pollination. The self-compatible pear 27 variety “Jinzhuili” is a naturally occurring bud mutant from “Yali”, a leading Chinese native 28 cultivar exhibiting typical gametophytic self-incompatibility. We collected the styles of ‘Yali’ 29 and ‘Jinzhuili’ at 0.5 and 2 h after self-pollination and then performed high-throughput 30 sequencing. According to the pathway enrichment analysis of the differentially expressed 31 genes, “plant-pathogen interaction” was the most represented pathway. Quantitative PCR was 32 used to validate these differential genes. The expression levels of genes related to pollen 33 growth and disease inhibition, such as LRR (LEUCINE-RICH REPEAT EXTENSIN), 34 resistance, and defensin, differed significantly between compatible and incompatible 35 pollination. Interestingly, at 0.5 h, most of these genes were upregulated in the compatible 36 pollination system compared with the incompatible pollination system. Calcium ion transport, 37 which requires ATPase, also demonstrated upregulated expression. In summary, the 38 self-incompatibility reaction was initiated when the pollen came into contact with the stigma. 39 Keywords: Pyrus species, Styles, RNA-seq, Pyrus bretschneideri Rehd, differentially 40 expressed genes 41 2 bioRxiv preprint doi: https://doi.org/10.1101/344044; this version posted June 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 Introduction 43 Self-incompatibility (SI) is the general term used to describe several genetic mechanisms 44 in angiosperms that prevent self-fertilization and thus encourage outcrossing and allogamy. 45 RNase-based gametophytic self-incompatibility (GSI) is the most phylogenetically 46 widespread, occurring in at least three families: Solanaceae, Rosaceae, and Scrophulariaceae 47 (Igic et al., 2004). Most fruit tree species belong to family Rosaceae and subfamily 48 Spiraeoideae, which includes the supertribe Spiraeoideae that consists of the tribes Maleae 49 (e.g., Malus and Pyrus) and Amygdaleae (e.g., Prunus), all of which exhibit typical 50 gametophytic self-incompatibility (Shulaev et al., 2008). Wu et al. (2012) reviewed the 51 research progress in GSI mechanisms in fruit crops such as pear, apple, almond, sweet cherry, 52 apricot, Japanese apricot, plum, and sour cherry (Wu et al., 2012). As pear was the first fruit 53 tree species in which S-ribonucleases were identified, the complete history of S-genotyping 54 and technical developments can be monitored by evaluating the reported experiments (Halász 55 & Hegedûs, 2006). The draft genome of pear (Pyrus bretschneideri) was generated using a 56 combination of BAC-by-BAC and next-generation sequencing (Wu et al., 2013b). 57 In Rosaceae, GSI is controlled by a single multi-allelic S locus that is composed of the 58 pistil-S and pollen-S genes. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase) 59 essential for discriminating self-pollen; however, the S-RNase system has not been fully 60 elucidated. SLF (S-locus F-box) genes appear to be involved in the determination of pollen-S 61 specificity, but the function of F-box genes in the SI reaction remains unknown. There is no 62 direct evidence of the interaction between SLF and different alleles/domains of the S-RNase 63 protein. McClure (2006) proposed the compartmentalization model of GSI, but no evidence 64 exists regarding when the vesicles begin to break down following pollination incompatibility 65 (McClure, 2006). In addition, a number of researchers suggested that S-RNase specifically 66 induces tip-localized reactive oxygen species (ROS) disruption, actin cytoskeleton 67 depolymerisation, and nuclear DNA degradation in the incompatible pollen tubes of pear (Liu 68 et al., 2007; Wang et al., 2010a). We also found that self S-RNase selectively blocks the 69 activity of Ca2+ channels at the apical pollen tube of pear (Qu et al., 2016). It has been 70 suggested that pollen rejection and incompatibility systems are interconnected and should be 71 regarded as different aspects of a single system that provide fine control over plant 72 fertilization (Cruz‐Garcia et al., 2003). 73 For fruit trees, including pear, a stable transgenic system is highly difficult to construct. 74 However, we were able to obtain ‘Jinzhuili’, which is a naturally occurring self-compatible 3 bioRxiv preprint doi: https://doi.org/10.1101/344044; this version posted June 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 75 mutant of ‘Yali’ (Pyrus bretschneideri Rehd.). Field pollination tests showed that >76% of 76 flowers set fruit and many pollen tubes grew down to the base of the style during ‘Jinzhuili’ 77 self-pollination and cross-pollination of ‘Yali’ (female) × ‘Jinzhuili’ (male). In contrast, fruit 78 setting did not occur and most pollen tubes were arrested at the upper part of the style during 79 ‘Yali’ self-pollination and cross-pollination of ‘Jinzhuili’ (female) × ‘Yali’ (male). These 80 results indicate that ‘Yali’ and ‘Jinzhuili’ exhibit a normal SI response in the style, rejecting 81 competent ‘Yali’ pollen but accepting ‘Jinzhuili’ pollen. Further molecular analyses revealed 82 that both ‘Yali’- and ‘Jinzhuili’-labelled S21-RNase and S34-RNase genes were transcribed 83 normally with identical mRNA sequences. Therefore, we suggest that ‘Jinzhuili’ has most 84 likely lost pollen SI activity, resulting in a style that behaves normally during the SI response, 85 similar to that of the wild-type ‘Yali’. However, Wu et al. (2013) identified five SLF (S-Locus 86 F-box) genes in ‘Yali’ but no nucleotide differences were observed in the SLF genes of 87 ‘Jinzhuili’ (Wu et al., 2013a). We selected ‘Jinzhuili’ and ‘Yali’, two cultivars of the same 88 species with the same genetic background but with some differing genetic mutations, to 89 perform high-throughput sequencing in order to explore the variation in gene expression 90 between compatible and incompatible pollination. 91 Present research on self-incompatibility in Rosaceae has predominantly focused on the 92 response of the pollen tubes in the style, but little data exists regarding the role of the stigma 93 at the start of pollination. Therefore, we selected the styles as the tissue with which to perform 94 high-throughput sequencing analysis at 0.5 h and 2 h after pollination. 95 Results 96 De novo assembled transcriptomes of self-compatible and self-incompatible styles 97 Five whole-plant RNA bulks derived from the styles of ‘Jinzhuili’ at 0.5 h (JZ_0.5h) and 98 2 h (JZ_2h) after self-pollination and the styles of ‘Yali’ without pollination (control, YL_CK), 99 and at 0.5 h (YL_0.5h) and 2 h (YL_2h) after self-pollination were utilized for transcriptome 100 sequencing analysis to identify genes that were differentially expressed between the 101 self-compatible and self-incompatible style and the pollen tube. RNA sequencing (RNA-Seq) 102 generated approximately 4.46 Gb raw reads from each of the five transcriptomes. After 103 filtering the reads, the clean reads were mapped to the reference genome using HISAT (Kim 104 et al., 2015). An average of 59.32% reads was mapped, and the uniformity of the mapping 105 result for each sample suggested that the samples were comparable. The mapping details are 106 shown in Table 1. After mapping the sequenced reads to the reference genome and 107 reconstructing the transcripts, we ultimately obtained 8,559 novel transcripts from all of the 108 samples, of which 5,768 constitute previously unknown splicing events for a known gene; 4 bioRxiv preprint doi: https://doi.org/10.1101/344044; this version posted June 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 109 1,185 are novel coding transcripts without any known features; and the remaining 1,606 are 110 long noncoding RNAs. 111 Differential expression statistics 112 There were 1,795 and 1,658 differentially expressed genes (DEGs) between the style of 113 the control and ‘Jinzhuili’ at 0.5 and 2 h after self-pollination, respectively. There were 3,842 114 and 3,787 DEGs between the style of the control and the style of ‘Yali’ at 0.5 and 2 h 115 following self-pollination, respectively.