High-quality genome assembly of 'Cuiguan' pear (Pyrus pyrifolia) as a reference genome for identifying regulatory genes and epigenetic modifications responsible for bud dormancy

Abstract

Dormancy-associated MADS-box (DAM) genes serve as crucial regulators of the endodormancy cycle in rosaceous plants. Although pear DAM genes have been identified previously, the lack of a high-quality reference genome and techniques to study gene function have prevented accurate genome-wide analysis and functional verification of such genes. Additionally, the contribution of other genes to the regulation of endodormancy release remains poorly understood. In this study, a high-quality genome assembly for 'Cuiguan' pear (Pyrus pyrifolia), which is a leading cultivar with a low chilling requirement cultivated in China, was constructed using PacBio and Hi-C technologies. Using this genome sequence, we revealed that pear DAM genes were tandemly clustered on Chr8 and Chr15 and were differentially expressed in the buds between 'Cuiguan' and the high-chilling-requirement cultivar 'Suli' during the dormancy cycle. Using a virus-induced gene silencing system, we determined the repressive effects of DAM genes on bud break. Several novel genes potentially involved in the regulation of endodormancy release were identified by RNA sequencing and H3K4me3 chromatin immunoprecipitation sequencing analyses of 'Suli' buds during artificial chilling using the new reference genome. Our findings enrich the knowledge of the regulatory mechanism underlying endodormancy release and chilling requirements and provide a foundation for the practical regulation of dormancy release in fruit trees as an adaptation to climate change.

References

[1] Lang, G. A., Early, J. D., Darnell, R. L. & Martin, G. C. Endo-, para-, and ecodormancy: physiological terminology and classification for dormancy research. Hortscience 22, 371-377 (1987).

[2] Saito, T. et al. Development of flower buds in the Japanese pear (Pyrus pyrifolia) from late autumn to early spring. Tree Physiol. 35, 653-662 (2015).

[3] Ito, A. et al. Comparative phenology of dormant Japanese pear (Pyrus pyrifolia) flower buds: a possible cause of ‘flowering disorder’. Tree Physiol. 38, 825-839 (2018).

[4] Legave, J. M. et al. A comprehensive overview of the spatial and temporal variability of apple bud dormancy release and blooming phenology in Western Europe. Int. J. Biometeorol. 57, 333-335 (2013).

[5] Bielenberg, D. G. et al. Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation. Tree Genet. Genomes 4, 495-507 (2008).

[6] Sasaki, R. et al. Functional and expressional analyses of PmDAM genes associated with endodormancy in Japanese apricot. Plant Physiol. 157, 485-497 (2011).

[7] Yamane, H. Regulation of bud dormancy and bud break in Japanese apricot (Prunus mume Siebold & Zucc.) and peach [Prunus persica (L.) Batsch]: a summary of recent studies. J. Jpn. Soc. Hort. Sci. 83, 187-202 (2014).

[8] Wu, R. M. et al. SVP-like MADS box genes control dormancy and bud break in apple. Fronti. Plant Sci. 8, 477 https://doi.org/10.3389/fpls.2017.00477 (2017).

[9] Saito, T. et al. Expression and genomic structure of the dormancy-associated MADS box genes MADS13 in Japanese pears (Pyrus pyrifolia Nakai) that differ in their chilling requirement for endodormancy release. Tree Physiol. 33, 654-667 (2013).

[10] Niu, Q. et al. Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia White Pear Group) flower bud. J. Exp. Bot. 67, 239 (2016).

[11] Ubi, B. E. et al. Molecular cloning of dormancy-associated MADS-box gene homologs and their characterization during seasonal endodormancy transitional phases of Japanese pear. J. Am. Soc. Hortic. Sci. 135, 174 (2010).

[12] Li, J. Z. et al. PpCBFs selectively regulate PpDAMs and contribute to the pear bud endodormancy process. Plant Mol. Biol. 99, 575-586 (2019).

[13] Yang, Q. et al. ABA-responsive ABRE-BINDING FACTOR3 activates DAM3 expression to promote bud dormancy in Asian pear. Plant Cell Environ. 43, 1360-1375 (2020).

[14] Singh, R. K. et al. A genetic network mediating the control of bud break in hybrid aspen. Nat. Commun. 9, 4173 (2018).

[15] Tylewicz, S. et al. Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication. Science 360, 212-215 (2018).

[16] Leida, C., Conesa, A., Llácer, G., Badenes, M. L. & Ríos, G. Histone modifications and expression of DAM6 gene in peach are modulated during bud dormancy release in a cultivar-dependent manner. N. Phytol. 193, 67-80 (2012).

[17] Santamaría, M. E., Rodríguez, R., Cañal, M. J. & Toorop, P. E. Transcriptome analysis of chestnut (Castanea sativa) tree buds suggests a putative role for epigenetic control of bud dormancy. Ann. Bot. 108, 485 (2011).

[18] Vimont, N. et al. ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy. Tree Genet. Genomes 16, 9 https://doi.org/10.1007/s11295-019-1395-9 (2019).

[19] Zhu, H. et al. Thermal-responsive genetic and epigenetic regulation of DAM cluster controlling dormancy and chilling requirement in peach floral buds. Hort. Res. 7, 114 (2020).

[20] Saito, T. et al. Histone modification and signalling cascade of the dormancy-associated MADS-box gene, PpMADS13-1, in Japanese pear (Pyrus pyrifolia) during endodormancy. Plant Cell Environ. 38, 1157-1566 (2015).

[21] Liu, G. et al. Transcriptomic analysis of ‘Suli’ pear (Pyrus pyrifolia White Pear Group) buds during the dormancy by RNA-Seq. BMC Genom. 13, 700 (2012).

[22] Feng, L., Ma, C., Wu, H., Qi, K. & Zhang, S. Comparative studies of chilling requirement and covering time of forcing cultivation of different pear cultivars in Nanjing. Chin. Agric. Sci. Bull. 29, 94-98 (2013).

[23] Wu, J. et al. The genome of the pear (Pyrus bretschneideri Rehd.). Genome Res. 23, 396 (2013).

[24] Linsmith, G. et al. Pseudo-chromosome-length genome assembly of a double haploid “Bartlett” pear (Pyrus communis L.). Gigascience 8: 1-9, https://doi.org/10.1093/gigascience/giz138 (2019).

[25] Dong, X. et al. De novo assembly of a wild pear (Pyrus betuleafolia) genome. Plant Biotechnol. J. 18, 581-595 (2020).

[26] Ou, C. et al. A de novo genome assembly of the dwarfing pear rootstock Zhongai 1. Sci. Data 6, 281 https://doi.org/10.1038/s41597-019-0291-3 (2019).

[27] Teng, Y. & Tanabe, K. Reconsideration on the origin of cultivated pears native to east asia. Acta Hortic. 634, 175-182, https://doi.org/10.17660/ActaHortic.2004.634.21 (2004).

[28] Seppey, M., Manni, M. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness. Methods Mol. Biol. 1962, 227-245 (2019).

[29] Zhang, L. et al. A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nat. Commun. 10, 1494 (2019).

[30] Verde, I. et al. The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat. Genet. 45, 487-494 (2013).

[31] Raymond, O. et al. The Rosa genome provides new insights into the domestication of modern roses. Nat. Genet. 50, 772-777 (2018).

[32] Edger, P. P. et al. Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity. Gigascience 7, 1-7 (2018).

[33] VanBuren, R. et al. A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome. Gigascience 7, giy094, https://doi.org/10.1093/gigascience/giy094 (2018).

[34] Tomato Genome, C. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485, 635-641 (2012).

[35] Yang, Q. et al. PpyGAST1 is potentially involved in bud dormancy release by integrating the GA biosynthesis and ABA signaling in ‘Suli’ pear (Pyrus pyrifolia White Pear Group). Environ. Exp. Bot. 162, 302-312 (2019).

[36] Kumar, L. & Futschik, M. E. Mfuzz: A software package for soft clustering of microarray data. Bioinformation 2, 5-7 (2007).

[37] Ou, S., Chen, J. & Jiang, N. Assessing genome assembly quality using the LTR Assembly Index (LAI). Nucleic Acids Res. 46, e126 (2018).

[38] Bao, L., Chen, K., Zhang, D., Li, X. & Teng, Y. An assessment of genetic variability and relationships within Asian pears based on AFLP (amplified fragment length polymorphism) markers. Sci. Hortic. 116, 374-380 (2008).

[39] Jiang, S. et al. Primitive genepools of Asian pears and their complex hybrid origins inferred from fluorescent sequence-specific amplification polymorphism (SSAP) markers based on LTR retrotransposons. PLoS ONE 11, e0149192 (2016).

[40] Yue, X. et al. Combined analyses of chloroplast DNA haplotypes and microsatellite markers reveal new insights into the origin and dissemination route of cultivated pears native to East Asia. Front. Plant Sci. 9, 591 (2018).

[41] Jiménez, S., Reighard, G. L. & Bielenberg, D. G. Gene expression of DAM5 and DAM6 is suppressed by chilling temperatures and inversely correlated with bud break rate. Plant Mol. Biol. 73, 157-167 (2010).

[42] Tian, J. et al. Population genomic analysis of gibberellin-responsive long noncoding RNAs in Populus. J. Exp. Bot. 67, 2467-2482 (2016).

[43] Bai, S. et al. BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol. J. 17, 1985-1997 (2019).

[44] Hao, L. et al. A constitutive and drought-responsive mRNA undergoes long-distance transport in pear (Pyrus betulaefolia) phloem. Plant Sci. 293, 110419 (2020).

[45] Gabay, G. et al. Transcriptome analysis and metabolic profiling reveal the key role of alpha-linolenic acid in dormancy regulation of European pear. J. Exp. Bot. 70, 1017-1031 (2019).

[46] Kolonin, M. G. & Finley, R. L. Jr. A role for cyclin J in the rapid nuclear division cycles of early Drosophila embryogenesis. Dev. Biol. 227, 661-672 (2000).

[47] Rinne, P. L. et al. Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-beta-glucanases to reopen signal conduits and release dormancy in Populus. Plant Cell 23, 130-146 (2011).

[48] Ahmad, M. et al. Genome wide identification and predicted functional analyses of NAC transcription factors in Asian pears. BNC Plant Biol. 18, 214 (2018).

[49] Ferrero, L. V., Viola, I. L., Ariel, F. D. & Gonzalez, D. H. Class I TCP transcription factors target the gibberellin biosynthesis gene GA20ox1 and the growth-promoting genes HBI1 and PRE6 during thermomorphogenic growth in Arabidopsis. Plant Cell Physiol. 60, 1633-1645 (2019).

[50] Lee, S. et al. Overexpression of PRE1 and its homologous genes activates gibberellin-dependent responses in Arabidopsis thaliana. Plant Cell Physiol. 47, 591-600 (2006).

[51] Wu, R. et al. Histone modification and activation by SOC1-like and drought stress-related transcription factors may regulate AcSVP2 expression during kiwifruit winter dormancy. Plant Sci. 281, 242-250 (2019).

[52] Li, S. et al. Histone acetylation cooperating with AREB1 transcription factor regulates drought response and tolerance in Populus trichocarpa. Plant Cell 31, 663-686 (2018).

[53] Azeez, A. et al. EARLY BUD-BREAK 1 and EARLY BUD-BREAK 3 control resumption of poplar growth after winter dormancy. Nat. Commun. 12, 1123, https://doi.org/10.1038/s41467-021-21449-0 (2021).

[54] Li, J. et al. Abscisic Acid (ABA) promotes the induction and maintenance of pear (Pyrus pyrifolia White Pear Group) flower bud endodormancy. Int. J. Mol. Sci. 19, 310 (2018).

[55] Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722-736 (2017).

[56] Kurtz, S. et al. Versatile and open software for comparing large genomes. Genome Biol. 5, R12 (2004).

[57] Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009).

[58] Servant, N. et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol. 16, 259 (2015).

[59] Burton, J. N. et al. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat. Biotechnol. 31, 1119-1125 (2013).

[60] Xu, Z. & Wang, H. LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res. 35, W265-W268 (2007).

[61] Han, Y. & Wessler, S. R. MITE-Hunter: a program for discovering miniature inverted-repeat transposable elements from genomic sequences. Nucleic Acids Res. 38, e199 (2010).

[62] Price, A. L., Jones, N. C. & Pevzner, P. A. De novo identification of repeat families in large genomes. Bioinformatics 21, i351-i358 (2005).

[63] Edgar, R. C. & Myers, E. W. PILER: identification and classification of genomic repeats. Bioinformatics 21, i152-i158 (2005).

[64] Wicker, T. et al. A unified classification system for eukaryotic transposable elements. Nat. Rev. Genet. 8, 973-982 (2007).

[65] Haas, B. J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol. 9, R7 (2008).

[66] Burge, C. & Karlin, S. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 268, 78-94 (1997).

[67] Stanke, M. & Waack, S. Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19, ii215-ii225 (2003).

[68] Majoros, W. H., Pertea, M. & Salzberg, S. L. TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics 20, 2878-2879 (2004).

[69] Blanco, E., Parra, G. & Guigó, R. Using geneid to identify genes. Curr. Protoc. Bioinforma. 18, 4.3.1-4.3.28 (2007).

[70] Korf, I. Gene finding in novel genomes. BMC Bioinform. 5, 59 (2004).

[71] Keilwagen, J. et al. Using intron position conservation for homology-based gene prediction. Nucleic Acids Res. 44, e89 (2016).

[72] Haas, B. J. et al. Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res. 31, 5654-5666 (2003).

[73] Shao, Y. et al. OsSPL3, an SBP-domain protein, regulates crown root development in rice. Plant Cell 31, 1257-1275 (2019).

[74] Chang, S., Puryear, J. & Cairney, J. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Report. 11, 113-116 (1993).

[75] Haring, M. et al. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant methods 3, 11 (2007).

[76] Li, W. et al. A robust chromatin immunoprecipitation protocol for studying transcription factor-DNA interactions and histone modifications in wood-forming tissue. Nat. Protoc. 9, 2180-2193 (2014).