Sequencing a Juglans regia × J. microcarpa hybrid yields high-quality genome assemblies of parental species

Abstract

Members of the genus Juglans are monecious wind-pollinated trees in the family Juglandaceae with highly heterozygous genomes, which greatly complicates genome sequence assembly. The genomes of interspecific hybrids are usually comprised of haploid genomes of parental species. We exploited this attribute of interspecific hybrids to avoid heterozygosity and sequenced an interspecific hybrid Juglans microcarpa × J. regia using a novel combination of single-molecule sequencing and optical genome mapping technologies. The resulting assemblies of both genomes were remarkably complete including chromosome termini and centromere regions. Chromosome termini consisted of arrays of telomeric repeats about 8 kb long and heterochromatic subtelomeric regions about 10 kb long. The centromeres consisted of arrays of a centromere-specific Gypsy retrotransposon and most contained genes, many of them transcribed. Juglans genomes evolved by a whole-genome-duplication dating back to the Cretaceous-Paleogene boundary and consist of two subgenomes, which were fractionated by numerous short gene deletions evenly distributed along the length of the chromosomes. Fractionation was shown to be asymmetric with one subgenome exhibiting greater gene loss than the other. The asymmetry of the process is ongoing and mirrors an asymmetry in gene expression between the subgenomes. Given the importance of J. microcarpa × J. regia hybrids as potential walnut rootstocks, we catalogued disease resistance genes in the parental genomes and studied their chromosomal distribution. We also estimated the molecular clock rates for woody perennials and deployed them in estimating divergence times of Juglans genomes and those of other woody perennials.

References

[1] Zhang, F. et al. Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube. Nat. Biotechnol. 35, 852-857 (2017).

[2] Schnable, P. S. et al. The B73 maize genome: complexity, diversity, and dynamics. Science 326, 1112-1115 (2009).

[3] Xu, Q. et al. The draft genome of sweet orange (Citrus sinensis) . Nat. Genet. 45, 59-66 (2013).

[4] Neale, D. B., Martínez-García, P. J., De La Torre, A. R., Montanari, S. & Wei, X. X. Tree genome sequencing: Novel insights into plant biology. Ann. Rev. Plant Biol. 68, 457-483 (2017).

[5] Stevens, K. A. et al. Genomic variation among and within six Juglans species . G3: Genes|Genomes|Genetics (2018). https://doi.org/10.1534/g3.118.200030

[6] Pan, A., Sun, Q., Manson, J. E., Willett, W. C. & Hu, F. B. Walnut consumption is associated with lower risk of type 2 diabetes in women. J. Nutr. 143, 512-518 (2013).

[7] Kris-Etherton, P. M. Walnuts decrease risk of cardiovascular disease: a summary of efficacy and biologic mechanisms. J. Nutr. 144, 547s-554s (2014).

[8] Poulose, S. M., Miller, M. G. & Shukitt-Hale, B. Role of walnuts in maintaining brain health with age. J. Nutr. 144, 561s-566s (2014).

[9] Browne, G. T. et al. Resistance to Species of Phytophthora identified among clones of Juglans microcarpa x J. regia . Hortsci 50, 1136-1142 (2015).

[10] Kluepfel, D. A. et al. Evaluation of wild walnut Juglans spp. for resistance to crown gall disease . Phytopathology 101, S92- S92 (2011).

[11] Martinez-Garcia, P. J. et al. The walnut (Juglans regia) genome sequence reveals diversity in genes coding for the biosynthesis of non-structural polyphenols . Plant J. 87, 507-532 (2016).

[12] Bai, W. N. et al. Demographically idiosyncratic responses to climate change and rapid Pleistocene diversification of the walnut genus Juglans (Juglandaceae) revealed by whole-genome sequences . New Phytol. 217, 1726-1736 (2018).

[13] Zimin, A. V. et al. Hybrid assembly of the large and highly repetitive genome of Aegilops tauschii, a progenitor of bread wheat, with the MaSuRCA mega-reads algorithm . Genome Res. 27, 787-792 (2017).

[14] Luo, M. C. et al. Genome sequence of the progenitor of the wheat D genome Aegilops tauschii . Nature 551, 498-502 (2017).

[15] Dvorak, J. et al. Structural variation and rates of genome evolution in the grass family seen through comparison of sequences of genomes greatly differing in size. Plant J. 95, 487-503 (2018).

[16] Zhu, T. et al. Analysis of Brachypodium genomes with genome-wide optical maps . Genome 61, 559-561 (2018).

[17] Luo, M. C. et al. Synteny analysis in Rosids with a walnut physical map reveals slow genome evolution in long-lived woody perennials. BMC Genom. 16, 707 (2015).

[18] Jaillon, O. et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449, 463-467 (2007).

[19] Albert, V. A. et al. The Amborella genome and the evolution of flowering plants . Science 342, 1467 (2013).

[20] Manos, P. S. & Stone, D. E. Evolution, phylogeny, and systematics of the Juglandaceae. Ann. Mo. Bot. Gard. 88, 231-269 (2001).

[21] Manchester, S. R. The fossil history of Juglandaceae. Monographs In Systematic Botany from the Missouri Botanical Garden. 21, 1-137 (1987).

[22] Manchester, S. R. Early history of the Juglandaceae. Plant Syst. Evol. 162, 231-250 (1989).

[23] Zhang, J. B. et al. Integrated fossil and molecular data reveal the biogeographic diversification of the Eastern Asian-Eastern North American Disjunct Hickory Genus (Carya Nutt.) . PLoS. ONE. https://doi.org/10.1371/journal.pone.0070449 (2013).

[24] Xiang, X. G. et al. Large-scale phylogenetic analyses reveal fagalean diversification promoted by the interplay of diaspores and environments in the Paleogene. Perspect. Plant Ecol. 16, 101-110 (2014).

[25] Dong, W. P. et al. Phylogenetic resolution in Juglans based on complete chloroplast genomes and nuclear DNA sequences . Front. Plant Sci. 8, 1148 (2017).

[26] Clarke, J. T., Warnock, R. C. M. & Donoghue, P. C. J. Establishing a time-scale for plant evolution. New Phytol. 192, 266-301 (2011).

[27] Jiao, Y. N. et al. A genome triplication associated with early diversification of the core eudicots. Genome Biol. 13, R3 (2012).

[28] Zeng, L. P. et al. Resolution of deep eudicot phylogeny and their temporal diversification using nuclear genes from transcriptomic and genomic datasets. New Phytol. 214, 1338-1354 (2017).

[29] Presting, G. G., Malysheva, L., Fuchs, J. & Schubert, I. Z. A TY3/GYPSY retrotransposon-like sequence localizes to the centromeric regions of cereal chromosomes. Plant J. 16, 721-728 (1998).

[30] Sharma, A. & Presting, G. G. Evolution of centromeric retrotransposons in grasses. Genome Biol. Evol. 6, 1335-1352 (2014).

[31] Luo, S. et al. The cotton centromere contains a Ty3-gypsy-like LTR retroelement. PLoS. ONE 7, 35261 (2012).

[32] Tek, A. L., Kashihara, K., Murata, M. & Nagaki, K. Functional centromeres in soybean include two distinct tandem repeats and a retrotransposon. Chrom. Res. 18, 337-347 (2010).

[33] Weber, B. & Schmidt, T. Nested Ty3-gypsy retrotransposons of a single Beta procumbens centromere contain a putative chromodomain . Chrom. Res. 17, 379-396 (2009).

[34] Lim, K. B. et al. Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species . Plant J. 49, 173-183 (2007).

[35] Tuskan, G. A. et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray) . Science 313, 1596-1604 (2006).

[36] Velasco, E. A. A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. PLoS. ONE 2, 326 (2007).

[37] Velasco, R. et al. The genome of the domesticated apple (Malus x domestica Borkh.) . Nat. Genet. 42, 833-839 (2010).

[38] Minio, A., Lin, J., Gaut, B. S. & Cantu, D. How Single molecule real-time sequencing and haplotype phasing have enabled reference-grade diploid genome assembly of wine grapes. Front. Plant Sci. 8, 826 (2017).

[39] Schmutz, J. et al. Quality assessment of the human genome sequency. Nature 429, 365-368 (2004).

[40] Ho, K. M. & Kasha, K. J. Genetic control of chromosome elimination during haploid formation in barley. Genetics 81, 263-275 (1975).

[41] Song, K. M., Lu, P., Tang, K. L. & Osborn, T. C. Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution . Proc. Natl Acad. Sci. USA 92, 7719-7723 (1995).

[42] Liu, B. et al. Rapid genomic changes in newly synthesized amphiploids of Triticum and Aegilops. I. changes in low-copy noncoding DNA sequences . Genome 41, 272-277 (1998).

[43] Shaked, H., Kashkush, K., Ozkan, H., Feldman, M. & Levy, A. A. Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy. Plant Cell 13, 1750-1759 (2001).

[44] Osborn, T. C. et al. Understanding mechanisms of novel gene expression in polyploids. Trends Genet. 19, 141-147 (2003).

[45] Oleszczuk, S. & Lukaszewski, A. J. The origin of unusual chromosome constitutions among newly formed allopolyploids. Am. J. Bot. 101, 318-326 (2014).

[46] Langham, R. J. et al. Genomic duplication, fractionation and the origin of regulatory novelty. Genetics 166, 935-945 (2004).

[47] Dvorak, J., Yang, Z.-L., You, F. M. & Luo, M. C. Deletion polymorphism in wheat chromosome regions with contrasting recombination rates. Genetics 168, 1665-1675 (2004).

[48] Cheng, F. et al. Biased gene fractionation and dominant gene expression among the subgenomes of Brassica rapa . PLoS. ONE 7, 36442 (2012).

[49] Renny-Byfield, S., Gong, L., Gallagher, J. P. & Wendel, J. F. Persistence of subgenomes in paleopolyploid cotton after 60 My of evolution. Mol. Biol. Evol. 32, 1063-1071 (2015).

[50] Schnable, J. C., Springer, N. M. & Freeling, M. Differentiation of the maize subgenomes by genome dominance and both ancient and ongoing gene loss. Proc. Natl Acad. Sci. USA 108, 4069-4074 (2011).

[51] Renny-Byfield, S., Rodgers-Melnick, E. & Ross-Ibarra, J. Gene fractionation and function in the ancient subgenomes of maize. Mol. Biol. Evol. 34, 1825-1832 (2017).

[52] Woodhouse, M. R. et al. Origin, inheritance, and gene regulatory consequences of genome dominance in polyploids (vol 111, pg 5283, 2014). Proc. Natl Acad. Sci. USA 111, 6527- 6527 (2014).

[53] Lam, E. T. et al. Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly. Nat. Biotechnol. 30, 771-776 (2012).

[54] Cao, H. Z. et al. Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology. Gigascience 3, 34 (2014).

[55] Dvorak, J., McGuire, P. E. & Cassidy, B. Apparent sources of the A genomes of wheats inferred from the polymorphism in abundance and restriction fragment length of repeated nucleotide sequences. Genome 30, 680-689 (1988).

[56] Chin, C. S. et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat. Methods 13, 1050-1054 (2016).

[57] Weisenfeld, N. I., Kumar, V., Shah, P., Church, D. M. & Jaffe, D. B. Direct determination of diploid genome sequences. Genome Res. 27, 757-767 (2017).

[58] Shelton, J. M. et al. Tools and pipelines for BioNano data: molecule assembly pipeline and FASTA super scaffolding tool. BMC Genom. 16, 734 (2015).

[59] Piro, V. C. et al. FGAP: an automated gap closing tool. BMC Res. Notes 7, 371 (2014).

[60] Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589-595 (2010).

[61] Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079 (2009).

[62] Li, H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27, 2987-2993 (2011).

[63] Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156-2158 (2011).

[64] Han, Y. J. & Wessler, S. R. MITE-Hunter: a program for discovering miniature inverted-repeat transposable elements from genomic sequences. Nucleic Acids Res. 38, 199 (2010).

[65] Wenke, T. et al. Targeted identification of short interspersed nuclear element families shows their widespread existence and extreme heterogeneity in plant genomes. Plant Cell 23, 3117-3128 (2011).

[66] Bao, W., Kojima, K. K. & Kohany, O. Repbase update, a database of repetitive elements in eukaryotic genomes. Mob. DNA 6, 11 (2015).

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

[68] Thiel, T., Michalek, W., Varshney, R. K. & Graner, A. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.) . Theor. Appl. Genet. 106, 411-422 (2003).

[69] Stanke, M. et al. AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res. 34, W435-W439 (2006).

[70] Cantarel, B. L. et al. MAKER: An easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Res. 18, 188-196 (2008).

[71] Simão, F. A. et al. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210-3212 (2016).

[72] Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279-D285 (2016).

[73] Li, P. C. et al. RGAugury: a pipeline for genome-wide prediction of resistance gene analogs (RGAs) in plants. BMC Genom. 17, 852 (2016).

[74] Wang, Y. P. et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 40, e49 (2012).

[75] Krzywinski, M. et al. Circos: An information aesthetic for comparative genomics. Genome Res. 19, 1639-1645 (2009).

[76] Lechner, M. et al. Proteinortho: detection of (co-)orthologs in large-scale analysis. BMC Bioinforma. 12, 124 (2011).

[77] Thompson, J. D., Higgins, D. G. & Gibson, T. J. Improved sensitivity of profile searches through the use of sequence weights and gap excision. Comput. Appl. Biosci. 10, 19-29 (1994).

[78] Wang, D., Zhang, Y., Zhang, Z., Zhu, J. & Yu, J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genom., Proteom., Bioinforma. 8, 77-80 (2010).

[79] Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511-U174 (2010).

[80] Zhang, T. Z. et al. Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement . Nat. Biotechnol. 33, 531-U252 (2015).

[81] International Peach Genome, 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).

[82] Argout, X. et al. The genome of Theobroma cacao . Nat. Genet. 43, 101-108 (2011).

[83] Daccord, N. et al. High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat. Genet. 49, 1099-1106 (2017).

[84] Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059-3066 (2002).

[85] Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268-274 (2015).

[86] Rambaut, A. FigTree v1.4.3. http://tree.bio.ed.ac.uk (2009).

[87] Manos, P. S. et al. Phylogeny of extant and fossil Juglandaceae inferred from the integration of molecular and morphological data sets. Syst. Biol. 56, 412-430 (2007).

[88] Chen, F. et al. Genomics: cracking the mysteries of walnuts. J. Genet. 98, (2019) in press.

[89] Ranwez, V., Harispe, S., Delsuc, F. & Douzery, E. J. MACSE: multiple alignment of coding sequences accounting for frameshifts and stop codons. PLoS. ONE 6, 22594 (2011).