A phased genome of the highly heterozygous ‘Texas’ almond uncovers patterns of allele-specific expression linked to heterozygous structural variants

Abstract

The vast majority of traditional almond varieties are self-incompatible, and the level of variability of the species is very high, resulting in a high-heterozygosity genome. Therefore, information on the different haplotypes is particularly relevant to understand the genetic basis of trait variability in this species. However, although reference genomes for several almond varieties exist, none of them is phased and has genome information at the haplotype level. Here, we present a phased assembly of genome of the almond cv. Texas. This new assembly has 13% more assembled sequence than the previous version of the Texas genome and has an increased contiguity, in particular in repetitive regions such as the centromeres. Our analysis shows that the ‘Texas’ genome has a high degree of heterozygosity, both at SNPs, short indels, and structural variants level. Many of the SVs are the result of heterozygous transposable element insertions, and in many cases, they also contain genic sequences. In addition to the direct consequences of this genic variability on the presence/absence of genes, our results show that variants located close to genes are often associated with allele-specific gene expression, which highlights the importance of heterozygous SVs in almond.

Acknowledgements

This work was supported by the Spanish Ministerio de Ciencia e Innovación PID2019-106374RB-I00 and PID2022-143167NB-I00 grants to J.C. and IJC2020-045949-I fellowship to R.C., and RTI2018-100795-B-I00 and PID2021-128885OB-I00 both funded by MCIN/AEI/10.13039/501100011033 and by ‘ERDF A way of making Europe’ to M.J.A. We also acknowledge the support from the CERCA Programme (‘Generalitat de Catalunya’) and the ‘Severo Ochoa Programme for Centres of Excellence in R&D’ 2016-2019 (SEV-2015-0533) and 2020-2023 (CEX2019-000902-S).

Author contributions

C.d.T. contributed to transcriptomic analyses and performed TIP detection in the almond population. V.R. performed the genome assembly and gene annotation. M.J.A., I.E., and P.A. contributed to obtain plant material and critically revised the manuscript. C.V. contributed to article writing and critically revised the manuscript. R.C. performed TE annotation and analyzed the data. J.M.C. and R.C. conceived the study, designed the experiments, and wrote the article. All authors revised the final version of the manuscript.

Data availability

Raw and processed RNA-seq datasets are available at NCBI GEO repository (accession number GSE254853). Long read data, Hi-C and genome assemblies have been submitted to ENA database (Study Code: ERP158378). In addition, the genomic datasets produced in this study (genome assemblies, gene, TE and SV annotations) are publicly available at zenodo (https://zenodo.org/records/10829948).

Conflict of interest statement

None declared.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Pérez de Los Cobos F, Coindre E, Dlalah N et al. Almond population genomics and non-additive GWAS reveal new insights into almond dissemination history and candidate genes for nut traits and blooming time. Hortic Res. 2023; 10 :uhad193.

[2] Sánchez-Pérez R, Pavan S, Mazzeo R et al. Mutation of a bHLH transcription factor allowed almond domestication. Science. 2019; 364 :1095-8.

[3] Alioto T, Alexiou KG, Bardil A et al. Transposons played a major role in the diversification between the closely related almond and peach genomes: results from the almond genome sequence. Plant J. 2020; 101 :455-72.

[4] D’Amico-Willman K, Ouma W, Meulia T et al. Whole-genome sequence and methylome profiling of the almond [Prunus dulcis (mill.) D.A. Webb] cultivar ‘nonpareil’. G3 (Bethesda). 2022; 12 :jkac065.

[5] Duval H, Coindre E, Ramos-Onsins SE et al. Development and evaluation of an AxiomTM 60K SNP Array for almond (Prunus dulcis). Plants (Basel). 2023; 12 :242.

[6] Verde I, Jenkins J, Dondini L et al. The peach v2.0 release: high-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity. BMC Genomics. 2017; 18 :225.

[7] Shirasawa K, Isuzugawa K, Ikenaga M et al. The genome sequence of sweet cherry (Prunus avium) for use in genomics-assisted breeding. DNA Res. 2017; 24 :499-508.

[8] Zheng T, Li P, Zhuo X et al. The chromosome-level genome provides insight into the molecular mechanism underlying the tortuous-branch phenotype of Prunus mume. New Phytol. 2022; 235 :141-56.

[9] Jiang F, Zhang J, Wang S et al. The apricot (Prunus armeniaca L.) genome elucidates Rosaceae evolution and beta-carotenoid synthesis. Hortic Res. 2019; 6 :128.

[10] Huang Z, Shen F, Chen Y et al. Chromosome-scale genome assembly and population genomics provide insights into the adaptation, domestication, and flavonoid metabolism of Chinese plum. Plant J. 2021; 108 :1174-92.

[11] Baek S, Choi K, Kim GB et al. Draft genome sequence of wild Prunus yedoensis reveals massive inter-specific hybridization between sympatric flowering cherries. Genome Biol. 2018; 19 :127.

[12] Jung S, Lee T, Cheng C-H et al. 15 years of GDR: new data and functionality in the genome database for Rosaceae. Nucleic Acids Res. 2019; 47 :D1137-45.

[13] Schnable P, Springer N. Progress toward understanding heterosis in crop plants. Annu Rev Plant Biol. 2013; 64 :71-88.

[14] Donoso JM, Eduardo I, Picañol R et al. High-density mapping suggests cytoplasmic male sterility with two restorer genes in almond × peach progenies. Hortic Res. 2015; 2 :15016.

[15] Ou S, Chen J, Jiang N. Assessing genome assembly quality using the LTR assembly index (LAI). Nucleic Acids Res. 2018; 46 :e126.

[16] Simão FA, Waterhouse RM, Ioannidis P et al. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31 :3210-2.

[17] Wang L, Wang Y, Zhang J et al. Comparative analysis of transposable elements and the identification of candidate Centromeric elements in the Prunus subgenus Cerasus and its relatives. Genes (Basel). 2022; 13 :641.

[18] Marimon N, Luque J, Arús P et al. Fine mapping and identification of candidate genes for the peach powdery mildew resistance gene Vr3. Hortic Res. 2020; 7 :175.

[19] de Tomás C, Bardil A, Castanera R et al. Absence of major epigenetic and transcriptomic changes accompanying an interspecific cross between peach and almond. Hortic Res. 2022; 9 :uhac127.

[20] Pulido M, Casacuberta JM. Transposable element evolution in plant genome ecosystems. Curr Opin Plant Biol. 2023; 75 :102418.

[21] Cingolani P, Platts A, Wang le L et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012; 6 :80-92.

[22] Shi L, Song J, Guo C et al. A CACTA-like transposable element in the upstream region of BnaA9.CYP78A 9 acts as an enhancer to increase silique length and seed weight in rapeseed. Plant J. 2019; 98 :524-39.

[23] Castanera R, Morales-Díaz N, Gupta S et al. Transposons are important contributors to gene expression variability under selection in rice populations. elife. 2023; 12 :RP86324.

[24] Zhang L, Hu J, Han X et al. A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nat Commun. 2019; 10 :1494.

[25] Fiol A, Jurado-Ruiz F, López-Girona E et al. An efficient CRISPR-Cas9 enrichment sequencing strategy for characterizing complex and highly duplicated genomic regions. A case study in the Prunus salicina LG3-MYB10 genes cluster. Plant Methods. 2022; 18 :105.

[26] Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin. 1987; 19 :11-5.

[27] Bolger A, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30 :2114-20.

[28] Chin C, Peluso P, Sedlazeck F et al. Phased diploid genome assembly with single molecule real-time sequencing. Nat Methods. 2016; 13 :1050-4.

[29] Aury J-M, Istace B. Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads. NAR Genom Bioinform. 2021; 3 :lqab034.

[30] Guan D, McCarthy S, Wood J et al. Identifying and removing haplotypic duplication in primary genome assemblies. Bioinformatics. 2020; 36 :2896-8.

[31] Zhang X, Zhang S, Zhao Q et al. Assembly of allele-aware, chromosomal-scale autopolyploid genomes based on hi-C data. Nat Plants. 2019; 5 :833-45.

[32] Tang H, Zhang X, Miao C et al. ALLMAPS: robust scaffold ordering based on multiple maps. Genome Biol. 2015; 16 :3.

[33] Grabherr MG, Haas BJ, Yassour M et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29 :644-52.

[34] Bryant D, Johnson K, DiTommaso T et al. A tissue-mapped axolotl De novo transcriptome enables identification of limb regeneration factors. Cell Rep. 2017; 18 :762-76.

[35] Ou S, Su W, Liao Y et al. Benchmarking transposable element annotation methods for creation of a streamlined, comprehensive pipeline. Genome Biol. 2019; 20 :275.

[36] Fu L, Niu B, Zhu Z et al. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012; 28 :3150-2.

[37] Zhang R-G, Li G-Y, Wang X-L et al. TEsorter: an accurate and fast method to classify LTR-retrotransposons in plant genomes. Hortic Res. 2022; 9 :uhac017.

[38] Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34 :3094-100.

[39] Heller D, Vingron M. SVIM-asm: structural variant detection from haploid and diploid genome assemblies. Bioinformatics. 2021; 36 :5519-21.

[40] Langmead B, Salzberg S. Fast gapped-read alignment with bowtie 2. Nat Methods. 2012; 9 :357-9.

[41] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15 :550.

[42] Poplin R, Ruano-Rubio V, DePristo MA et al. Scaling accurate genetic variant discovery to tens of thousands of samples bioRxiv. 2018;201178.

[43] Danecek P, Auton A, Abecasis G et al. 1000 genomes project analysis group. The variant call format and VCFtools. Bioinformatics. 2011; 27 :2156-8.

[44] Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26 :841-2.

[45] Kim D, Paggi JM, Park C et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019; 37 :907-15.