A gap-free and haplotype-resolved lemon genome provides insights into flavor synthesis and huanglongbing (HLB) tolerance

Abstract

The lemon (Citrus limon; family Rutaceae) is one of the most important and popular fruits worldwide. Lemon also tolerates huanglongbing (HLB) disease, which is a devastating citrus disease. Here we produced a gap-free and haplotype-resolved chromosome-scale genome assembly of the lemon by combining Pacific Biosciences circular consensus sequencing, Oxford Nanopore 50-kb ultra-long, and high-throughput chromatin conformation capture technologies. The assembly contained nine-pair chromosomes with a contig N50 of 35.6 Mb and zero gaps, while a total of 633.0 Mb genomic sequences were generated. The origination analysis identified 338.5 Mb genomic sequences originating from citron (53.5%), 147.4 Mb from mandarin (23.3%), and 147.1 Mb from pummelo (23.2%). The genome included 30 528 protein-coding genes, and most of the assembled sequences were found to be repetitive sequences. Several significantly expanded gene families were associated with plant–pathogen interactions, plant hormone signal transduction, and the biosynthesis of major active components, such as terpenoids and flavor compounds. Most HLB-tolerant genes were expanded in the lemon genome, such as 2-oxoglutarate (2OG)/Fe(II)-dependent oxygenase and constitutive disease resistance 1, cell wall-related genes, and lignin synthesis genes. Comparative transcriptomic analysis showed that phloem regeneration and lower levels of phloem plugging are the elements that contribute to HLB tolerance in lemon. Our results provide insight into lemon genome evolution, active component biosynthesis, and genes associated with HLB tolerance.

Acknowledgements

The authors thank Dr Yongping Duan for invaluable suggestions for elaborating the manuscript. This work was supported by the Guangxi Major Project of Science and Technology (Guike AA18118027), the Postdoctoral Project of Hainan Yazhou Bay Seed Laboratory Program (B21Y10203), and the Scientific Research and Development Fund of the College of Agriculture, Guangxi University (EE101731).

Author contributions

M.Z. and X.Z. conceived the study and participated in its design and coordination. Y.B., X.Z., and M.Z. drafted the manuscript. W.Y., X.Z., B.W., J.X., A.S., C.A.P., and B.C. reviewed and revised the manuscript. Y.B., Z.Z., X.C., M.J., and X.Z. performed the genome and transcriptomic analysis. Y.B., Z.Z., and W.Y. carried out the experiments. All authors read and approved the final manuscript.

Data availability

RNA-sequencing data have been deposited in the NCBI Bioproject database under accession number PRJNA812325. The genome sequence data have been deposited in the CNCB Genome Warehouse under accession numbers GWHCBFQ00000000.1.

Conflict of interest

The authors declare that they have no conflict of interest.

References

[1] Chaturvedi D, Shrivastava Suhane RRN . Basketful benefit of Citrus limon. Int Res J Pharm. 2016; 7: 1-4.

[2] Makni M, Jemai R, Kriaa W et al. Citrus limon from Tunisia: phytochemical and physicochemical properties and biological activities . Biomed Res Int. 2018; 2018: 1-10.

[3] Del Río JA, Fuster MD, Gómez P et al. Citrus limon: a source of flavonoids of pharmaceutical interest . Food Chem. 2004; 84: 457-61.

[4] Singh N, Yarla NS, Siddiqi NJ et al. Features, pharmacological chemistry, molecular mechanism and health benefits of lemon. Med Chem. 2021; 17: 187-202.

[5] Xi W, Lu J, Qun J et al. Characterization of phenolic profile and antioxidant capacity of different fruit parts from lemon (Citrus limon Burm.) cultivars . J Food Sci Technol 2017; 54: 1108-18.

[6] Singh N, Jaiswal J, Tiwari P et al. Phytochemicals from Citrus limon juice as potential antibacterial agents . Open Bioactive Compounds J. 2020; 8: 1-6.

[7] Dala-Paula BM, Plotto A, Bai J et al. Effect of huanglongbing or greening disease on orange juice quality, a review. Front Plant Sci. 2019; 9: 1976.

[8] Liao H, Burns JK . Gene expression in Citrus sinensis fruit tissues harvested from huanglongbing-infected trees: comparison with girdled fruit . J Exp Bot. 2012; 63: 3307-19.

[9] Federico M, Uratsu SL, Ute A et al. Transcriptome profiling of citrus fruit response to Huanglongbing disease. PLoS One. 2012; 7: e38039.

[10] Fan J, Chen C, Yu Q et al. Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to ‘ Candidatus Liberibacter asiaticus.’ infection . Mol Plant Microbe Interact. 2012; 25: 1396-407.

[11] Folimonova SY, Robertson CJ, Garnsey SM et al. Examination of the responses of different genotypes of citrus to huanglongbing (citrus greening) under different conditions. Phytopathology. 2009; 99: 1346-54.

[12] Xia Z, Huang D, Zhang S et al. Chromosome-scale genome assembly provides insights into the evolution and flavor synthesis of passion fruit (Passiflora edulis Sims). Hortic Res. 2021; 8: 14.

[13] Wu GA, Prochnik S, Jenkins J et al. Sequencing of diverse mandarin, pummelo, and orange genomes reveals a complex history of admixture during citrus domestication. Nat Biotechnol. 2014; 32: 656-62.

[14] Fan J, Chen C, Achor DS et al. Differential anatomical responses of tolerant and susceptible citrus species to the infection of ‘ Candidatus Liberibacter asiaticus . Physiol Mol Plant Pathol. 2013; 83: 69-74.

[15] Guardo MD, Moretto M, Moser M et al. The haplotype-resolved reference genome of lemon (Citrus limon L. Burm f.). Tree Genet Genomes. 2021; 17: 46.

[16] Ma J, Wing RA, Bennetzen JL et al. Plant centromere organization: a dynamic structure with conserved functions. Trends Genet. 2007; 23: 134-9.

[17] Zhang J, Zhang X, Tang H et al. Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L. Nat Genet. 2018; 50: 1565-73.

[18] Liao P, Hemmerlin A, Bach TJ et al. The potential of the mevalonate pathway for enhanced isoprenoid production. Biotechnol Adv. 2016; 34: 697-713.

[19] Rohmer M, Rohmer M . The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae, and higher plants. Nat Prod Rep. 1999; 16: 565-74.

[20] Liu L, Shao Z, Zhang M et al. Regulation of carotenoid metabolism in tomato. Mol Plant. 2015; 8: 28-39.

[21] Cunningham FX, Gantt E . Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol. 1998; 49: 557-83.

[22] Albrecht U, Bowman KD . Transcriptional response of susceptible and tolerant citrus to infection with ‘ Candidatus Liberibacter asiaticus’ . Plant Sci. 2012; 185-186: 118-30.

[23] Lewis NG, Yamamoto E . Lignin: occurrence, biogenesis, and biodegradation. Annu Rev Plant Physiol 1990; 41: 455-96.

[24] Achor DS, Etxeberria E, Wang N et al. Sequence of anatomical symptom observations in citrus affected with huanglongbing disease. Plant Pathol J. 2010; 9: 56-64.

[25] Wang P, Yu J, Jin S et al. Genetic basis of high aroma and stress tolerance in the oolong tea cultivar genome. Hortic Res. 2021; 8: 107.

[26] Xu X, Yuan H, Yu X et al. The chromosome-level Stevia genome provides insights into steviol glycoside biosynthesis . Hortic Res. 2021; 8: 129.

[27] Hodges AW, Spreen TH . Economic impacts of citrus greening (HLB) in Florida, 2006/07-2010/11. University of Florida, Gainesville, FL, 2012.

[28] Bourquin V, Nishikubo N, Abe H et al. Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues. Plant Cell. 2002; 14: 3073-88.

[29] Koh EJ, Zhou L, Williams DS et al. Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with ‘ Candidatus Liberibacter asiaticus . Protoplasma. 2012; 249: 687-97.

[30] Yordanov YS, Regan B, Busov V . Members of the lateral organ boundaries domain transcription factor family are involved in the regulation of secondary growth in Populus. Plant Cell. 2010; 22: 3662-77.

[31] Li W, Hartung JS, Levy L . Quantitative real-time PCR for detection and identification of ‘ Candidatus Liberibacter’ species associated with citrus huanglongbing . J Microbiol Methods. 2006; 66: 104-15.

[32] Lee JA, Halbert SE, Dawson WO et al. Asymptomatic spread of huanglongbing and implications for disease control. Proc Natl Acad Sci USA. 2015; 112: 7605-10.

[33] Servant N, Varoquaux N, Lajoie BR et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol. 2015; 16: 259.

[34] Cheng H, Concepcion GT, Feng X et al. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021; 18: 170-5.

[35] Li H . Minimap2: pairwise alignment for nucleotide sequences. arXiv 2017.

[36] Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25: 1754-60.

[37] Xu M, Guo L, Gu S et al. TGS-GapCloser: a fast and accurate gap closer for large genomes with low coverage of error-prone long reads. GigaScience 2020; 9: giaa094.

[38] Xu G, Xu T, Zhu R et al. Lr_Gapcloser: a tiling path-based gap closer that uses long reads to complete genome assembly. GigaScience 2019; 8: giy157.

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

[40] Tarailo-Graovac M, Chen N . Using repeat masker to identify repetitive elements in genomic sequences. Curr Protoc Bioinformatics. 2009; 25: 4.10.1-14.

[41] Flynn JM, Hubley R, Rosen J et al. Repeatmodeler2 for automated genomic discovery of transposable element families. Proc Natl Acad Sci USA. 2020; 117: 9451-7.

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

[43] 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.

[44] Birney E, Clamp M, Durbin R . GeneWise and Genomewise. Genome Res. 2004; 14: 988-95.

[45] Stanke M, Tzvetkova A, Morgenstern B . AUGUSTUS at EGASP: using EST, protein and genomic alignments for improved gene prediction in the human genome. Genome Biol. 2006; 7: S11-S11.8.

[46] Abrusan G, Grundmann N, DeMester L et al. TEclass - a tool for automated classification of unknown eukaryotic transposable elements. Bioinformatics. 2009; 25: 1329-30.

[47] Zheng Y, Jiao C, Sun H et al. iTAK: a program for genome-wide prediction and classification of plant transcription factors, transcriptional regulators, and protein kinases. Mol Plant. 2016; 9: 1667-70.

[48] Li L, Stoeckert CJ, Roos DS . OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003; 13: 2178-89.

[49] Katoh K, Standley DM . MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30: 772-80.

[50] Nguyen LT, Schmidt HA, Haeseler AV et al. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015; 32: 268-74.

[51] Alexandros S. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30: 1312-3.

[52] Han MV, Thomas GWC, Lugo-Martinez J et al. Estimating gene gain and loss rates in the presence of error in genome assembly and annotation using CAFE 3. Mol Biol Evol. 2013; 30: 1987-97.

[53] Martin M . Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011; 17: 10-2.

[54] Trapnell C, Sauvageau M, Goff L et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2012; 31: 46-53.

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

[56] Ginestet C . ggplot2: elegant graphics for data analysis. J R Stat Soc. 2011; 174: 245-6.

[57] Rodriguez A, Laio A . Clustering by fast search and find of density peaks. Science. 2014; 344: 1492-6.

[58] Kumar S, Stecher G, Tamura K . MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33: 1870-4.

[59] Letunic I, Bork P . Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019; 47: W256-9.

[60] Hu B, Jin J, Guo A et al. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015; 31: 1296-7.