Pan-genome and transcriptome analyses provide insights into genomic variation and differential gene expression profiles related to disease resistance and fatty acid biosynthesis in eastern black walnut (Juglans nigra)

Abstract

Walnut (Juglans) species are used as nut crops worldwide. Eastern black walnut (EBW, Juglans nigra), a diploid, horticultural important woody species is native to much of eastern North America . Although it is highly valued for its wood and nut, there are few resources for understanding EBW genetics. Here, we present a high-quality genome assembly of J. nigra based on Illumina, Pacbio, and Hi- C technologies. The genome size was 540.8 Mb, with a scaffold N50 size of 35.1 Mb, and 99.0% of the assembly was anchored to 16 chromosomes. Using this genome as a reference, the resequencing of 74 accessions revealed the effective population size of J. nigra declined during the glacial maximum. A single whole-genome duplication event was identified in the J. nigra genome. Large syntenic blocks among J. nigra, Juglans regia, and Juglans microcarpa predominated, but inversions of more than 600 kb were identified. By comparing the EBW genome with those of J. regia and J. microcarpa, we detected InDel sizes of 34.9 Mb in J. regia and 18.3 Mb in J. microcarpa, respectively. Transcriptomic analysis of differentially expressed genes identified five presumed NBS-LRR (NUCLEOTIDE BINDING SITE- LEUCINE-RICH REPEAT) genes were upregulated during the development of walnut husks and shells compared to developing embryos. We also identified candidate genes with essential roles in seed oil synthesis, including FAD (FATTY ACID DESATURASE) and OLE (OLEOSIN). Our work advances the understanding of fatty acid bioaccumulation and disease resistance in nut crops, and also provides an essential resource for conducting genomics-enabled breeding in walnut.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32070372, 41471038, and 31800372), the Operating Services of Qinling National Forest Ecosystem Research Station financed by Ministry of Science and Technology of China, Natural Science Basis Research Plan in Shaanxi Province of China (2019JQ-641), Shaanxi Academy of Science Research Funding Project (2019 K-06), and Science Foundation for Distinguished Young Scholars of Shaanxi Province (2023-JC-JQ-22). This work was also supported in part by the United States Department of Agriculture Forest Service. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.

Author contributions

P.Z. and S.Z. conceived and designed the study. F.Y., F.H., and P.Z. collected the samples. F.H., H.Z., and F.Y. took the morphology picture and collected the transcriptome materials of black walnut. H.Z., F.Y., F.H., and P.Z. assembled the genome, and performed gene annotation, gene family, and expression profiles. H.Z., F.Y., M.Y., K.W., H.Y., P.Z., and S.Z. supported the software. F.Y., H.Z., and P.Z. performed the comparative genome analysis. H.Y., H.Z., F.H., and P.Z. performed the population genomic analysis, F.Y. and H.Z. performed the whole-genome duplication and LTRs analysis. H.Z. and P.Z. wrote the draft manuscript and then P.Z., K.W., and S.Z. edited and revised the English writing of this manuscript. All authors contributed to and approved the final manuscript.

Data availability

The whole genome sequence data including Illumina short reads, Nanopore long reads, Hi-C interaction reads, and transcriptome data have been deposited in the NCBI, under accession numbers: PRJNA801766 (SRR17842367 and SRR17841629). The Illumina whole-genome resequencing data of J. nigra in this study have been deposited in China National GeneBank (CNGB) Nucleotide Sequence Archive database under project accession number CNP0001209 (https://db.cngb.org/search/project/CNP0001209/). The transcriptome data of six tissues and organs (leaves, female flowers, immature fruits, mature fruits, bark, and young stem) and husk, shell, and embryo (three developmental stages) have been deposited in the NCBI, under accession num-bers: PRJNA799697 (SRR17728328, SRR17728315, SRR17728293, SRR17727056, SRR17715789, SRR17714911, SRR17822893, SRR178 417850, RR17817728, SRR17798583, SRR17794307, SRR17777323, SRR17775560, SRR17775247, and SRR17761587).

Conflict of interest statement

None declared.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Wang Y, Xin H, Fan P et al. The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine . Plant J. 2021; 105: 1495-506.

[2] Rao G, Zhang J, Liu X et al. De novo assembly of a new Olea europaea genome accession using nanopore sequencing. Hortic Res. 2021; 8: 64.

[3] Ji F, Ma Q, Zhang W et al. A genome variation map provides insights into the genetics of walnut adaptation and agronomic traits. Genome Biol. 2021; 22: 300.

[4] Martínez-García PJ, Crepeau MW, Puiu D et al. The walnut (Juglans regia) genome sequence reveals diversity in genes coding for the biosynthesis of non-structural polyphenols . Plant J. 2016; 87: 507-32.

[5] Marrano A, Britton M, Zaini PA et al. High-quality chromosome-scale assembly of the walnut (Juglans regia L.). Gigascience. 2020; 9: giaa050.

[6] Zhang J, Zhang W, Ji F et al. A high-quality walnut genome assembly reveals extensive gene expression divergences after whole-genome duplication. Plant Biotechnol J. 2020; 18: 1848-50.

[7] Yan F, Xi RM, She RX et al. Improved de novo chromosome-level genome assembly of the vulnerable walnut tree Juglans mandshurica reveals gene family evolution and possible genome basis of resistance to lesion nematode. Mol Ecol Resour. 2021; 21: 2063-76.

[8] Zhu T, Wang L, You FM et al. Sequencing a Juglans regia × J. microcarpa hybrid yields high-quality genome assemblies of parental species. Hortic Res. 2019; 6: 55.

[9] Zhang BW, Xu LL, Li N et al. Phylogenomics reveals an ancient hybrid origin of the Persian walnut. Mol Biol Evol. 2019; 36: 2451-61.

[10] Nicolescu VN, Rédei K, Vor T et al. A review of black walnut (Juglans nigra L.) ecology and management in Europe . Trees. 2020; 34: 1087-112.

[11] Sitz RA, Luna EK, Ibarra Caballero J et al. Eastern black walnut (Juglans nigra L.) originating from native range varies in their response to inoculation with Geosmithia morbida . Front For Glob Chang. 2021; 4: 627911.

[12] Fang H, Liu X, Dong Y et al. Transcriptome and proteome analysis of walnut (Juglans regia L.) fruit in response to infection by Colletotrichum gloeosporioides . BMC Plant Biol. 2021; 21: 249.

[13] Feng S, Fang HC, Liu X et al. Genome-wide identification and characterization of long non-coding RNAs conferring resistance to Colletotrichum gloeosporioides in walnut (Juglans regia). BMC Genomics. 2021; 22: 15.

[14] Ning DL, Wu T, Xiao LJ et al. Chromosomal-level assembly of Juglans sigillata genome using Nanopore, BioNano, and hi-C analysis. GigaScience. 2020; 9: giaa006.

[15] Huang Y, Xiao L, Zhang Z et al. The genomes of pecan and Chinese hickory provide insights into Carya evolution and nut nutrition. GigaScience. 2019; 8: giz036.

[16] Xing Y, Liu Y, Zhang Q et al. Hybrid de novo genome assembly of Chinese chestnut (Castanea mollissima). GigaScience. 2019; 8: giz112.

[17] Urbach JM, Ausubel FM . The NBS-LRR architectures of plant R-proteins and metazoan NLRs evolved in independent events. Proc Natl Acad Sci U S A. 2017; 114: 1063-8.

[18] Li X, Cai K, Zhang Q et al. The Manchurian walnut genome: insights into Juglone and lipid biosynthesis. Gigascience. 2020; 11: giac057.

[19] Zhong Y, Yin H, Sargent DJ et al. Species-specific duplications driving the recent expansion of NBS-LRR genes in five Rosaceae species. BMC Genomics. 2015; 16: 77.

[20] Guo L, Winzer T, Yang X et al. The opium poppy genome and morphinan production. Science. 2018; 362: 343-7.

[21] Belisario A, Scotton M, Santori A et al. Variability in the Italian population of Gnomonia leptostyla, homothallism and resistance of Juglans species to anthracnose. Forest Pathol. 2008; 38: 129-45.

[22] Zhou R, Dong Y, Liu X et al. JrWRKY21 interacts with JrPTI5L to activate the expression of JrPR5L for resistance to Colletotrichum gloeosporioides in walnut. Plant J. 2022; 111: 1152-66.

[23] H An, K Yang . Resistance gene analogs in walnut (Juglans regia) conferring resistance to Colletotrichum gloeosporioides . Euphytica. 2014; 197: 175-190.

[24] Jiang N, Cui J, Shi Y et al. Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato-Phytophthora infestans interaction. Hortic Res. 2019; 6: 28.

[25] Montecchio L, Fanchin G, Simonato M et al. First record of thousand cankers disease fungal pathogen Geosmithia morbida and walnut twig beetle Pityophthorus juglandis on Juglans regia in Europe. Plant Dis. 2014; 98: 1445.

[26] Hadziabdic D, Vito LM, Windham MT et al. Genetic differentiation and spatial structure of Geosmithia morbida, the causal agent of thousand cankers disease in black walnut (Juglans nigra). Curr Genet. 2014; 60: 75-87.

[27] Gazis R, Poplawski L, Klingeman W et al. Mycobiota associated with insect galleries in walnut with thousand cankers disease reveals a potential natural enemy against Geosmithia morbida. Fungal Biol. 2018; 122: 241-53.

[28] Ramasamy RK, Luo MC, Leslie CA et al. Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa × J. regia hybrids. Hortic Res. 2021; 8: 111.

[29] Huang R, Zhou Y, Zhang J et al. Transcriptome analysis of walnut (Juglans regia L.) embryos reveals key developmental stages and genes involved in lipid biosynthesis and polyunsaturated fatty acid metabolism . J Agric Food Chem. 2021; 69: 377-96.

[30] Liu K, Zhao S, Wang S et al. Identification and analysis of the FAD gene family in walnuts (Juglans regia L.) based on transcriptome data . BMC Genomics. 2020; 21: 299.

[31] Chen Y, Chen Y, Shi C et al. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience. 2018; 7: 1-6.

[32] Koren S, Walenz BP, Berlin K et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017; 27: 722-36.

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

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

[35] Burton JN, Adey A, Patwardhan RP et al. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat Biotechnol. 2013; 31: 1119-25.

[36] 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.

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

[38] Li H . Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Genomics. 2013; 1303: 3997.

[39] Wu TD, Watanabe CK . GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics. 2005; 21: 1859-75.

[40] Krzywinski M, Schein J, Birol I et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009; 19: 1639-45.

[41] Stanke M, Keller O, Gunduz I et al. AUGUSTUS: Ab initio prediction of alternative transcripts. Nucleic Acids Res. 2006; 34: W435-9.

[42] Slater GSC, Birney E . Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005; 6: 31-40.

[43] Holt C, Yandell M . MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinformatics. 2011; 12: 491.

[44] Bairoch A, Apweiler R . The SWISS-PROT protein sequence database and its supplement TrEMBL. Nucleic Acids Res. 2000; 28: 45-8.

[45] Pruitt KD, Tatusova T, Maglott DR . NCBI reference sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2005; 33: D501-4.

[46] Kanehisa M, Goto S . KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28: 27-30.

[47] Zdobnov E, Apweiler R . InterProScan. An integration platform for the signature-recognition methods in InterPro. Bioinformatics. 2001; 17: 847-8.

[48] Ashburner M, Ball CA, Blake JA et al. Gene ontology: tool for the unification of biology. Nat Genet. 2000; 25: 25-9.

[49] Finn RD, Tate J, Mistry J et al. The Pfam protein families database. Nucleic Acids Res. 2008; 36: D281-8.

[50] Bao W, Kojima KK, Kohany O . Repbase update, a database of repetitive elements in eukaryotic genomes. Mob DNA. 2015; 6: 11.

[51] Tarailo-Graovac M, Chen N . Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinformatics. 2009; 25: 10.

[52] Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999; 27: 573-80.

[53] Edgar RC, Myers EW . PILER: identification and classification of genomic repeats. Bioinformatics. 2005; 21: i152-8.

[54] Ellinghaus D, Kurtz S, Willhoeft U . LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons. BMC Bioinformatics. 2008; 9: 18.

[55] Wang Y, Tang H, DeBarry JD et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012; 40: e49.

[56] Camacho C, Coulouris G, Avagyan V et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009; 10: 421.

[57] Quinlan AR . BEDTools: the Swiss-Army tool for genome feature analysis. Curr Protoc Bioinformatics. 2014; 47: 1-34.

[58] Chen C, Chen H, Zhang Y et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13: 1194-202.

[59] McKenna A, Hanna M, Banks E et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20: 1297-303.

[60] Li H, Handsaker B, Wysoker A et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25: 2078-9.

[61] Purcell S, Neale B, Todd-Brown K et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007; 81: 559-75.

[62] Kumar S, Stecher G, Li M et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35: 1547-9.

[63] Alexander DH, Novembre J, Lange K . Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 2009; 19: 1655-64.

[64] Montana G, Clive JH . Statistical software for gene mapping by admixture linkage disequilibrium. Brief Bioinformatics. 2007; 8: 393-5.

[65] Patterson N, Price AL, Reich D . Population structure and eigenanalysis. PLoS Genet. 2006; 2: e190.

[66] Enright AJ, Van Dongen S, Ouzounis CA . An efficient algorithm for large-scale detection of protein families. Nucleic Acids Res. 2002; 30: 1575-84.

[67] Stamatakis A . RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies. Bioinformatics. 2014; 30: 1312-3.

[68] Bie DT, Cristianini N, Demuth JP et al. CAFE: a computational tool for the study of gene family evolution. Bioinformatics. 2006; 22: 1269-71.

[69] Yang ZH . PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007; 24: 1586-91.

[70] Zhou Z-W, Yu Z-G, Huang X-M . GenomeSyn: a bioinformatics tool for visualizing genome synteny and structural variations. J Genet Genomics. 2022; 22: 00104-7.

[71] Wang D, Zhang Y, Zhang Z et al. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genom Proteom Bioinf. 2010; 8: 77-80.

[72] Kaori I, Murphy D . Application of ggplot2 to Pharmacometric Graphics. CPT: Pharmacomet Syst. 2013; 2: e79.

[73] Nattestad M, Schatz MC . Assemblytics: a web analytics tool for the detection of variants from an assembly. Bioinformatics. 2016; 32: 3021-3.

[74] Marçais G, Delcher AL, Phillippy AM et al. MUMmer4: a fast and versatile genome alignment system. PLoS Compu Biol. 2018; 14: e1005944.

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

[76] Trapnell C, Williams BA, Pertea G et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28: 511-5.

[77] Anders S, Huber W . Differential expression of RNA-seq data at the gene level - the DESeq package. Mol Biol. 2012; 11: e1005944.

[78] Lescot M, Déhais P, Thijs G et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002; 30: 325-7.