A chromosome-scale and haplotype-resolved genome assembly of tetraploid blackberry (Rubus L. subgenus Rubus Watson)

Abstract

Blackberries (Rubus spp.) are globally consumed and well known for their rich anthocyanin and antioxidant content and distinct flavors. Improving blackberries has been challenging due to genetic complexity of traits and limited genomic resources. The blackberry genome has been particularly challenging to assemble due to its polyploid nature. Here, we present the first chromosome-scale and haplotype-phased assembly for the primocane-fruiting, thornless tetraploid blackberry selection BL1 (Rubus L. subgenus Rubus Watson). The genome assembly was generated using Oxford Nanopore Technology and Hi-C scaffolding, resulting in a 919 Mb genome distributed across 27 pseudochromosomes, with an N50 of 35.73 Mb. This assembly covers >92% of the genome length and contains over 98% of complete BUSCOs. Approximately, 58% of the assembly consists of repetitive sequences, with long terminal repeats being the most abundant class. A total of 87,968 protein-coding genes were predicted, of which, 82% were functionally annotated. Genome mining and RNA-Seq analyses identified possible candidate genes and transcription factors related to thornlessness and the key structural genes and transcription factors for anthocyanin biosynthesis. Activator genes including PAP1 and TTG1 and repressor genes such as ANL2 and MYBPA1 play an important role in the fine tuning of anthocyanin production during blackberry development. Resequencing of seven tetraploid blackberry cultivars/selections with different horticultural characteristics revealed candidate genes that could impact fruiting habit and disease resistance/susceptibility. This tetraploid reference genome should provide a valuable resource for accelerating genetic analysis of blackberries and facilitating the development of new improved cultivars with enhanced horticultural and nutritional traits.

Acknowledgements

We thank the following sequencing companies for genome and transcriptome sequencing or contig scaffolding: NextOmics Biosciences (Wuhan, China) for Oxford Nanopore Sequencing, Illumina sequencing, and RNA-Seq; CD Genomics (Shirley, NY, USA) for Illumina sequencing and RNA-Seq, and PhaseGenomics (Seattle, WA, USA) for Hi-C and scaffolding. All genome and transcriptome assemblies and analyses were performed on the University of Florida’s HiPerGator supercomputer, supported by the UFIT Research Computing unit (Gainesville, FL, USA). We sincerely thank Dr. Jaroslav Doležel (Institute Experimental Botany, Olomouc, Czeck Republic) for providing ‘Polanka’ soybean (Glycine max Merr.) and ‘Stupické polní rané’ tomato (Solanum lycopersicum L.), which served as internal standards for flow cytometrical analysis.

Author Contributions

ZD secured funding, initiated and supervised the project, and finalized the manuscript. SP collected blackberry leaf tissue samples, isolated genomic DNA for genome sequencing or resequencing, isolated RNA for transcriptome sequencing, and prepared blackberry leaf tissues for Hi-C genome scaffolding; ZP coordinated genome and transcriptome sequencing with sequencing companies; SBP performed chromosome squashing, conducted genome size estimation using flow cytometry, and analyzed the expression of anthocyonin biosynthesis genes; DP assembled, annotated, and analyzed the blackberry genome and transcriptomes, identified candidate genes, conducted phylogeny analysis, and wrote the manuscript; All authors revised and reviewed the manuscript.

Data availability

The assembly and annotation files of the BL1 blackberry genome have been uploaded to the Rosaceae database (accession number: tfGDR1070). All raw sequencing data are available in the NCBI under BioProject: PRJNA1125996.

Conflict of interest statement

The authors declare that they have no conflict of interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Clark JR, Stafne ET, Hall HK. et al. Blackberry breeding and genetics. Plant Breed Rev. 2007; 29:19-144

[2] Foster TM, Bassil NV, Dossett M. et al. Genetic and genomic resources for Rubus breeding: a roadmap for the future. Hortic Res. 2019; 6:116

[3] Morgan KL. Market trends for US berry crops: implications for Florida blueberry, blackberry, and raspberry. EDIS. 2022; 2022: 1-5

[4] Thompson MM. Survey of chromosome numbers in Rubus (Rosaceae: Rosoideae). Ann Mo Bot Gard. 1997; 84:128-64

[5] Hummer KE, Bassil NV, Alice LA. Rubus ploidy assessment. Acta Hortic. 2016; 1133:81-7

[6] Meng R, Finn C. Determining ploidy level and nuclear DNA content in Rubus by flow cytometry. J Am Soc Hortic Sci. 2002; 127: 767-75

[7] Castro P, Stafne ET, Clark JR. et al. Genetic map of the primocane-fruiting and thornless traits of tetraploid black-berry. Theor Appl Genet. 2013; 126:2521-32

[8] Chizk TM, Clark JR, Johns C. et al. Genome-wide association identifies key loci controlling blackberry postharvest quality. Front Plant Sci. 2023; 14:1182790

[9] Hall HK, Cohen D, Skirvin RM. The inheritance of thornlessness from tissue culture-derived “thornless evergreen” blackberry. Euphytica. 1986a; 35:891-8

[10] Du X, Qian M. Flavor chemistry of small fruits:blackberry, raspberry, and blueberry. In: Flavor and Health Benefits of Small Fruits, 2010, 27-43

[11] Strik B, Buller G. Reducing thorn contamination in machine harvested “Marion” blackberry. Acta Hortic. 2002; 585:677-81

[12] Clark JR, Perkins-Veazie P. “APF-45” primocane-fruiting black-berry. HortScience. 2011; 46:670-3

[13] Lopez-Medina J, Moore JN, McNew RW. A proposed model for inheritance of primocane fruiting in tetraploid erect blackberry. J Am Soc Hortic Sci. 2000; 125:217-21

[14] Browne FB, Brannen PM, Scherm H. et al. Yield response to orange cane blotch of blackberry grown in the Georgia coastal plain. Plant Dis. 2020; 104:161-7

[15] Hall HK. Blackberry breeding. Plant Breed Rev. 1990;249-312

[16] Jennings DL, Daubeny HA, Moore JN. Blackberries and raspber-ries (Rubus). Acta Hortic. 1991; 290:331-89

[17] Jibran R, Dzierzon H, Bassil N. et al. Chromosome-scale scaffold-ing of the black raspberry (Rubus occidentalis L.) genome based on chromatin interaction data. Hortic Res. 2018; 7:8

[18] VanBuren R, Bryant D, Bushakra JM. et al. The genome of black raspberry (Rubus occidentalis). Plant J. 2016; 87:535-47

[19] Vanburen R, Wai CM, Colle M. et al. A near complete, chromosome-scale assembly of the black raspberry (Rubus occi-dentalis) genome. Gigascience. 2018;7:1, giy094-21

[20] Davik J, Roen D, Lysoe E. et al. A chromosome-level genome sequence assembly of the red raspberry (Rubus idaeus L.). PLoS One. 2022; 17:e0265096-15

[21] Price RJ, Davik J, Fernandéz F. et al. Chromosome-scale genome sequence assemblies of the ‘Autumn Bliss’ and ‘Malling Jewel’ cultivars of the highly heterozygous red raspberry (Rubus idaeus L.) derived from long-read Oxford nanopore sequence data. PLoS One. 2023; 18:e0285756

[22] Wang L, Lei T, Han G. et al. The chromosome-scale refer-ence genome of Rubus chingii Hu provides insight into the biosynthetic pathway of hydrolyzable tannins. Plant J. 2021; 107: 1466-77

[23] Br ˚una T, Aryal R, Dudchenko O. et al. A chromosome-length genome assembly and annotation of blackberry (Rubus argutus, cv. “Hillquist”). G3 (Bethesda). 2023;13:jkac289

[24] Han H, Barbey CR, Fan Z. et al. Telomere-to-telomere and haplotype-phased genome assemblies of the heterozygous octoploid ‘Florida Brilliance’ strawberry (Fragaria x ananassa). bioRxiv. 2022:10.05.509768

[25] Mao J, Wang Y, Wang B. et al. High-quality haplotype-resolved genome assembly of cultivated octoploid strawberry. Hortic Res. 2023;10:uhad002

[26] SongY, PengL, LiuY. et al. Phased gap-free genome assem-bly of octoploid cultivated strawberry illustrates the genetic and epigenetic divergence among subgenomes. Hortic Res. 2024;11:uhad252

[27] Zhang Z, Yang T, Liu Y. et al. Haplotype-resolved genome assembly and resequencing provide insights into the origin and breeding of modern rose. Nat Plants. 2024; 10:1659-71

[28] Edger PP, VanBuren R, Colle M. et al. Single-molecule sequenc-ing and optical mapping yields an improved genome of wood-land strawberry (Fragaria vesca) with chromosome-scale conti-guity, GigaScience, 2018;7:gix124

[29] Hibrand Saint-Oyant L, Ruttink T, Hamama L. et al. Ahigh-quality genome sequence of Rosa chinensis to elucidate orna-mental traits. Nat. Plants. 2018; 4:473-84

[30] Daccord N, Celton JM, Linsmith G. et al. High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat Genet. 2017; 49:1099-106

[31] Dong X, Wang Z, Tian L. et al. De novo assembly of a wild pear (Pyrus betuleafolia) genome. Plant Biotechnol J. 2020; 18:581-95

[32] Shirasawa K, Itai A, Isobe S. Genome sequencing and analysis of two early-flowering cherry (Cerasus × kanza-kura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’. DNA Res. 2021;28:dsab026

[33] Wang J, Liu W, Zhu D. et al. Chromosome-scale genome assem-blyofsweet cherry (Prunus avium L.) cv. Tieton obtained using long-read and hi-C sequencing. Hortic Res. 2020; 7:122

[34] 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

[35] Zhang A, Zhou H, Jiang X. et al. The draft genome of a flat peach (Prunus persica L. cv. ‘124 Pan’) provides insights into its good fruit flavor traits. Plants (Basel). 2021; 10:538

[36] Wang Y, Xie J, Zhang H. et al. The genome of Prunus humilis provides new insights to drought adaptation and population diversity. DNA Res. 2022; 29:1-10

[37] Liu C, Feng C, Peng W. et al. Chromosome-level draft genome of a diploid plum (Prunus salicina). GigaScience. 2021a; 9:1-11

[38] Groppi A, Liu S, Cornille A. et al. Population genomics of apri-cots unravels domestication history and adaptive events. Nat Commun. 2021; 12:1-16

[39] Liu X-J, Wang X-R, Tang H-R. et al. The complete chloroplast genome sequence of a hybrid blackberry (Rubus spp.) cultivar. Mitochondrial DNA B. 2021b; 6:2103-4

[40] Qiao F, Guo W, Dai W. et al. The complete chloroplast genome sequence of Rubus peltatus Maxim. (Rosaceae). Mitochondrial DNA B. 2022; 7:1056-7

[41] Zhu Y-A, Li C, Wang S. et al. The complete chloroplast genome of Rubus setchuenensis, an edible and medicinal dual-purpose wild plant. Mitochondrial DNA B Resour. 2022; 7:228-30

[42] Chen Q, Wang Y, Wang X. et al. The complete chloroplast genome sequence of Rubus lambertianus var. glaber : sequence divergence and phylogenetic application. Mitochondrial DNA B. 2020; 5:1261-2

[43] Agehara S, Lin S-Y, Deng Z. Choosing the right blackberry cultivar in subtropical Florida. EDIS. 2020; 2020:6

[44] Marin MV, Wang N-Y, Seijo TE. et al. Pseudocercospora pancratii causing leaf spots on commercial blackberry ( Rubus sp.) in Florida. Plant Dis. 2022a; 107:131-5

[45] Marin MV, Seijo T, Druffel KL. et al. First report of leaf rust on blackberry (Rubus spp.) caused by Kuehneola uredinis in Florida. Plant Dis. 2022b; 106:2528

[46] Pastrana AM, Kirkpatrick SC, Kong M. et al. Fusarium oxysporum f. sp. mori, a new Forma Specialis causing Fusarium wilt of blackberry. Plant Dis. 2017; 101:2066-72

[47] Ali ME, Hudson O, Waliullah S. et al. First report of downy mildew on blackberry caused by Peronospora sparsa in Georgia, U.S.A. Plant Dis. 2020; 104:996

[48] Solano-Báez AR, Leyva-Mir SG, Camacho-Tapia M. et al. First report of Podosphaera aphanis causing powdery mildew on wild blackberry species (Rubus species) in Mexico. Plant Dis. 2022; 106:1749

[49] Stevanovi ćM, VojvodićM, Kovačevi ćS. et al. First report of powdery mildew of blackberry caused by Podosphaera aphanis in Serbia. Plant Dis. 2021; 105:503

[50] Afanador-Kafuri L, Mejía JF, González A. et al. Identifying and analyzing the diversity of resistance gene analogs in Colombian Rubus genotypes. Plant Dis. 2015; 99:994-1001

[51] DeYoung BJ, Innes RW. Plant NBS-LRR proteins in pathogen sensing and host defense. Nat Immunol. 2006; 7: 1243-9

[52] McHale L, Tan X, Koehl P. et al. Plant NBS-LRR proteins: adapt-able guards. Genome Biol. 2006; 7:212

[53] Kusch S, Panstruga R. Mlo-based resistance: an apparently uni-versal “weapon” to defeat powdery mildew disease. Mol Plant-Microbe Interact. 2017; 30:179-89

[54] Kusch S, Pesch L, Panstruga R. Comprehensive phylogenetic analysis sheds light on the diversity and origin of the MLO family of integral membrane proteins. Genome Biol Evol. 2016; 8: 878-95

[55] van Damme M, Huibers RP, Elberse J. et al. Arabidopsis DMR6 encodes a putative 2OG-Fe(II) oxygenase that is defense-associated but required for susceptibility to downy mildew. Plant J. 2008; 54:785-93

[56] Zeilmaker T, Ludwig NR, Elberse J. et al. DOWNY MILDEW RESIS-TANT 6 and DMR6-LIKE OXYGENASE 1 are partially redundant but distinct suppressors of immunity in Arabidopsis. Plant J. 2015; 81:210-22

[57] Giacomelli L, Zeilmaker T, Giovannini O. et al. Simultane-ous editing of two DMR6 genes in grapevine results in reduced susceptibility to downy mildew. Front Plant Sci. 2023; 14: 1242240

[58] Djennane S, Gersch S, Le-Bohec F. et al. CRISPR/Cas9 editing of downy mildew resistant 6 (DMR6-1) in grapevine leads to reduced susceptibility to Plasmopara viticola. JExp Bot. 2024; 75:2100-12

[59] Hasley JAR, Navet N, Tian M. CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance. PLoS One. 2021; 16:e0253245

[60] de Toledo Thomazella DP, Seong K, Mackelprang R. et al. Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance. Proc Natl Acad Sci USA. 2021; 118:e2026152118

[61] Kieu N, Lenman M, Wang ES. et al. Mutations introduced in sus-ceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes. Sci Rep. 2021; 11: 1-12

[62] Parajuli S, Huo H, Gmitter FG. et al. Editing the CsDMR6 gene in citrus results in resistance to the bacterial disease citrus canker. Hortic Res. 2022; 9:11-3

[63] Tripathi JN, Ntui VO, Shah T. et al. CRISPR/Cas9-mediated editing of DMR6 orthologue in banana (Musa spp.) confers enhanced resistance to bacterial disease. Plant Biotechnol J. 2021; 19:1291-3

[64] Müller D, Leyser O. Auxin, cytokinin and the control of shoot branching. Ann Bot. 2011; 107:1203-12

[65] Coyner MA, Skirvin RM, Norton MA. et al. Thornlessness in blackberries: a review. Small Fruit Rev. 2005; 4:83-106

[66] Stanton M, Gauthier NW. Blackberry rosette (double blossom). Plant Pathol Fact Sheet. 2017;1-3. https://plantpathology.ca.uky. edu/files/ppfs-fr-s-03.pdf

[67] Hall HK, Quazi MH, Skirvin RM. Isolation of a pure thorn-less loganberry by meristem tip culture. Euphytica. 1986b; 35: 1039-44

[68] Pattanaik S, Patra B, Singh SK. et al. An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis. Front Plant Sci. 2014; 5:1-8

[69] Payne CT, Zhang F, Lloyd AM. GL 3 encodes a bHLH pro-tein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics. 2000; 156: 1349-62

[70] Zhang F, Gonzalez A, Zhao M. et al. A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development. 2003; 130:4859-69

[71] Oshima Y, Mitsuda N. The MIXTA-like transcription factor MYB16 is a major regulator of cuticle formation in vegetative organs. Plant Signal Behav. 2013; 8:e26826

[72] Khadgi A, Weber CA. Genome-wide association study (GWAS) for examining the genomics controlling prickle production in red raspberry (Rubus idaeus L.). Agronomy. 2020; 11:27

[73] Liu X, Wang T, Bartholomew E. et al. Comprehensive analysis of NAC transcription factors and their expression during fruit spine development in cucumber (Cucumis sativus L.). Hortic Res. 2018; 5:31

[74] Lan T, Zheng Y, Su Z. et al. OsSPL10, a SBP-Box gene, plays a dual role in salt tolerance and trichome formation in rice (Oryza sativa L.). G3|Genes|Genomes|Genetics. 2019; 9:4107-14

[75] Stukkens Y, Bultreys A, Grec S. et al. NpPDR1, a pleiotropic drug resistance-type ATP-binding cassette transporter from Nicotiana plumbaginifolia, plays a major role in plant pathogen defense. Plant Physiol. 2005; 139:341-52

[76] Nagel J, Culley LK, Lu Y. et al. EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol. Plant Cell. 2008; 20:186-200

[77] Lee SB, Go YS, Bae HJ. et al. Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglobules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola. Plant Physiol. 2009; 150: 42-54

[78] Kirch T, Simon R, Grünewald M. et al. The Dornröschen/enhancer of shoot regeneration1 gene of Arabidopsis acts in the control of meristem cell fate and lateral organ development. Plant Cell. 2003; 15:694-705

[79] Banno H, Ikeda Y, Niu QW. et al. Overexpression of Arabidop-sis ESR1 induces initiation of shoot regeneration. Plant Cell. 2001; 13:2609-18

[80] Pandey S, Goel R, Bhardwaj A. et al. Transcriptome analysis pro-vides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum Dunal. Sci Rep. 2018; 8:1-12

[81] Rabby MG, Hossen MM, Kamal MM. et al. Genome-wide identi-fication and functional analysis of lysine histidine transporter (LHT) gene families in maize. Genet Res (Camb). 2022; 2022:1-10

[82] Shaikhali J, Davoine C, Björklund S. et al. Redox regulation of the MED28 and MED32 mediator subunits is important for development and senescence. Protoplasma. 2016; 253:957-63

[83] Bouché F, Lobet G, Tocquin P. et al. FLOR-ID: an interac-tive database of flowering-time gene networks in Arabidopsis thaliana. Nucleic Acids Res. 2016;44:D1167-71

[84] Shang L, Tao J, Song J. et al. CRISPR/Cas9-mediated mutations of FANTASTIC FOUR gene family for creating early flowering mutants in tomato. Plant Biotechnol J. 2024; 22:1-11

[85] Fan-Chiang H-J, Wrolstad R. Anthocyanin pigment composition of blackberries. J Food Sci. 2006;70:C198-202

[86] LeeH, WangZ, DengZ. et al. Assessment of six blackberry cul-tivars using a combination of metabolomics, biological activity, and network pharmacology approaches. Antioxidants (Basel). 2024; 13:319

[87] Clark JR, Salgado A. ‘Prime-Ark® Traveler’ primocane-fruiting thornless blackberry for the commercial shipping market. HortScience. 2016; 51:1287-93

[88] Clark JR. Prime-Ark® Freedom primocane-fruiting thornless blackberry. HortScience. 2014; 49:1097-101

[89] Moore JN, Clark JR. “Kiowa” blackberry. HortScience. 1996;31:750b-0

[90] Clark JR. “Osage” thornless blackberry. HortScience. 2013; 48: 909-12

[91] Dolezel J, Greilhuber J, Suda J. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2007; 2: 2233-44

[92] Rothleutner JJ, Friddle MW, Contreras RN. Ploidy levels, relative genome sizes, and base pair composition in cotoneaster. JAm SocHorticSci. 2016; 141:457-66

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

[94] Ruan J, Li H. Fast and accurate long-read assembly with wtdbg2. Nat Methods. 2020; 17:155-8

[95] Di Genova A, Buena-Atienza E, Ossowski S. et al. Efficient hybrid de novo assembly of human genomes with WENGAN. Nat Biotechnol. 2021; 39:422-30

[96] LiuH, WuS, LiA. et al. SMARTdenovo: a de novo assembler using long noisy reads. GigaByte. 2021; 2021:1-9

[97] Chen Y, Nie F, Xie S-Q. et al. Efficient assembly of nanopore reads via highly accurate and intact error correction. Nat Com-mun. 2021; 12:60

[98] Kolmogorov M, Yuan J, Lin Y. et al. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019; 37:540-6

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

[100] Faust GG, Hall IM. SAMBLASTER: fast duplicate marking and structural variant read extraction. Bioinformatics. 2014; 30: 2503-5

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

[102] Durand NC, Robinson JT, Shamim MS. et al. Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell Syst. 2016; 3:99-101

[103] Bickhart DM, Rosen BD, Koren S. et al. Single-molecule sequenc-ing and chromatin conformation capture enable de novo refer-ence assembly of the domestic goat genome. Nat Genet. 2017; 49: 643-50

[104] Smit AFA. Repeat-Masker Open-3.0, 2004. Institute for Systems Biology, Seattle, WA, USA. http://www.Repeatmasker.org

[105] Han Y, Wessler SR. MITE-Hunter: a program for discover-ing miniature inverted-repeat transposable elements from genomic sequences. Nucleic Acids Res. 2010; 38:e199-9

[106] Xu Z, Wang H. LTR_FINDER: an efficient tool for the pre-diction of full-length LTR retrotransposons. Nucleic Acids Res. 2007;35:W265-8

[107] Gremme G, Steinbiss S, Kurtz S. GenomeTools: a comprehen-sive software library for efficient processing of structured genome annotations. IEEE ACM Trans Comput Biol Bioinform. 2013; 10:645-56

[108] Ou S, Jiang N. LTR_retriever: a highly accurate and sensitive program for identification of long terminal repeat retrotrans-posons. Plant Physiol. 2018; 176:1410-22

[109] Hoff KJ, Lange S, Lomsadze A. et al. BRAKER1: unsupervised RNA-seq-based genome annotation with GeneMark-ET and AUGUSTUS. Bioinformatics. 2016; 32:767-9

[110] Gabriel L, Hoff KJ, Br ˚una T. et al. TSEBRA: transcript selector for BRAKER. BMC Bioinformatics. 2021; 22:1-12

[111] Jin JJ, Yu WB, Yang JB. et al. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020; 21:241-31

[112] Tillich M, Lehwark P, Pellizzer T. et al. GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017;45:W6-11

[113] Zheng S, Poczai P, Hyvönen J. et al. Chloroplot: an online program for the versatile plotting of organelle genomes. Front Genet. 2020; 11:576124

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

[115] Andrews S. 2017 FastQC: a quality control tool for high throughput sequence data, Babraham Institute, Babraham, Cambridge, UK. 2010

[116] Dobin A, Davis CA, Schlesinger F. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29:15-21

[117] Pertea M, Pertea GM, Antonescu CM. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015; 33:290-5

[118] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009; 26:139-40

[119] Altschul SF, Gish W, Miller W. et al. Basic local alignment search tool. J Mol Biol. 1990; 215:403-10

[120] Li H, Durbin R. Fast and accurate long-read alignment with burrows-wheeler transform. Bioinformatics. 2010; 26:589-95

[121] Cingolani P, Platts A, Wang LL. 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

[122] Emms DM, Kelly S. Orthofinder: phylogenetic orthology infer-ence for comparative genomics. Genome Biol. 2019; 20:1-14

[123] Eddy S. Hmmer User’s Guide. Vol. 2. Dep Genet Washingt Univ Sch Med; 1992:13

[124] Ludwiczak J, Winski A, Szczepaniak K. et al. DeepCoil—a fast and accurate prediction of coiled-coil domains in protein sequences. Bioinformatics. 2019; 35:2790-5

[125] Edgar RC. Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32: 1792-7

[126] Paradis E, Schliep K. Ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019; 35:526-8

[127] Yu G, Smith DK, Zhu H. et al. Ggtree: an R package for visualiza-tion and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol Evol. 2017; 8:28-36

[128] Tian F, Yang DC, Meng YQ. et al. PlantRegMap: chart-ing functional regulatory maps in plants. Nucleic Acids Res. 2020;48:D1104-13

[129] Madden T. The BLASTSequence Analysis Tool. NCBI Handb, National Center for Biotechnology Information (US), Bethesda, MD, USA. 2003