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The breeding of yellow dragon fruit (Selenicereus megalanthus, 2n=4x=44) is still seriously hampered due to the lack of a reference genome. Here, we discuss the high-quality chromosome-level genomic assembly of yellow dragon fruit based on Hi-C, ATAC, and tissue-specific RNA-seq data, and link phenotypic traits to genomic data. We declare yellow dragon fruit to be homotetraploid with a genome size of 7.16 Gb (containing 27,246 high-confidence genes) that evolved primarily from a diploid ancestor, whose ancestry is currently unknown. In addition to generative genome assembly, we explored 3D chromatin organization, revealing different amounts of structural variation (SV) in the genome, compartment A (648 and 519), compartment B (728 and 1064), topology-related domain TAD (3376 and 2031), and diploid and polyploid dragon fruit, respectively. Overall, the AP2, WRKY18/60/75, MYB63/116, PHL2 and GATA8 motifs in the two dragon fruit species enriched the TAD boundary. By linking open chromatin genome structure to function, we identified major changes in betain biosynthesis pathways in diploid and polyploid dragon fruit. In addition, SmeADH1 [Chr11, cell A (135400000-135500000), genes within the TAD region (135480000-135520000)] had higher gene expression, while HuDOPA [Chr11, compartment A (87100000-87200000)] had lower expression and genes within the TAD region (87160000-87200000)] as key regulators of yellow and red color on polyploid and diploid dragon fruit peels, respectively. In addition, the higher expression of HuCYP76AD1 gene in diploid dragon fruit and the lower expression of SmeCYP76AD1 in polyploid dragon fruit may cause differences in oxidase processes, resulting in the production of betalain and betaxanthin, respectively. In addition, our results not only reveal the types of motifs that play a potential role in trait patterns, but we further found that motif counts in TAD boundaries may affect gene expression within the TAD region of diploid and polyploid dragon fruit. Comparison of diploid and polyploid dragon fruit species with 3D euchromatin structure not only helps to advance molecular breeding efforts in genomic organization, SV, compartmentalization (A and B).
outcome
Key innovations
Despite great advances in genomics tools and the growing demand for dragon fruit, the high-quality reference genome of the homotetraploid yellow dragon fruit (S. megalanthus, 2n=4x=44) is still lacking, hindering its molecular breeding. Therefore, we strive to adopt modern genomics tools to gain a deeper understanding of the genome of yellow dragon fruit. The focus of innovation is:
1. We performed long-read sequencing using PacBio-HiFi and discussed the high-quality chromosome-level genome sequence and assembly of homotetraploid (S. megalanthus; 2n=4x=44; ~7.16Gb).
2. We address centuries-old misconceptions about the classification and evolution of yellow dragon fruit and declare it to be highly heterozygous homotetraploid (AAAB).
3. Using both Hi-C and RNA-seq, we identified putative genes associated with different phenotypic traits in the red and yellow dragon fruit genomes. An integrated approach is more valuable than RNA-seq alone because it combines spatial genomic organization with gene expression, provides insights into gene regulation and interactions, fast-tracks the discovery of target genes, and reduces the time and effort required for breeding programs. Previously, this ensemble approach was only used by Liu et al. （DOI：10.1126/science.adg3797）。
4. By utilizing comparative genomics of diploid and homotetraploid dragon fruit, we identified chromosomal organization, chromatin structure, genomic compartmentalization (A and B), topology-related domain (TAD) boundaries, and structural variation (SV) in both genomes.
Detailed summary
Dragon fruit or pitaya belongs to the cactus family (Skinskin), an ancient family that originated ~65 million years ago. The genus is divided into 28 species; Among them, S. megalanthus (2n=4x=44) and S. undatus (2n=22) (formerly Hylocereus undatus) are the most commercialized and consumerly valuable plant groups, with attractive fruit appearance, color, and flavor, rich in vitamins, antioxidants, minerals, phytochemicals, dietary fiber, and cancer prevention. Despite the tremendous advances in genomics tools and the growing demand for dragon fruit, the high-quality reference genome of the homotetraploid yellow dragon fruit (S. megalanthus, 2n=4x=44) is still lacking, hindering its molecular breeding. In addition, there is an urgent need to map the phenotypic variation between diploid red dragon fruit (red peel, thin stem/branch, and green fin-like part) and tetraploid yellow dragon fruit (yellow peel, thick stem/branch, and spines) to accelerate molecular breeding programs. Therefore, we reported high-quality chromosome-level genome assembly of yellow dragon fruit and linked phenotypic traits to genomic data based on tissue-specific Hi-C, ATAC, and RNA-seq data. We declare yellow dragon fruit to be a homotetraploid with high heterozygosity (AAAB), with an estimated genome of 7.16Gb containing 27,246 genes, which evolved mainly from a diploid ancestor, which is still unknown.
In addition to genome assembly, we compared and explored the 3D chromatin organization between the genomes of diploid red dragon fruit (2n=22) and homotetraploid yellow dragon fruit (2n=4x=44), and identified chromosomal organization, chromatin structure, genomic compartments (A and B), topology-associated domain (TADs) boundaries, and structural variants (SVs) in both genomes. Overall, the TAD boundaries of the two dragon fruits were enriched with the motifs of AP2, WRKY18/60/75, MYB63/116, PHL2 and GATA8. In addition, by linking open chromatin genome structure to function, we identified major changes in the shikimic acid pathway that regulates "betain biosynthesis" in two dragon fruits. Expression analysis based on RNA-seq showed high expression of SmeDOPA gene in the TAD region (115360,000-1154000000) [Chr08, chamber A (115300000-115500000)] and low expression of HuDOPA gene in the TAD region (97520000-975600000) [Chr08, chamber A (97500000-97600000)] As a key regulator of yellow and red color of polyploid and diploid dragon fruit peels. In addition, our results not only found a potential role for motif types in trait patterns, but also further revealed that motif counts at TAD boundaries may affect gene expression within the TAD region of diploid and polyploid dragon fruit. In short, our comparison of genomic resources between diploid and polyploid dragon fruit species contributes not only to the development of molecular breeding (e.g., GAB) worldwide, but also to the understanding of genomic organization, SVs, regionalization (A and B), and TADs. This has the potential to enhance future TADs-based trait improvements.
chart
Selenicereusmegalanthus)基因緰组装
Table 2: Statistical table of the number and length of compartments
Figure 1: Classification and evolution of the misunderstood polyploid S. megalanthus. Comparative genomics and phenomics of diploid and polyploid dragon fruit. Phenotypic characteristics of a) yellow and b) red dragon fruit (white flesh). c) Schematic diagram of the misunderstood classification of yellow dragon fruit for 100 years. The genomic resources we assembled provide insights into the evolution of yellow dragon fruit (97% homotetraploid, 3% heteropolyploidy). d) It shows the interchromosomal compatibility of the newly assembled yellow dragon fruit genome. e) Comparative genomics of diploid and tetraploid dragon fruit. Comprehensive analysis of 3D genomics, TAD boundaries, and RNA-seq data revealed specific genes associated with the respective phenotypic traits (fruit color) of both species. This method makes it possible to skip the laborious MAS breeding method.
Figure 2: Genomic characterization of the homotetraploid yellow dragon fruit genome (S. megalanthus). The ring represents 11 chromosomes, a) represents the GC content distribution, b) gene density distribution, c) total repeat density distribution, d) long terminal repeat (LTR) distribution, e) LINE density distribution, and f) dna-transposable element distribution.
Figure 3: Contig distribution map on homotetraploid chromosomes, differentiation time of S. megalanthus, estimation of gene family expansion and contraction. a) Gray indicates the length of each chromosome, and other colors indicate contig with different length ranges. b) Differentiation time of yellow dragon fruit from other plant species. The number at the node position represents the time of divergence of the plant species from its ancestors millions of years ago (MYA). The differentiation time supported by the highest posterior density (HPD) of 95% is also mentioned in parentheses. The branch length of a phylogenetic tree simply indicates the number of base substitutions or genetic distance at each point. c) Expansion and contraction of gene families in plant species. Green indicates gene families added to the genome during the evolution of the species, and red indicates the loss of gene families in that plant species.
Figure 4: 3D genome structure analysis of diploid and polyploid dragon fruit. a) Single chromosomal compartment. b) Single chromosomal compartment of S. megalanthus. [The upper halves of Figures 4a and 4b represent the distribution of chamber A/B values.] The blue bar indicates the active (A) portion of the genome, while the red bar indicates the inactive (B) portion of the genome. The lower half of the diagram represents the correlation matrix converted from a single chromosome interaction matrix. The color bar indicates the correlation coefficient]. c) Single-chromosomal TADs of S. undatus. The horizontal axis represents the position on the reference genome (unit: Mb). The upper part of the diagram represents the interaction of individual chromosomes at a specific location. In the lower part of the figure, the blue line is the insulation score and the gray line is the TAD boundary. d) The number of genes within the TAD boundary of diploid dragon fruit. e) The number of genes within the TAD boundary of polyploid dragon fruit. [The horizontal axes in Figures 4d and 4e represent the number of genes within the TAD (Inter) and the TAD border.] The significance test result in **** represents 0.0001≥p]. f) The number of genes within the TAD boundary of polyploid dragon fruit. The horizontal axis represents the number of genes within the TAD (Inter) and the TAD border. The significance test result in represents 0.0001≥p. g) Single-chromosomal TADs of S. megalanthus. The horizontal axis represents the position on the reference genome (unit: Mb). The upper part of the diagram represents the interaction of individual chromosomes at a specific location. In the lower part of the figure, the blue line is the insulation score and the gray line is the TAD boundary. h) Circular plot of cis/straddle significant interaction sites in the whole genome of diploid dragon fruit. i) Circular plot of polyploid dragon fruit genome-wide cis/straddle significant interaction sites. In Figures 4h and 4i, the names and numbers of chromosomes are presented in a clockwise direction in the figure. Red indicates the number of genes on each chromosome. Lines from dark blue to light blue indicate p-values on each chromosome from large to small and cis-significant interaction sites. The red bar indicates significant trans interaction sites with other chromosomes. Blue from dark to light indicates the number of reads that support interactions from large to small. j) S. undatus first 5 motif sequences. k) S. megalanthus first 5 motif sequences. In Figures 4j and 4k, the horizontal axis represents the length of the motif in bp, and the vertical axis represents the frequency distribution of the ATGC bases.
Figure 5: Genome-wide comparative analysis of diploid and polyploid dragon fruit species. a) Interaction heat map at 200 kb resolution showing the differences in the polyploid and diploid dragon fruit genomes (differences in S. megalanthus minus differences in interactions in S. undatus). The color bars, including red, indicate that S. megalanthus has a greater interaction intensity than S. undatus. Blue indicates the opposite effect, and higher values indicate differences in the intensity of interactions at this site across the diploid and polyploid genomes. The horizontal and vertical axes indicate the location of the genome. b) Simplified heat map of single-chromosome interactions between two dragon fruit species at 200-kb resolution. The red bar graph indicates that the interaction intensity of Nepeta grandiflora is greater than that of Nepeta flora. The higher the value of the blue bar, the greater the difference in the intensity of the two interactions. c) Comparison chart and compartment saddle diagram. The red contrast on the diagram shows compartment A, and the blue bar represents compartment B. In the saddle plot, the colors represent the interaction signals of the diploid and polyploid dragon fruit compartments AA, BB, and AB. d) Differential inter-chamber gene density distribution between diploid and polyploid dragon fruit. The horizontal axis represents the four different types of compartments, and the vertical axis represents the distribution of gene density within the region. e) Venn diagram of the common and distinct TAD boundaries of diploid (S. undatus) and polyploid (S. megalanthus) dragon fruits. f) Venn diagram of the common and unique cis-significant interactions between the two species. g) Venn diagrams of cross-salient interactions, including common and unique interactions between the two species h) GO-annotated genes in differential compartments of diploid and polyploid species. The horizontal axis indicates the number of genes annotated to the GO term. The vertical axis represents three broad classes of GO terms, including biological processes, cellular components, and molecular functions. i) Distribution of gene expression between genomic compartments. On the horizontal axis, it is divided into S. undatus and S. megalanthus. The vertical axis represents the distribution of gene expression in the A2A, A2B, B2A, and B2B compartments. Note: The significance test result **** represents 0.0001≥p. However, "ns" indicates no significant results. j) Differential compartment gene density distribution and intercompartmental gene switching between diploid and polyploid dragon fruit (S. undatus-S. megalanthus). The transverse axis represents the four compartment switches, and the transverse axis represents the gene number distribution for the conserved compartments (A2A and B2B) and the switch compartments (A2B and B2A).
Figure 6: Genomic structural variation of homotetraploid polyploid dragon fruit. The blue bar represents the reference genome of the yellow dragon fruit, while the orange bar represents the diploid genome of the red-skinned dragon fruit. The blue bar (reference sequence) indicates the genome of S. undatus. The orange bar (query sequence) represents the genome of the yellow dragon fruit (S. megalanthus).
Figure 7: Genome-wide ATAC-seq analysis of diploid and polyploid dragon fruit. a) ATAC-seq signal enrichment map of the TSS region of the S. undatus and S. megalanthus genomes. The horizontal axis of the heat map is 3 kb upstream and downstream of TSS, and the vertical axis is the gene. The horizontal axis of the trend graph is 3KB upstream and downstream of TSS, and the vertical axis is the signal enrichment of genomic locations. b) DAR clustering map of diploid and polyploid dragon fruit. The horizontal axis is the name of the dragon fruit species, and the vertical axis is the DAR obtained by comparing the two dragon fruits. The colors in the plot indicate the magnitude of the DAR signal values for each species. c) Distribution of DAR and DAR. The map shows the location of the DAR on the genome and the proportion of the DAR at that location. d) GO terms in acquired and lost DAR-related genes located in the promoter region. Genes are clustered according to their nomenclature and show their function on the left. e) Comparison of DAR-related genes with differentially expressed genes to identify overlapping genes in the promoter region. f) Box plot of DAR-associated gene expression. g) Multi-omics data visualization showed significant differences in chromatin accessibility between diploid and polyploid dragon fruit species.
Figure 8: Ligation of tissue-specific gene expression to open chromatin of diploid and polyploid dragon fruit. a) Except for a few genes, the genes related to betain synthesis of diploid dragon fruit had higher similarity with the genome sequence of yellow dragon fruit under the same number of chromosomes, and there were also differences in copy number. Genes may play a potential role in red-skinned dragon fruit (S. undatus), participating in the regulation of pathways and producing enzymes that spontaneously bind to cyclodopaine amino groups, ultimately forming betain pigment, i.e., reddish-purple betaanthin. In homotetraploid dragon fruit, genes belonging to the SmeABA2, SmeFG2/3, and SmeHD-ZIP families exert a potential role with amino groups to spontaneously condense betaine to produce yellow betain, but not b) genes that directly regulate shikimic acid pathway (Sme_10G0002470, Sme_7G0004800) in yellow dragon fruit are expressed in higher levels than genes expressed in red-skinned dragon fruit (HU10G00264, HU07G00551). 3 times higher. Homologous copies of the CYP736A12 gene are located in compartments A and B of the yellow dragon fruit genome. During the evolution and polyploidization of yellow dragon fruit, the duplication of CYP736A12 gene copies and the reduction of CYP36A12 gene expression in the A/B compartment of yellow dragon fruit eventually led to the loss of the ability of yellow dragon fruit to convert tyrosine oxidase to L-DOPA, and the production of betaflavin in turn. c) TFs and the top 5 TFs identified in the 40-Kb region of the polyploid dragon fruit TAD boundary. d) TFs and the first 5 TFs identified in the 40-Kb region of the diploid dragon fruit TAD boundary are shown in the figure.
Dr.Qamar近年来所发表的论文
1. Zaman QU, L Hui, MF Nazir, G Wang, V Garg, M Ikram, A Raza, W Lv, D Khan, AA Khokhar, Z You, A Chitikineni, B Usman, C Jianpeng, X Yang, S Zuo, P Liu, S Kumar, M Guo, ZX Zhu, G Dwivedi, YH Qin, RK Varshney*, HF Wang*. Chromosome-level genome assembly of autotetraploid Selenicereus megalanthus and gaining genomic insights into the evolution of trait patterning in diploid and polyploid pitaya species. Submitted to Preprint: https://doi.org/10.1101/2024.06.23.600268. [Top期刊，IF:10.1，按照发表时的分区和影响因子，下同].
2. Zaman QU, A Raza, L Chao, JL Juste, MGK Jones, HF Wang*, R K Varshney. Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops. Trends in Biotechnology, 42(5): 560-574. https://doi.org/10.1016/j.tibtech.2023.10.007 [中科院一区Top期刊，IF:17.3].
3. Zaman QU, A Raza, RA Gill, MA Hussain, HF Wang*, RK Varshney. 2023. New possibilities for trait improvement via mobile CRISPR-RNA. Trends in Biotechnology, 41(11), 1335-1338. (https://doi.org/10.1016/j.tibtech.2023.05.001). [中科院一区Top期刊，IF: 21.942].
4. Zaman QU, V Garg, A Raza, MF Nazir, L Hui, D Khan, AA Khokhar, MA Hussain, H-F Wang*, R K Varshney. Unique regulatory network of dragon fruit mitigates the effect of vanadium pollutant and environmental factors simultaneously. Physiologia Plantarum, e14416. https://doi.org/10.1111/ ppl.14416 [中科院二区，IF:5.4].
5. Zaman QU, MA Hussain, LU Khan, L Hui, D Khan, AA Khokhar, JP Cui, A Raza, HF Wang*. 2023. Genome-wide identification and expression profiling of APX gene family under multifactorial stress combinations and melatonin-mediated tolerance in pitaya. Scientia Horticulturae 321, 112312. [中科院二区Top期刊，IF: 4.3].
6. Zaman QU, MA Hussain, LU Khan, JP Cui, L Hui, D Khan, W Lv, HF Wang*. 2023. Genome-wide identification and expression pattern of GRAS gene family in pitaya (Selenicereus undatus L.). Biology 2023, 12(1),11[中科院一区，IF: 5.168].
7. Zaman QU, LU Khan, MA Hussain, A Ali, L Hui, AA Khokhar, D Khan, HF Wang*. Characterizing the HMA gene family in dragon fruit (Selenicereus undatus L.) and revealing their response to multifactorial stress combinations and melatonin-mediated tolerance. South African Journal of Botany, 163, 145-156. [I.F:3.1].
8. Zaman QU, W Chu, M Hao, Y Shi, D Mei, J Batley, B Zhang, C Li, Q Hu. 2021. Characterization of SHATTERPROOF homoeologs and CRISPR-Cas9-mediated genome editing enhances pod-shattering resistance in Brassica napus L. CRISPR Journal 4(3):360-370. [I.F:4.321]
9. Zaman QU, W Chu, M Hao, Y Shi, M Sun, S Sang, D Mei, H Cheng, J Liu, C Li, Q Hu. 2019. CRISPR/Cas9-mediated multiplex genome editing of JAGGED gene in Brassica napus L. Biomolecules 9,725. [I.F: 6.064].
10. Wang H*, QU Zaman*, W Huang, D Mei, J Liu, W Wang, B Ding, M Hao, L Fu, H Cheng, Q Hu. 2019. QTL and candidate genes identification for silique length based on high-dense genetic map in Brassica napus L. Frontiers in Plant Sciences 10,1579. [IF: 6.627].
11. Zaman QU, C Li, C Hongtao, H Qiong. 2019. Genome editing opens a new era of genetic improvement in polyploid crops. Crop Journal 7(2):141-150. [IF: 6.6].
12.Khokhar AA, L Hui, D Khan, W Lv, MA Hussain, QU Zaman, HF Wang. Comprehensive characterization of SBP genes revealed their role under multifactorial stress combinations dragon fruit (Selenicereus undatus L.). Plant Stress, 10, 100294. https://doi.org/10.1016/j.stress.2023.100294 [IF: 6].
13.. Darya Khan, L Hui, AA Khokhar, MA Hussain, W Lv, QU Zaman, HF Wang*. 2024. Functional characterization of MATE gene family under abiotic stresses and melatonin-mediated tolerance in pitaya (Selenicereus undatus L.). Plant Stress, 11, 100300. [IF: 6]. https://doi.org/10.1016/j.stress.2023.100300
14. Sang S, Y Wang, G Yao, T Ma, X Sun, Y Zhang, N Su, X Tan, HMK Abbas, S Ji, QU Zaman. A Critical Review of Conventional and Modern Approaches to Develop Herbicide-Resistance in Rice. Physiologia Plantarum, 176(2), e14254. https://doi.org/10.1111/ppl.14254. [IF: 6.4].
15. Hui L, D Khan, AA Khokhar, B Usman, QU Zaman, HF Wang*. Genome-wide identification and expression pattern of the MADS BOX gene family in pitaya (Selenicereus undatus L.). Plant Stress, 12, 100492. https://doi.org/10.1016/j.stress.2024.100492. [IF: 6].
16. Alam O, LU Khan, A Khan, F Mehwish, MA Husain, WU Khan, QU Zaman, HF Wang. Genome-wide identification and expression analysis of the Dof gene family in dragon fruit (Selenicereus undatus) under multiple abiotic stresses. Functional Plant Biology, 51, FP23269. doi.org/10.1071/FP23269. [IF: 3].
Members' demeanor
Group photo of the biodiversity and phylogenetic genomics team
王华锋教授(左一)Rajiv K. Varshneya-Faras教授
RESEARCH MAP OF PROFESSOR WANG HUAFENG AND DR. QAMAR DRAGON FRUIT BASE
Prof. Huafeng Wang's lab (Hainan University) has a good cooperative relationship with Prof. Rajeev K. Varshney (Murdoch University, Australia). Prof. Rajeev K. Varshney has opened up their lab resources to support our Pitaya genome sequencing project.
Rajiv K. Varshaneya-Faras教授
DR. QAMAR
文章标题：Chromosome-level genome assembly of autotetraploid Selenicereus megalanthus and gaining genomic insights into the evolution of trait patterning in diploid and polyploid pitaya species
作者信息：Qamar U Zaman, Liu Hui, Mian Faisal Nazir, Guoqing Wang, Vanika Garg, Muhammad Ikram, Ali Raza，Wei Lv, Darya Khan, Aamir Ali Khokhar, Zhang You, Annapurna Chitikineni, Babar Usman, Cui Jianpeng, Xulong Yang, Shiyou Zuo, Peifeng Liu, Sunjeet Kumar, Mengqi Guo, Zhi-Xin Zhu, Girish Dwivedi, Yong-Hua Qin, Rajeev K. Varshney, Hua-Feng Wang
Original link: https://www.biorxiv.org/content/10.1101/2024.06.23.600268v1
The pictures in the article and the cover picture are from the original article