Leaf margin phenotype and the genetic diversity of collected pineapple germplasm: a validation of the P locus on Spiny/Pipe leaf phenotype

Leaf margin phenotype and the genetic diversity of collected pineapple germplasm: a validation of the P locus on Spiny/Pipe leaf phenotype | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Leaf margin phenotype and the genetic diversity of collected pineapple germplasm: a validation of the P locus on Spiny/Pipe leaf phenotype Nguyen Nhat Truong, Luong Bao Duy, Bui Thanh Liem This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7242245/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Pineapple ( Ananas comosus (L.) Merr.) is a commercially important tropical fruit, with global production exceeding 28 million metric tons. Breeding programs require the assessment of genetic diversity and trait-specific markers across diverse germplasms. This study evaluated 60 pineapple accessions, including cultivars, mutants, and breeding lines, using 20 SSR markers and investigated the relationship between leaf spine traits and the presence of the P locus. The SSR analysis revealed moderate genetic diversity, with an average of 2.65 alleles per locus and a mean polymorphism information content (PIC) of 0.70. Clustering analysis revealed distinct groupings based on genetic background. A strong negative correlation (rho = -0.848, p value < 2.2e-16) was observed between the presence of the P locus and the leaf spiny phenotype, supported by a significant Wilcoxon rank sum test ( p value = 7.667e-11), which suggested an inverse relationship. These results validate the P locus control of the spine/pipe leaf phenotype and demonstrate the utility of SSR markers and the P locus in marker-assisted selection, providing insights into the genetic control of spineless leaf traits in pineapple. Ananas genetic diversity spiny leaf P locus Figures Figure 1 Introduction Pineapple ( Ananas comosus (L.) Merr.) is one of the most important tropical fruits, which plays a key role in agricultural production worldwide. Pineapple production exceeds 28 million metric tons and expected to reach 32.2 million metric tons by 2026, significantly contributing to the income of countries such as Indonesia, the Philippines, Costa Rica, Thailand, Brazil, and China (Shahbandeh 2025 ). The economic value of pineapple is not only in fresh use but also in processed products (Li et al. 2022 ). Under the global climate change and high demand for pineapple quality, the evaluation of genetic diversity and trait exploitation on collected pineapple germplasms, including wild types, cultivars, and breeding lines, is crucial for breeding programs that improve quality and climate adaptation (Wang et al. 2017 ; Li et al. 2022 ; Yow et al. 2022 ; Abike Chalotte Adjé et al. 2024 ). SSR molecular markers are powerful tools for discriminating the diversity and clustering individuals, which can be used as potential parents in breeding programs (Abdalah et al. 2018 ; Ismail et al. 2020 ; Nashima et al. 2020 ). One of the major traits of the pineapple is the spine/pipe on leaf margins. The spiny leaves cause problems in growing plants, harvesting, and transporting fruits on big farms. The pineapples with pipe leaves are preferred for cultivation and production. However, spiny/pipe leaf traits are widely diverse in pineapples. This trait varies from leaves of pipe (no spines) to spines on the leaf tip, spines on one marginal side, to spines fully on both sides. Spiny leaves, common in wild varieties, make handling difficult and increase labor costs, while pipe varieties, a result of domestication, are favored for their ease of harvesting (Sanewski 2020 ; de Lira Júnior et al. 2021 ). Recent studies of genes controlling spiny/pipe leaf trait on P locus have shown that AcWOX3 on the chromosome 8 may play a key role in in the formation of leaf margin spine beside other modifer genes (Sanewski 2020 ; Nashima et al. 2022 ). This study focused on assessing genetic diversity using SSR markers and exploring the relationship between leaf spine traits and P locus, aiming to provide a scientific basis for marker-assisted breeding programs. Materials and Methods A collection of pineapples at Southern Horticultural Research Institute (Vietnam) used in this study including 28 cultivars, 9 mutants, 25 breeding line from different parents (Queen x MD2, MD2 x Queen, DL8 x MD2). Leaves from these pineapple plants were collected for leaf margin phenotyping and DNA extraction for SSR and target gene analysis. Leaf spiny/pipe phenotyping by placing a section of the leaf (15 cm from the leaf tip) on a Canon LiDE300 scanner at 300dpi resolution. The score applied based on the presence of leaf spines were 0 = no spines, 1 = leaf tip spines, 2 = spines on one margin side, 3 = full spines on both margin sides (Fig. 1B). PCR-based SSR markers were performed in a total reaction volume of 15 µL containing 7.5 µL Magic PCR Mix 2X with Hot Start Polymerase (Phu Sa Genomics), 10 pmol each forward and reverse primer (Table 1 ) (Shoda et al. 2013 ; Ismail et al. 2020 ), and 10 ng of genomic DNA. Primers for P locus were followed by description in Nashima et al. (Nashima et al. 2022 ). PCR conditions were set at 95 0 C for 5 min, 30 cycles of 95 0 C for 10 sec, 60 0 C for 40s, and final extension at 72 0 C for 2 min. Pig-tail primers were used as Pig-tail primers were used as described by Shoda et al. (Shoda et al. 2013 ). Genomic DNA was extracted following La et al. (Thang et al. 2020 ), 20 mg of young leaves ground in 200 µL NaOH 0,5 M with stainless beads using Bioblock Scentific at 60 Hz 1 min, then adding 350 µL Tris 1M pH 8.0 and centrifuge at 13000 rpm in 3 min. DNA extracts can be stored at -20 o C and used for PCR reaction. SSR marker genotyping data analysis was performed using GenAlEx 6.51b2 (Peakall and Smouse 2012 ). Data visualization, correlation, and Mann–Whitney test on spiny trait and target gene were performed on R software with packages ‘ ComplexHeatmap ’, ‘ circlize’, ‘grid’ . Results and Discussion The genetic diversity of 60 pineapple accessions was assessed using 20 polymorphic SSR markers, revealing a mean number of alleles (Na) of 2.65 per locus, with a range from 1.00 (TsuAC035_Pigtail) to 4.00 (TsuAC023_Pigtail) (Table 1 ). Observed heterozygosity (Ho) averaged 0.31 (0.00–0.83), while expected heterozygosity (He) was 0.74 (0.45–0.91), indicating moderate genetic diversity. The polymorphism information content (PIC) averaged 0.70 (0.26–0.91), with Acom9.9 and TsuAC004_Pigtail showing the highest values (0.89 and 0.91, respectively). Shannon’s information index (I) averaged 1.22 (range: 0.64–1.63), reflecting variability across loci. These genetic parameters indicate that pineapple accessions have moderate to high diversity. A cluster dendrogram based on SSR data (Fig. 1) illustrated the genetic relationships among accessions, with clear clusters influenced by genetic background. Pineapple accessions revealed the presence of a P locus that causes the pipe leaf trait, except in Thom_Trung Quoc, which needs to be further validated. Moreover, the P locus presence in the breeding lines and the leaf spine/pipe phenotype were both closely linked in a cause – effect manner. The parents carrying the P locus and pipe leaf phenotype produced a hybrid that also carried the P locus and leaf spineless (DL08 x MD2) (Fig. 1A). Similarly, the hybrids from the Queen x MD2 or MD2 x Queen crosses all expressed the genetic motif of leaf spine/pipe trait and P locus following their parental leaf phenotype (Fig. 1A). Statistical analysis showed a strong negative Spearman’s rank correlation (rho = -0.848, p < 2.2e-16) between the spiny leaf trait and P locus, suggesting an inverse relationship. The Wilcoxon rank sum test further confirmed a significant difference in spiny leaf trait distribution between groups with and without the P locus (W = 790, p = 7.667e-11), indicating distinct phenotypic patterns. The SSR analysis demonstrated moderate genetic diversity in the pineapple germplasm, consistent with previous studies (Chen et al. 2019 ; Ismail et al. 2020 ; Nashima et al. 2020 ), with high PIC values (> 0.7) at loci like Acom9.9 and TsuAC004_Pigtail indicating their utility for marker-assisted selection. The dendrogram (Fig. 1) likely reflects clustering by genetic type, such as Cultivars, Mutants, and Breeding lines, supporting the role of SSRs in assessing population structure (Ming et al. 2015 ). The low Ho compared to He suggests strong selection of hybrid lines for pineapple cultivation. The strong negative correlation (rho = -0.848) between spiny leaf trait and P locus, coupled with the significant Wilcoxon test result ( p = 7.667e-11), indicates that more leaf spines are strongly associated with the presence of P locus, and vice versa . This evidence was confirmed on MD2_WT (wild type) vs MD2_Mutant (mutated MD2), where MD2_Mutant lost its P locus, causing spiny leaves compared to the pipe leaf of MD2 wild type. This inverse relationship may reflect pleiotropic effects or tightly linkage between loci controlling spine density and presence P locus, possibly involving AcWOX3 or QTLs on chromosome 23 (Sanewski 2020 ; Nashima et al. 2022 ). However, the unexpected distribution suggests environmental or epigenetic influences, as noted in prior studies (Sanewski et al., 2019). These findings highlight the need for genome-wide association studies to figure out causal variants, enhancing breeding strategies for spineless cultivars of pineapples. Conclusion In summary, twenty SSR markers were used to assess genetic diversity of 60 accessions of pineapples showed high diversity in the germplasm. The expected heterozygosity (He) is higher than the observed heterozygosity (Ho) suggesting that the pineapple population is highly hybrid. The P locus was tightly associated with spine/pipe leaf phenotype and the high genetic diversity of the pineapple population may imply the use of the P locus as an effective molecular marker for selection pineapple varieties with or without leaf spine in breeding programs. Declarations ACKNOWLEDGEMENTS The authors thank Nguyen Vu Gia Han, and Nguyen Truong Tho (Institute of Food and Biotechnology, Can Tho University) for field trip support. Author contributions Bui Thanh Liem conceived and designed the project, analyzed the data, and wrote the original draft. Nguyen Nhat Truong and Luong Bao Duy performed the experiments. All authors read and approved the final manuscript. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Conflicts of Interest The authors declare no conflict of interest. Data Availability The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. References Abdalah M, Miccah SS, Ayub N, Emmarold EM, George M, Kusirieli L, Aron G, Lilian M, Andrew K, Emmanuel M, Theodosy JM (2018) Diversity and genetic identity of pineapple [Ananas comosus (L.) Merr.] in Tanzania based on microsatellite markers. Afr J Biotechnol 17(26):811–817 Abike Chalotte Adjé O, Missihoun AA, Sedah P, Sagbadja HA, Enoch Gbenato A-D, Clément A (2024) Genetic diversity and structure of Benin pineapple (Ananas comosus (L) Merr.) germplasm collection using Simple Sequence Repeat (SSR) markers. J Plant Mol Breed 11(2):94–106 Chen LY, VanBuren R, Paris M, Zhou H, Zhang X, Wai CM, Yan H, Chen S, Alonge M, Ramakrishnan S, Liao Z, Liu J, Lin J, Yue J, Fatima M, Lin Z, Zhang J, Huang L, Wang H, Hwa TY, Kao SM, Choi JY, Sharma A, Song J, Wang L, Yim WC, Cushman JC, Paull RE, Matsumoto T, Qin Y, Wu Q, Wang J, Yu Q, Wu J, Zhang S, Boches P, Tung CW, Wang ML, d’Eeckenbrugge C, Sanewski G, Purugganan GM, Schatz MD, Bennetzen MC, Lexer JL, C. and, Ming R (2019) The bracteatus pineapple genome and domestication of clonally propagated crops. Nature Genetics [online], 51 (10), 1549–1558. Available from: http://dx.doi.org/10.1038/s41588-019-0506-8 Ismail SN, Ghani NSA, Razak SFA, Abidin RAZ, Yusof MFM, Zubir MN, Zainol R (2020) Genetic diversity of pineapple (Ananas comosus) germplasm in malaysia using simple sequence repeat (ssr) markers. Trop Life Sci Res 31(3):15–27 Li D, Jing M, Dai X, Chen Z, Ma C, Chen J (2022) Current status of pineapple breeding, industrial development, and genetics in China. Euphytica [online], 218 (6), 1–17. Available from: https://doi.org/10.1007/s10681-022-03030-y de Lira Júnior JS, Bezerra JEF, de Andrade DEGT (2021) Genetic control of leaf spinescence in BRS Imperial, Pérola, and Pico de Rosa pineapple cultivars. Crop Breed Appl Biotechnol 21(1):1–7 Ming R, VanBuren R, Wai CM, Tang H, Schatz MC, Bowers JE, Lyons E, Wang ML, Chen J, Biggers E, Zhang J, Huang L, Zhang L, Miao W, Zhang J, Ye Z, Miao C, Lin Z, Wang H, Zhou H, Yim WC, Priest HD, Zheng C, Woodhouse M, Edger PP, Guyot R, Guo HB, Guo H, Zheng G, Singh R, Sharma A, Min X, Zheng Y, Lee H, Gurtowski J, Sedlazeck FJ, Harkess A, McKain MR, Liao Z, Fang J, Liu J, Zhang X, Zhang Q, Hu W, Qin Y, Wang K, Chen LY, Shirley N, Lin YR, Liu LY, Hernandez AG, Wright CL, Bulone V, Tuskan GA, Heath K, Zee F, Moore PH, Sunkar R, Leebens-Mack JH, Mockler T, Bennetzen JL, Freeling M, Sankoff D, Paterson AH, Zhu X, Yang X, Smith JAC, Cushman JC, Paull RE, Yu Q (2015) The pineapple genome and the evolution of CAM photosynthesis. Nature Genetics 2015 47:12 [online], 47 (12), 1435–1442. Available from: https://www.nature.com/articles/ng.3435 [Accessed 16 Jul 2025] Nashima K, Hosaka F, Terakami S, Kunihisa M, Nishitani C, Moromizato C, Takeuchi M, Shoda M, Tarora K, Urasaki N, Yamamoto T (2020) Ssr markers developed using next-generation sequencing technology in pineapple, Ananas comosus (L.) merr. Breed Sci 70(3):415–421 Nashima K, Shirasawa K, Isobe S, Urasaki N, Tarora K, Irei A, Shoda M, Takeuchi M, Omine Y, Nishiba Y, Sugawara T, Kunihisa M, Nishitani C, Yamamoto T (2022) Gene prediction for leaf margin phenotype and fruit flesh color in pineapple (Ananas comosus) using haplotype-resolved genome sequencing. Plant J 110(3):720–734 Peakall R, Smouse PE (2012) GenALEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28(19):2537–2539 Sanewski GM (2020) DArTseq Molecular Markers Associated with the Spiny-Tip Leaf Margin in Pineapple (Ananas comosus L). Trop Plant Biology 13(1):91–116 Shahbandeh M (2025) Global pineapple production 2023, by leading countries [online]. Statista. Available from: https://www.statista.com/statistics/298517/global-pineapple-production-by-leading-countries/ Shoda M, Urasaki N, Sakiyama S, Terakami S, Hosaka F, Shigeta N, Nishitani C, Yamamoto T (2013) DNA profiling of pineapple cultivars in Japan discriminated by SSR markers. Breed Sci 62(4):352–359 Thang LC, Luan HM, Liem BT, SO SÁNH TÍNH HIỆU QUẢ VÀ KINH TẾ CỦA BA PHƯƠNG PHÁP LY TRÍCH ADN TRÊN CÂY LÚA (2020) J Vietnam Agricultural Sci Technol 6(115):46–49 Wang Jsheng, He J, hu, Chen H, rui, Chen Y, yuan, Qiao F (2017) Genetic Diversity in Various Accessions of Pineapple [Ananas comosus (L.) Merr.] Using ISSR and SSR Markers. Biochemical Genetics [online], 55 (5–6), 347–366. Available from: https://link.springer.com/article/ 10.1007/s10528-017-9803-z [Accessed 16 Jul 2025] Yow AG, Bostan H, Ruggieri V, Mengist MF, Curaba J, Young R, Gillitt N, Iorizzo M (2022) Improved High-Quality Genome Assembly and Annotation of Pineapple (Ananas comosus) Cultivar MD2 Revealed Extensive Haplotype Diversity and Diversified FRS/FRF Gene Family. Genes 13(52):1–20 Tables Table 1 Diversity information parameters on the 20 polymorphic SSR markers SSR locus Na Ho He I PIC Acom9.9 3.00 0.13 0.89 1.28 0.89 Acom22.22 3.00 0.58 0.69 1.44 0.65 Acom39.5 3.00 0.60 0.61 1.43 0.53 Acom68.3 2.00 0.25 0.74 0.96 0.74 Acom82.8 2.00 0.00 0.85 0.85 0.85 APCT136B 2.00 0.27 0.83 0.97 0.82 TsuAC004 3.00 0.50 0.60 1.49 0.51 TsuAC008 3.00 0.83 0.64 1.32 0.59 TsuAC010 3.00 0.28 0.81 1.38 0.80 TsuAC013 2.00 0.13 0.76 0.77 0.76 TsuAC018 3.00 0.08 0.45 1.56 0.26 TsuAC023 2.00 0.20 0.86 0.86 0.86 TsuAC030 3.00 0.23 0.63 1.55 0.55 TsuAC035 3.00 0.38 0.76 1.33 0.74 TsuAC004_Pigtail 3.00 0.02 0.91 1.22 0.91 TsuAC008_Pigtail 2.00 0.05 0.83 0.90 0.83 TsuAC010_PigTail 3.00 0.47 0.81 1.43 0.79 TsuAC018_PigTail 3.00 0.42 0.78 1.28 0.77 TsuAC023_PigTail 4.00 0.80 0.54 1.63 0.43 TsuAC035_PigTail 1.00 0.00 0.77 0.64 0.77 Mean 2.65 0.31 0.74 1.22 0.70 Notes: Na = Number of alleles; Ho = observed heterozygosity; He = expected Heterozygosity, I = Shannon’s information index, and PIC = polymorphic information content. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Feb, 2026 Reviews received at journal 24 Feb, 2026 Reviews received at journal 20 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviews received at journal 24 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers invited by journal 08 Sep, 2025 Editor assigned by journal 30 Jul, 2025 Submission checks completed at journal 30 Jul, 2025 First submitted to journal 29 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7242245","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":512027359,"identity":"6f9c24a9-791d-4b4f-93ff-5988a5708875","order_by":0,"name":"Nguyen Nhat Truong","email":"","orcid":"","institution":"Southern Horticultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Nguyen","middleName":"Nhat","lastName":"Truong","suffix":""},{"id":512027360,"identity":"5e59714e-29a6-40af-ab99-370c53a30024","order_by":1,"name":"Luong Bao Duy","email":"","orcid":"","institution":"Can Tho University","correspondingAuthor":false,"prefix":"","firstName":"Luong","middleName":"Bao","lastName":"Duy","suffix":""},{"id":512027361,"identity":"106f8d07-c52b-459a-bdc7-418f6e5e96a0","order_by":2,"name":"Bui Thanh Liem","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBACAwbmhgMMDDYQHg9RWtgYQVrSIKqJ1gKkDpOgxVy+sfHAx5zz8vYSCYwP3rYxJG4npMWyjbHh4Mxttw17JBKYDecCtexsIOSwY4wNh3m33U7gkUhgk+ZtYzA2OECMlr/bzoG0sP8mXgvjtgNgW5iBWuQIarFsS2w42Lst2bDnzMNmyTnnJAhrMWc+fPjDz2128uztyQc/vCmz4SGoBQmAI0iCePWjYBSMglEwCnADAMBJP3Gbs0QwAAAAAElFTkSuQmCC","orcid":"","institution":"Can Tho University","correspondingAuthor":true,"prefix":"","firstName":"Bui","middleName":"Thanh","lastName":"Liem","suffix":""}],"badges":[],"createdAt":"2025-07-29 10:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7242245/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7242245/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91362610,"identity":"fc9916c4-c5e9-48e5-80fd-8943620949df","added_by":"auto","created_at":"2025-09-15 16:44:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":414423,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic diversity and leaf margin phenotype\u003c/p\u003e\n\u003cp\u003e(A) clustering and heatmap of genetic background, leaf spiny/pipe type and the presence of \u003cem\u003eP\u003c/em\u003elocus; (B) Leaf spiny/pipe phenotype; (C) \u003cem\u003eP\u003c/em\u003e locus is the key factor controlling spiny/pipe leaf trai, deletion mutation of \u003cem\u003eP\u003c/em\u003e locus leads to the reappearance of leaf spines in the MD2 mutant, Queen is the most common cultivar with fully spiny leaves used as reference.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7242245/v1/a81e54635706693ebcf6742a.jpeg"},{"id":91363135,"identity":"4f21984f-8b0f-409c-95a4-6ec3c9048f4e","added_by":"auto","created_at":"2025-09-15 16:52:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":849364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7242245/v1/7398c6eb-8da6-4c1b-b262-fc3350252540.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Leaf margin phenotype and the genetic diversity of collected pineapple germplasm: a validation of the P locus on Spiny/Pipe leaf phenotype","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePineapple (\u003cem\u003eAnanas comosus\u003c/em\u003e (L.) Merr.) is one of the most important tropical fruits, which plays a key role in agricultural production worldwide. Pineapple production exceeds 28\u0026nbsp;million metric tons and expected to reach 32.2\u0026nbsp;million metric tons by 2026, significantly contributing to the income of countries such as Indonesia, the Philippines, Costa Rica, Thailand, Brazil, and China (Shahbandeh \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The economic value of pineapple is not only in fresh use but also in processed products (Li et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Under the global climate change and high demand for pineapple quality, the evaluation of genetic diversity and trait exploitation on collected pineapple germplasms, including wild types, cultivars, and breeding lines, is crucial for breeding programs that improve quality and climate adaptation (Wang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yow et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Abike Chalotte Adj\u0026eacute; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). SSR molecular markers are powerful tools for discriminating the diversity and clustering individuals, which can be used as potential parents in breeding programs (Abdalah et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ismail et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nashima et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne of the major traits of the pineapple is the spine/pipe on leaf margins. The spiny leaves cause problems in growing plants, harvesting, and transporting fruits on big farms. The pineapples with pipe leaves are preferred for cultivation and production. However, spiny/pipe leaf traits are widely diverse in pineapples. This trait varies from leaves of pipe (no spines) to spines on the leaf tip, spines on one marginal side, to spines fully on both sides. Spiny leaves, common in wild varieties, make handling difficult and increase labor costs, while pipe varieties, a result of domestication, are favored for their ease of harvesting (Sanewski \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; de Lira J\u0026uacute;nior et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent studies of genes controlling spiny/pipe leaf trait on \u003cem\u003eP\u003c/em\u003e locus have shown that \u003cem\u003eAcWOX3\u003c/em\u003e on the chromosome 8 may play a key role in in the formation of leaf margin spine beside other modifer genes (Sanewski \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nashima et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This study focused on assessing genetic diversity using SSR markers and exploring the relationship between leaf spine traits and \u003cem\u003eP\u003c/em\u003e locus, aiming to provide a scientific basis for marker-assisted breeding programs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eA collection of pineapples at Southern Horticultural Research Institute (Vietnam) used in this study including 28 cultivars, 9 mutants, 25 breeding line from different parents (Queen x MD2, MD2 x Queen, DL8 x MD2). Leaves from these pineapple plants were collected for leaf margin phenotyping and DNA extraction for SSR and target gene analysis. Leaf spiny/pipe phenotyping by placing a section of the leaf (15 cm from the leaf tip) on a Canon LiDE300 scanner at 300dpi resolution. The score applied based on the presence of leaf spines were 0\u0026thinsp;=\u0026thinsp;no spines, 1\u0026thinsp;=\u0026thinsp;leaf tip spines, 2\u0026thinsp;=\u0026thinsp;spines on one margin side, 3\u0026thinsp;=\u0026thinsp;full spines on both margin sides (Fig.\u0026nbsp;1B).\u003c/p\u003e\u003cp\u003ePCR-based SSR markers were performed in a total reaction volume of 15 \u0026micro;L containing 7.5 \u0026micro;L Magic PCR Mix 2X with Hot Start Polymerase (Phu Sa Genomics), 10 pmol each forward and reverse primer (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Shoda et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ismail et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and 10 ng of genomic DNA. Primers for \u003cem\u003eP\u003c/em\u003e locus were followed by description in Nashima et al. (Nashima et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). PCR conditions were set at 95\u003csup\u003e0\u003c/sup\u003eC for 5 min, 30 cycles of 95\u003csup\u003e0\u003c/sup\u003eC for 10 sec, 60\u003csup\u003e0\u003c/sup\u003eC for 40s, and final extension at 72\u003csup\u003e0\u003c/sup\u003eC for 2 min. Pig-tail primers were used as Pig-tail primers were used as described by Shoda et al. (Shoda et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Genomic DNA was extracted following La et al. (Thang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), 20 mg of young leaves ground in 200 \u0026micro;L NaOH 0,5 M with stainless beads using Bioblock Scentific at 60 Hz 1 min, then adding 350 \u0026micro;L Tris 1M pH 8.0 and centrifuge at 13000 rpm in 3 min. DNA extracts can be stored at -20\u003csup\u003eo\u003c/sup\u003eC and used for PCR reaction. SSR marker genotyping data analysis was performed using GenAlEx 6.51b2 (Peakall and Smouse \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Data visualization, correlation, and Mann\u0026ndash;Whitney test on spiny trait and target gene were performed on R software with packages \u0026lsquo;\u003cem\u003eComplexHeatmap\u003c/em\u003e\u0026rsquo;, \u0026lsquo;\u003cem\u003ecirclize\u0026rsquo;, \u0026lsquo;grid\u0026rsquo;\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe genetic diversity of 60 pineapple accessions was assessed using 20 polymorphic SSR markers, revealing a mean number of alleles (Na) of 2.65 per locus, with a range from 1.00 (TsuAC035_Pigtail) to 4.00 (TsuAC023_Pigtail) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Observed heterozygosity (Ho) averaged 0.31 (0.00\u0026ndash;0.83), while expected heterozygosity (He) was 0.74 (0.45\u0026ndash;0.91), indicating moderate genetic diversity. The polymorphism information content (PIC) averaged 0.70 (0.26\u0026ndash;0.91), with Acom9.9 and TsuAC004_Pigtail showing the highest values (0.89 and 0.91, respectively). Shannon\u0026rsquo;s information index (I) averaged 1.22 (range: 0.64\u0026ndash;1.63), reflecting variability across loci. These genetic parameters indicate that pineapple accessions have moderate to high diversity. A cluster dendrogram based on SSR data (Fig.\u0026nbsp;1) illustrated the genetic relationships among accessions, with clear clusters influenced by genetic background. Pineapple accessions revealed the presence of a \u003cem\u003eP\u003c/em\u003e locus that causes the pipe leaf trait, except in Thom_Trung Quoc, which needs to be further validated. Moreover, the \u003cem\u003eP\u003c/em\u003e locus presence in the breeding lines and the leaf spine/pipe phenotype were both closely linked in a cause \u0026ndash; effect manner. The parents carrying the \u003cem\u003eP\u003c/em\u003e locus and pipe leaf phenotype produced a hybrid that also carried the P locus and leaf spineless (DL08 x MD2) (Fig.\u0026nbsp;1A). Similarly, the hybrids from the Queen x MD2 or MD2 x Queen crosses all expressed the genetic motif of leaf spine/pipe trait and \u003cem\u003eP\u003c/em\u003e locus following their parental leaf phenotype (Fig.\u0026nbsp;1A).\u003c/p\u003e\u003cp\u003eStatistical analysis showed a strong negative Spearman\u0026rsquo;s rank correlation (rho = -0.848, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16) between the spiny leaf trait and \u003cem\u003eP\u003c/em\u003e locus, suggesting an inverse relationship. The Wilcoxon rank sum test further confirmed a significant difference in spiny leaf trait distribution between groups with and without the \u003cem\u003eP\u003c/em\u003e locus (W\u0026thinsp;=\u0026thinsp;790, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.667e-11), indicating distinct phenotypic patterns.\u003c/p\u003e\u003cp\u003eThe SSR analysis demonstrated moderate genetic diversity in the pineapple germplasm, consistent with previous studies (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ismail et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nashima et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with high PIC values (\u0026gt;\u0026thinsp;0.7) at loci like Acom9.9 and TsuAC004_Pigtail indicating their utility for marker-assisted selection. The dendrogram (Fig.\u0026nbsp;1) likely reflects clustering by genetic type, such as Cultivars, Mutants, and Breeding lines, supporting the role of SSRs in assessing population structure (Ming et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The low Ho compared to He suggests strong selection of hybrid lines for pineapple cultivation. The strong negative correlation (rho = -0.848) between spiny leaf trait and \u003cem\u003eP\u003c/em\u003e locus, coupled with the significant Wilcoxon test result (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.667e-11), indicates that more leaf spines are strongly associated with the presence of \u003cem\u003eP\u003c/em\u003e locus, and \u003cem\u003evice versa\u003c/em\u003e. This evidence was confirmed on MD2_WT (wild type) vs MD2_Mutant (mutated MD2), where MD2_Mutant lost its \u003cem\u003eP\u003c/em\u003e locus, causing spiny leaves compared to the pipe leaf of MD2 wild type. This inverse relationship may reflect pleiotropic effects or tightly linkage between loci controlling spine density and presence \u003cem\u003eP\u003c/em\u003e locus, possibly involving AcWOX3 or QTLs on chromosome 23 (Sanewski \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nashima et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the unexpected distribution suggests environmental or epigenetic influences, as noted in prior studies (Sanewski et al., 2019). These findings highlight the need for genome-wide association studies to figure out causal variants, enhancing breeding strategies for spineless cultivars of pineapples.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, twenty SSR markers were used to assess genetic diversity of 60 accessions of pineapples showed high diversity in the germplasm. The expected heterozygosity (He) is higher than the observed heterozygosity (Ho) suggesting that the pineapple population is highly hybrid. The \u003cem\u003eP\u003c/em\u003e locus was tightly associated with spine/pipe leaf phenotype and the high genetic diversity of the pineapple population may imply the use of the \u003cem\u003eP\u003c/em\u003e locus as an effective molecular marker for selection pineapple varieties with or without leaf spine in breeding programs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Nguyen Vu Gia Han, and Nguyen Truong Tho (Institute of Food and Biotechnology, Can Tho University) for field trip support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBui Thanh Liem conceived and designed the project, analyzed the data, and wrote the original draft. Nguyen Nhat Truong and Luong Bao Duy performed the experiments. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdalah M, Miccah SS, Ayub N, Emmarold EM, George M, Kusirieli L, Aron G, Lilian M, Andrew K, Emmanuel M, Theodosy JM (2018) Diversity and genetic identity of pineapple [Ananas comosus (L.) 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Breed Sci 62(4):352\u0026ndash;359\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThang LC, Luan HM, Liem BT, SO S\u0026Aacute;NH T\u0026Iacute;NH HIỆU QUẢ V\u0026Agrave; KINH TẾ CỦA BA PHƯƠNG PH\u0026Aacute;P LY TR\u0026Iacute;CH ADN TR\u0026Ecirc;N C\u0026Acirc;Y L\u0026Uacute;A (2020) J Vietnam Agricultural Sci Technol 6(115):46\u0026ndash;49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Jsheng, He J, hu, Chen H, rui, Chen Y, yuan, Qiao F (2017) Genetic Diversity in Various Accessions of Pineapple [Ananas comosus (L.) Merr.] Using ISSR and SSR Markers. \u003cem\u003eBiochemical Genetics\u003c/em\u003e [online], 55 (5\u0026ndash;6), 347\u0026ndash;366. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://link.springer.com/article/\u003c/span\u003e\u003cspan address=\"https://link.springer.com/article/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10528-017-9803-z\u003c/span\u003e\u003cspan address=\"10.1007/s10528-017-9803-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [Accessed 16 Jul 2025]\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYow AG, Bostan H, Ruggieri V, Mengist MF, Curaba J, Young R, Gillitt N, Iorizzo M (2022) Improved High-Quality Genome Assembly and Annotation of Pineapple (Ananas comosus) Cultivar MD2 Revealed Extensive Haplotype Diversity and Diversified FRS/FRF Gene Family. Genes 13(52):1\u0026ndash;20\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDiversity information parameters on the 20 polymorphic SSR markers\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSSR locus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHo\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHe\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePIC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcom9.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcom22.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcom39.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcom68.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcom82.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPCT136B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC004_Pigtail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC008_Pigtail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC010_PigTail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC018_PigTail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC023_PigTail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTsuAC035_PigTail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e2.65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.31\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.74\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1.22\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.70\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eNotes: Na\u0026thinsp;=\u0026thinsp;Number of alleles; Ho\u0026thinsp;=\u0026thinsp;observed heterozygosity; He\u0026thinsp;=\u0026thinsp;expected\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHeterozygosity, I\u0026thinsp;=\u0026thinsp;Shannon\u0026rsquo;s information index, and PIC\u0026thinsp;=\u0026thinsp;polymorphic information content.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology-reporter","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pmbr","sideBox":"Learn more about [Plant Molecular Biology Reporter](http://link.springer.com/journal/11105)","snPcode":"11105","submissionUrl":"https://submission.nature.com/new-submission/11105/3","title":"Plant Molecular Biology Reporter","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ananas, genetic diversity, spiny leaf, P locus","lastPublishedDoi":"10.21203/rs.3.rs-7242245/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7242245/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePineapple (\u003cem\u003eAnanas comosus\u003c/em\u003e (L.) Merr.) is a commercially important tropical fruit, with global production exceeding 28\u0026nbsp;million metric tons. Breeding programs require the assessment of genetic diversity and trait-specific markers across diverse germplasms. This study evaluated 60 pineapple accessions, including cultivars, mutants, and breeding lines, using 20 SSR markers and investigated the relationship between leaf spine traits and the presence of the \u003cem\u003eP\u003c/em\u003e locus. The SSR analysis revealed moderate genetic diversity, with an average of 2.65 alleles per locus and a mean polymorphism information content (PIC) of 0.70. Clustering analysis revealed distinct groupings based on genetic background. A strong negative correlation (rho = -0.848, \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16) was observed between the presence of the \u003cem\u003eP\u003c/em\u003e locus and the leaf spiny phenotype, supported by a significant Wilcoxon rank sum test (\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;7.667e-11), which suggested an inverse relationship. These results validate the \u003cem\u003eP\u003c/em\u003e locus control of the spine/pipe leaf phenotype and demonstrate the utility of SSR markers and the P locus in marker-assisted selection, providing insights into the genetic control of spineless leaf traits in pineapple.\u003c/p\u003e","manuscriptTitle":"Leaf margin phenotype and the genetic diversity of collected pineapple germplasm: a validation of the P locus on Spiny/Pipe leaf phenotype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 16:44:34","doi":"10.21203/rs.3.rs-7242245/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-25T05:57:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T03:40:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T17:41:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130212634149056860847888347043493713575","date":"2026-02-06T00:02:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317754074847224321015218840733964976426","date":"2026-02-05T09:52:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T20:12:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120838148853641004628557550868094932600","date":"2025-10-19T20:00:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194232391868396519291129943929617341680","date":"2025-09-21T10:17:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137092021794677065504319595448356922346","date":"2025-09-17T05:18:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188807455822847324572072606266627512110","date":"2025-09-17T02:18:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-08T10:25:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T06:02:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-30T06:01:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Molecular Biology Reporter","date":"2025-07-29T10:30:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology-reporter","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pmbr","sideBox":"Learn more about [Plant Molecular Biology Reporter](http://link.springer.com/journal/11105)","snPcode":"11105","submissionUrl":"https://submission.nature.com/new-submission/11105/3","title":"Plant Molecular Biology Reporter","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1993f61f-38f6-445d-8055-f70ba2a9dac5","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T04:53:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-15 16:44:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7242245","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7242245","identity":"rs-7242245","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}