Genetic Diversity Between and Within Species of Opuntia Spp. Revealed by Issr Molecular Marker

Genetic Diversity Between and Within Species of Opuntia Spp. Revealed by Issr Molecular Marker | 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 Genetic Diversity Between and Within Species of Opuntia Spp. Revealed by Issr Molecular Marker Joseilson Moreira de Araújo, Mailson Monteiro do Rêgo², Elizanilda Ramalho do Rêgo² This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6567776/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Opuntia spp. forage palm is a viable solution for animal feed in arid and semi-arid regions due to its easy adaptation to this type of environment, its physiological characteristics and its low production cost. Given its importance, it is essential to learn more about its genetic characteristics. Molecular markers are one of the most widely used techniques. Within this context, the aim of this study was to assess the genetic diversity of 40 genotypes of Opuntia spp. from the Active Germplasm Bank (AGB) of the National Semi-Arid Institute (INSA), using Inter Simple Sequence Repeats (ISSR) molecular markers. Deoxyribonucleic acid (DNA) was extracted from the accessions using the DNeasy Plant Mini® Kit (Qiagen). Eleven ISSR oligonucleotide primers were used. This technique amplified 839 fragments, distributed over 91 loci. Nine groups were constructed from the genetic distances of the 40 accessions. It was found that there is high genetic diversity between and within the species evaluated, helping to broaden the genetic base of the segregating populations, suggesting intraspecific crossing between accessions 87 and 117 and interspecific crossing with accessions 1 x 119, 86 x ?(101), 86 x 135, 88 x 135, ?(101) x 135. issr genetic resources genetic improvement cactaceae Figures Figure 1 Figure 2 Figure 3 INTRODUTION The forage palm, Opuntia spp. belongs to the Cactaceae family, has 130 genera and more than 2,000 species. Its centre of origin is Mexico (Nefzaoui 2016), but it is distributed throughout the different regions of the world, as it easily adapts to adverse weather and climate conditions, from areas with high temperatures to the lowest. This type of crop is economically important for regions with arid and semi-arid climates, guaranteeing income generation and also being used for human and animal food. The use of palm fruit for human consumption has been common in Mexico since the period before Spanish colonisation (Reinolds 2008). The forage palm (Opuntia and Nopalea) is an important food in livestock farming, presenting itself as an alternative for the arid and semi-arid regions of north-eastern Brazil, as it is adapted to the climatic conditions of the region and has a special physiological aspect in terms of absorption, use and loss of water, withstanding prolonged periods of drought and being able to reach yields of up to 40 tonnes of dry matter per hectare per harvest (Santos et al. 2006 ). The main use of O. fícus-indica in north-eastern Brazil is as fodder under drought conditions (Paiva et al. 2016 ). As it is rich in water and fibre, it is used to feed cattle, goats and sheep (Bezerra et al. 2022 ). The Opuntia genus has a high genetic diversity. Of the more than 30 cultivated species, the economically important ones are: O. fícus-indica L., O. albicarpa Scheinvar, O. amyclaea Ten, O. cochenillifera (L.) Miller, O. hyptiacantha FAC Weber, O. megacantha Salm Dyck, O. robusta Wendl and O. streptacantha Lem (Pimenta Barrios 1994; Kiesling 1998 ; Mondragon Jacobo 2001; Las Casas et al. 2017). The high genetic diversity of Opuntias is conserved in active germplasm banks on all continents. In the Eastern Hemisphere, ex situ conservation has been carried out by Tunisia, Morocco, South Africa and Italy, while in the West, by Argentina, Brazil, Chile, the USA and Mexico (the largest centre of diversity). In Brazil, efforts have been directed towards the conservation of accessions for fodder, with 1,417 accessions, including accessions from various countries, varieties, hybrids and segregants from controlled crosses (Mondragón & Chessa 2017 ). Germplasm characterisation is essential for providing information on the characters of each accession, promoting its classification by estimating genetic diversity within and between groups (Nefzaoui 2017). Segundo Dennis et al., ( 2007 ) At the molecular level, there are different molecular markers for assessing the genetic variability of species, characterising germplasm, conserving it, improving it genetically and/or carrying out population genetic analyses. There are several types of molecular markers based on the polymerase chain reaction (PCR), such as random amplified polymorphic DNA (RAPD) (Williams et al. 1990 ), amplified fragment length polymorphisms (AFLPs) (Vos et al. 1995 ). Microsatellites (SSR), which are generally tandem regions of 6 bp (Lovin et al., 2009; Alencar, 2024 ) and amplification of inter simple sequence repeats (ISSR) (Zietkiewicz et al. 1994 ). The ISSR marker is a microsatellite-based technique that is very useful for genome studies. The primers used in ISSR amplifications are based on SSR sequences (di, tri, tetra, or penta nucleotide repeats), anchored to genomic sequences that flank each side of the targeted simple sequence repeats (SSRs) (5‘ or 3’), using two to four arbitrary and often degenerate nucleotides. Unlike SSRs, this technique does not require prior sequence information and generates a large number of polymorphisms (Zietkiewicz et al. 1994 ). ISSR markers are considered particularly useful for studying closely related individuals that require low levels of polymorphism (Zietkiewicz et al. 1994 ). In addition to ISSR, RAPD and ITS molecular markers have also proved to be efficient in accessing genetic diversity and discriminating between forage palm accessions (Bezerra et al. 2022 ). In this context, the aim of this study was to assess the genetic diversity of 40 Opuntia spp. genotypes from the Active Germplasm Bank of the National Semi-Arid Institute, using ISSR molecular markers. MATERIAL AND METHODS The experiment was carried out in two stages, the first at the Molecular Biology Laboratory of the National Semi-Arid Institute (INSA - from Portuguese " Instituto Nacional do Semiárido" ) in the municipality of Campina Grande-PB, and the second at the Molecular Biology Laboratory of the Centre for Agrarian Sciences at Universidade Federal da Paraíba. A total of 40 accessions from INSA's Active Germplasm Bank (BGA) were used, divided into 8 species of Opuntias. The collected cladodes were stored in a covered and ventilated area so that they could undergo a 15-day ‘curing’ period, after which they were planted in high-density polypropylene pots with a capacity of 5 litres and kept under a 60% shade roof. 25 days after transplanting, the roots were collected using tweezers and stored in 2.0 ml Eppendorf tubes. The roots were washed in distilled water and then placed on paper towels to absorb the liquid in Petri dishes. After the roots were clean and dry, they were placed in brown paper BGAs containing 15 g of silica gel for 72 hours to dehydrate. After this period, the roots were placed in 2 ml Eppendorf tubes along with 3 ceramic beads for maceration in the Bead Ruptor 4 OMNI for 5 minutes. The beads were removed and the material was transferred to new 2 ml Eppendorf tubes labelled with the access number and stored in an upright freezer at -20ºC. DNA extraction and purification Qiagen method DNA was extracted from the samples macerated in the Bead Ruptor using the DNeasy Plant Mini® Kit (Qiagen), following the manufacturer's recommendations: inserting 200 mg of the macerate into an Eppendorf tube, adding 400µl of AP1 buffer + 4 µl of RNAse A, using the Vortex Na3600 North Scientific sample shaker to homogenise the solution and taken to a bain marie for 10 minutes at 65ºC, inverting the tube every 3 minutes; 130 µl of P3 buffer was then added, after which the samples were placed on ice for 5 minutes. After this process, the samples were centrifuged using a Biocen 22R machine at 14,000 rpm for 5 minutes. After centrifugation, there was a separation between the liquid and solid parts, where the supernatant was collected and applied to the QIAshredder Mini spine (lilac colour) and the material was centrifuged at 14,000 rpm for 2 minutes. The filtered material was transferred to a new tube and 600 µl of AW1 buffer was added, mixing the liquids with the micropipette. 650 µl of the sample was transferred to another DNeasy Mini Spin column (white) and the material was centrifuged at 8,000 rpm for 1 minute. The material discarded in this process was the filtered liquid, and only the column filter remained. The previous operation was repeated using the rest of the sample. After filtering the rest of the sample, the DNeasy Mini Spin column was inserted into a new 2 ml tube and 500 µl AW2 was added, the eluate was centrifuged for 1 minute at 8000 rpm, then the filtered material was discarded. 500 µl of AW2 buffer was added and the material was centrifuged again at 14,000 rpm for 2 minutes. The DNeasy Mini Spin column was inserted into a new 1.5 ml tube, adding 100 µl of TAE and left to rest for 5 minutes. The material was then centrifuged at 8000 rpm for 1 minute and the previous step was repeated. The process was completed when the resulting DNA pellet was resuspended in 200 µl of TAE. DNA Quantification and Quality The DNA samples extracted as above were subjected to 1% agarose gel electrophoresis using TBE 1X buffer (tris, boric acid and EDTA) to check the quantity and quality of the DNA, as shown in Fig. 1. For electrophoresis, 10µl of each DNA sample was taken and inserted into a 200µl tube, 5µl of loading buffer and 2µl of Sybr Green were added, the final concentration was 17µl, this mixture was applied to the wells of the agarose gel and this in turn was placed in the electrophoresis vat, which was connected to the KASVI K33-300V electrophoresis source at 120 Volts for 30 minutes. After the run, the gel was transferred to the Gel Logic photodocumenter, Carestream MI SE and the image captured. A standard marker of known molecular weight (InvitrogenTM 1 Kb Plus DNA Ladder) was used (Fig. 1). Figura 1 Electrophoretic profile in 1% agarose gel of 19 forage palm accessions (Opuntia spp.), to check the quantity and quality of DNA, M − 100 bp molecular weight marker (InvitrogenTM 1 Kb Plus DNA Ladder). Samples that showed a band pattern with a high quantity were diluted in a ratio of 5 µl of DNA to 45 µl of AE buffer; samples that showed no bands were extracted again. Oligonucleotide primers (ISSR). Eleven ISSR oligonucleotide primers were used, the names and 5‘ − 3’ sequences of which can be found in (Table 1). Tabela 1 ISSR (Inter Simple Sequence Repeats) primer oligonucleotides and their respective sequences. Primers Sequence (5’-3’) UBC807 AGAGAGAGAGAGAGAGT UBC808 AGAGAGAGAGAGAGAGC UBC809 AGAGAGAGAGAGAGAGG UBC810 GAGAGAGAGAGAGAGAT UBC811 GAGAGAGAGAGAGAGAC UBC825 ACACACACACACACACT UBC827 ACACACACACACACACG UBC834 AGAGAGAGAGAGAGAGYT UBC840 GAGAGAGAGAGAGAGAYT UBC841 GAGAGAGAGAGAGAGAYC UBC842 GAGAGAGAGAGAGAGAYG PCR reactions using ISSR The Polymerase Chain Reactions (PCR) were carried out using samples with a final volume of 25 µL, composed of 23 µL of a mixture (18.6 µL of Mili-Q water + 1 µL of reaction buffer [200 mM Tris-HCl pH 8, 4, 500 mM KCl] + 1.5 mM MgCl2, 100 µM of each of the four dNTPs + 0.3 µM primer + 0.6 unit of Taq polymerase (Invitrogen)) and 2 µL of DNA (10ng µL-1). A GeneAmp PCR System 9700 thermal cycler was used to carry out the amplification cycles, with the following temperatures and times: 5 minutes at 95°C, followed by 35 cycles of 45 seconds at 95°C (DNA denaturation); 35 seconds at 50°C (primer pairing to the DNA template) and 50 seconds at 72°C (primer extension), followed by 7 minutes at 72°C (final fragment extension) and the last cycle at 4ºC for 10 min. At the end of the PCR process, the amplified DNA samples received 5µl of loading buffer and 2µl of Sybr Green, increasing the final volume of the samples to 32µl and mixed by pipetting. 15µl of this mixture was applied to the wells of a 2.0% agarose gel, The gel was inserted into the electrophoresis machine and TBE 1X buffer (tris, boric acid and EDTA) was added until the gel was completely covered and the gel was subjected to an electric current of 85 V for one hour and twenty minutes, after which the gel was placed in the Gel Logic Carestream MI SE photodocumenter, where it was photographed. The molecular weights of the PCR products were estimated using a 100 bp DNA standard (InvitrogenTM 1 Kb Plus DNA Ladder) (Fig. 2 ). For each primer, the number of amplified bands, number of polymorphic loci, number of monomorphic loci, polymorphic information content (PIC), marker index (MI), resolving power (Rp) and expected heterozygosity (He) values expressed in Table 2 were estimated. The PIC was calculated using the formula described by Serrote et al. (2019): PIC = 1 - (p2 + q2), where PIC is the polymorphic information content of the primer, p the frequency of bands present and q the frequency of bands absent. For dominant markers, the maximum value for PIC is 0.5 for p = 0.5 (Serrote et al., 2019). MI and Rp were estimated as proposed by Gilbert et al. (1999), MI = PIC x polymorphic bands and Rp = ∑▒Ib, where Ib represents the band information, estimated as follows: Ib = 1 - (2*|0.5 - p|), where p is the proportion of accessions containing band I. The percentage of polymorphic loci (PLP) was also estimated. The expected heterozygosity was estimated considering the Hardy-Weinberg equilibrium (2pq), where p is the frequency of bands present and q is the frequency of bands absent. The allelic diversity analyses were carried out using Microsoft® Excel for Mac software version 15.25. Table 2 Markers and their sequences, number of amplified bands (NBA), number of polymorphic loci (NLP), number of monomorphic loci (NLM), percentage of polymorphic loci (PLP), polymorphic information content (PIC), resolving power (Rp), marker index (MI) and expected heterozygosity (He). Primer Sequence (5’-3’) NBA NLP NLM PLP (%) PIC* R p MI He UBC807 (AG) 8 T 69 9 2 81,8 0,2419 3,4500 2,1774 0,2645 UBC808 (AG) 8 C 84 7 1 87,5 0,3653 4,2000 2,5572 0,3872 UBC809 (AG) 8 G 126 10 4 71,4 0,3007 6,3000 3,0071 0,3488 UBC810 (GA) 8 T 25 4 2 66,7 0,1848 1,2500 0,7392 0,1866 UBC811 (GA) 8 C 150 9 3 75,0 0,3398 7,5000 3,0581 0,4297 UBC825 (AC) 8 T 60 4 0 100 0,4150 3,0000 1,6600 0,4688 UBC827 (AC) 8 G 19 2 1 66,7 0,2479 0,9500 0,4958 0,2665 UBC834 (AG) 8 YT 59 4 0 100 0,4478 2,9500 1,7913 0,4655 UBC840 (GA) 8 YT 44 7 1 87,5 0,2347 2,2000 1,6428 0,2372 UBC841 (GA) 8 YC 163 10 2 83,3 0,3849 8,1500 3,8490 0,4485 UBC842 (GA) 8 YG 40 6 3 66,7 0,1931 2,0000 1,1583 0,1975 Total 839 72 19 Average 76,27 6,55 1,73 80,60 0,31 3,81 2,01 0,34 *PIC equals observed heterozygosity when the molecular marker is dominant (Serrote et al., 2019). RESULTS AND DISCUSSION The DNA samples were subjected to PCR using 11 ISSR primers, resulting in 839 bands, distributed in 91 loci, of which 72 are polymorphic and 19 monomorphic, a polymorphism of 79.12%. Primers 825 and 834 showed 100% polymorphic loci. The PIC values for the 11 ISSR primers were moderately high, ranging from 0.1848 to 0.4478, with an average value of 0.31, indicating that the primers used in this study were effective and polymorphic (Table 2 ). The resolving power ranged from 0.9500 to 8.1500 with the highest value in UBC841 and the lowest in UBC827 and the average value was 3.81, the marker index ranged from 0.4958 to 3.8490 with the highest value in UBC841 and the lowest in UBC827 with an average of 2.01, while the highest expected heterozygosity was found in UBC825 0.4688 and the lowest was recorded in UBC810 0.1866 with an average value of 0.34. Based on the data collected, it was possible to create the dendrogram (Fig. 3), using the method proposed by Ward, to analyse the genetic variability between and within species of the 40 Opuntia spp. accessions evaluated, which were allocated into nine different clusters according to genetic similarity and using the cut-off point (Mojena 1977). Cluster I was the largest of all, made up of seven accessions from different regions of Mexico, including 101, which is unidentified in the BGA and originates from the state of Guanajuato. Accessions 116, 119 and 120 originate from Zacatecas, 112 from the state of Coahuila, one of Mexico's 31 states, located in the north of the country, bordering the US state of Texas to the north and the Mexican states of Nuevo León to the east, San Luis Potosí and Zacatecas to the south and Durango and Chihuahua to the west, accession 117, which also makes up this cluster, was collected in the city of Chapingo, located in the State of Mexico, accession 98, which originated in San Luis Potosí, all these accessions belong to the O. megacantha species. Finally, accession 100, also unidentified in the BGA, is part of this group and was collected in the Chapingo region, a town near Texcoco in the State of Mexico. Accessions 100 and 101, which have not been identified in the BGA and which were grouped together with the O. megacantha accessions in this cluster, may also belong to this species, but more in-depth studies are needed to confirm this, especially cytogenetic analyses. Cluster II was made up of accessions 87, 88 and 86, all collected in the Zacatecas region of Mexico, 111, originating in Coahuila and 113 also collected in Zacatecas, belonging to the same species, O. megacantha, which is to be expected. Clusters I and II are made up of 13 accessions of the same species (O. megacantha), with genetic variability between them, which will allow them to be crossed and a segregating population to be obtained (Borém & Miranda 2017 ). Cluster III was made up of accessions 6 and 9 of the species O. cochenilifera, from the state of Michoacán and the city of Chapingo, respectively, and also 8 and 14, both of the species O. atropes, the first of which was collected in Chapingo and the second in Guanajuato. This grouping between the species O. cochenilifera and O. atropes is possibly related to the ploidy level of these species, which are diploid (2n = 22). O. atropes, O. cochenilifera and O. undulata ‘African Elephant Ear’ have 2n = 2x = 22 chromosomes (Silva et al. 2022 ). Cluster IV includes accessions 19, 16, 17 and 18, all from the species O. ficus-indica, the first and last two from Chapingo, and the second collected in Coahuila. Cluster V grouped together the following accessions: 4 and 5, from the O. ficus-indica species, 94, from the O. robusta species and also accessions 1 and 2, which are O. ficus-indica collected in different regions of Mexico, the first three in Chapingo, the fourth in the city of Texcoco and the last in the state of Nuevo León. The grouping of these two distinct species may be related to the level of ploidy. According to Segura (2007), both O. ficus-indica and O. robusta are considered octoploids, with equal DNA content in 2C cells = 4.98 pg, corroborating the results found in this study. Cluster VI is made up of accessions 123, 121, 122 and 125, all of the O. megacantha species, and accession 126, of the O. joconostle species, all from different regions of Mexico: the first originated in San Martín de las Pirámides, a city in the State of Mexico, the second and third in Zacatecas, the fourth in Chapingo and the last in San Luis Potosí. In flow cytometry analyses, O. megacantha Salm-Dyck has (2C = 4.43 pg), while O. joconostle FAC Weber has (2C = 5.01 pg), and they are octaploid species (Segura 2007). O. jonocostle and O. megacantha are the most widespread, domesticated and economically important species (Valadez-Moctezuma, 2023 ), and possibly the process of domestication of these species over the years has contributed to altering their physicochemical characteristics (Nava, 2014 ), for example, the firmness and permeability of cell membranes have increased with the level of domestication, making them more similar and therefore grouped in the same cluster. Nava ( 2014 ) also reported a gradient of domestication, in which O. streptacantha is closest to the wild species, while O. hyptiacantha and O. megacantha are intermediate species and O. albicarpa and O. ficus-indica are the most domesticated. Cluster VII consists of accessions 63, 58 and 67, belonging to the species O. cochenilifera and coming from the IPA in Arcoverde-PE, Brazil. Accession 76, of the species O. atropes, was already in the area of the INSA farm in Campina Grande-PB, Brazil. This formation was possibly due to the fact that both species are diploid, as occurred in cluster III. Cluster VIII, formed only by accession 135 of the species O. dillenii, which originated in the municipality of Soledade-Pb, Brazil, is the most genetically distant species when compared to the other accessions evaluated in this study. The ploidy level of O. dillenii is 2n = 44 (Castro 2012 ). Lastly, cluster IX, formed by accessions 85, O. atropes originating from Guanajuato in Mexico and the other accessions 54, 55, 56, all of the species O. undulata which originate from the IPA in Arcoverde-PE, Brazil. They were possibly grouped together because all the individuals assessed also had the same ploidy level (2n = 2x = 22) (Silva 2022). CONCLUSIONS Based on the results obtained in this study, it was found that there is genetic variability between and within the eight species that make up the 40 accessions of INSA's active germplasm bank (BGA) evaluated in this study. Based on the genetic diversity assessed, we suggest accessions 1, 86, 87, 88, 101, 117, 119 and 135 as potential genitors for the Forage Palm Genetic Improvement Programme of the INSA, UFPB, UFV, Empaer, Emparn and Embrapa Semiárido consortium. Declarations Conflicting interests The authors declare that there is no conflict of interest. Author Contribution The corresponding author Ms J.M.A., was directly responsible for the conception of the manuscript, and also carried out the entire research, drafting and publication process.The author M.M.R., contributed substantially to the production of the manuscript as advisor and reviewer.The author E.R.R., contributed as a reviewer in the analysis and interpretation of two data.The authors declare that there is no conflict of interest.The authors state that they have not received funding. Financing The authors state that they have not received funding. References ALENCAR, L. D. Desvendando a diversidade genética, plastomas e filogenômica de Coleocephalocereus. 2024. Tese de Doutorado. [sn]. Bezerra, J. D. C., de Andrade, A. P., do Rêgo, M. M., da Silva, D. S., do Nascimento Júnior, J. R. S., dos Santos Araújo, F., … de Medeiros, A. N. (2022). Genetic diversity and relationships among Nopalea sp. and Opuntia spp. accessions revealed by RAPD, ISSR and ITS molecular markers. 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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-6567776","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":455554450,"identity":"3228839c-8be0-4217-8a74-e3f52f2d85c0","order_by":0,"name":"Joseilson Moreira de Araújo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYBACxgYgkVAAYiaCmDYJYGGICD4tBnAtaQkMbHARfACsAGz+YYgWBjxamNvbHz54YFAnZ86e3Pbgw5/zefzy3YkfHhgwyPOLHcDusJ4zxgYJBoeNLXsethvObLtdLNnGu1kC6DDDmbMTsGuZkcMGVHAgccONxDZp3obbiRuO8W4AaUkwuI1LS/rzHwkGdRAtf/6cA2nZ/AO/lgQzoCwzRAsD2wGQlm34bQH6RQLkF4MzD9ske9uSE2e25W6zSDCQwOkXQ2CIffxRUSdncDz9mcSPP3aJ/cxnN9/8UWEjzy+NQ0sDVmEGBgkc4gwM8jhlRsEoGAWjYBTAAACTp2ToGByg3wAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Paraíba","correspondingAuthor":true,"prefix":"","firstName":"Joseilson","middleName":"Moreira","lastName":"de Araújo","suffix":""},{"id":455554451,"identity":"7aa13c86-7fa8-4f1e-9f56-20acf98deeaa","order_by":1,"name":"Mailson Monteiro do Rêgo²","email":"","orcid":"","institution":"Federal University of Paraíba","correspondingAuthor":false,"prefix":"","firstName":"Mailson","middleName":"Monteiro do","lastName":"Rêgo²","suffix":""},{"id":455554452,"identity":"a018ef28-a505-4ce0-b127-a02775b2ce63","order_by":2,"name":"Elizanilda Ramalho do Rêgo²","email":"","orcid":"","institution":"Federal University of Paraíba","correspondingAuthor":false,"prefix":"","firstName":"Elizanilda","middleName":"Ramalho do","lastName":"Rêgo²","suffix":""}],"badges":[],"createdAt":"2025-04-30 20:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6567776/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6567776/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82769815,"identity":"9680eddd-0b87-4721-9332-5bd005c262d3","added_by":"auto","created_at":"2025-05-15 06:01:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7562,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoretic profile in 1% agarose gel of 19 forage palm accessions (Opuntia spp.), to check the quantity and quality of DNA, M - 100 bp molecular weight marker (InvitrogenTM 1 Kb Plus DNA Ladder).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6567776/v1/74497be163a18e066c8ed431.jpg"},{"id":82769819,"identity":"3e5cb886-dd9c-4949-84c0-1e88191ee64c","added_by":"auto","created_at":"2025-05-15 06:01:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9663,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoretic profile in 2% agarose gel of 19 forage palm accessions (Opuntia spp.) generated with the ISSR UBC841 primer oligonucleotide. M - molecular weight marker ladder 100 bp.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6567776/v1/ec5a5c770253a078091de6d3.jpg"},{"id":82769817,"identity":"182f5985-a34c-4356-bb88-6ab5840b986d","added_by":"auto","created_at":"2025-05-15 06:01:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68436,"visible":true,"origin":"","legend":"\u003cp\u003eDendrogram constructed on the basis of Jaccard's coefficient of genetic similarity (1912) in a population of 40 Opuntia spp. accessions using 11 ISSR markers.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6567776/v1/e2890fca90c198a41302d36b.jpg"},{"id":85035451,"identity":"93f45c34-30a1-46de-9bf2-162d77b782ca","added_by":"auto","created_at":"2025-06-20 08:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":649739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6567776/v1/34e3e197-6a27-4db0-a62e-dbd8a5236833.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenetic Diversity Between and Within Species of Opuntia Spp. Revealed by Issr Molecular Marker\u003c/p\u003e","fulltext":[{"header":"INTRODUTION","content":"\u003cp\u003eThe forage palm, Opuntia spp. belongs to the Cactaceae family, has 130 genera and more than 2,000 species. Its centre of origin is Mexico (Nefzaoui 2016), but it is distributed throughout the different regions of the world, as it easily adapts to adverse weather and climate conditions, from areas with high temperatures to the lowest. This type of crop is economically important for regions with arid and semi-arid climates, guaranteeing income generation and also being used for human and animal food.\u003c/p\u003e \u003cp\u003eThe use of palm fruit for human consumption has been common in Mexico since the period before Spanish colonisation (Reinolds 2008).\u003c/p\u003e \u003cp\u003eThe forage palm (Opuntia and Nopalea) is an important food in livestock farming, presenting itself as an alternative for the arid and semi-arid regions of north-eastern Brazil, as it is adapted to the climatic conditions of the region and has a special physiological aspect in terms of absorption, use and loss of water, withstanding prolonged periods of drought and being able to reach yields of up to 40 tonnes of dry matter per hectare per harvest (Santos et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main use of O. f\u0026iacute;cus-indica in north-eastern Brazil is as fodder under drought conditions (Paiva et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As it is rich in water and fibre, it is used to feed cattle, goats and sheep (Bezerra et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Opuntia genus has a high genetic diversity. Of the more than 30 cultivated species, the economically important ones are: O. f\u0026iacute;cus-indica L., O. albicarpa Scheinvar, O. amyclaea Ten, O. cochenillifera (L.) Miller, O. hyptiacantha FAC Weber, O. megacantha Salm Dyck, O. robusta Wendl and O. streptacantha Lem (Pimenta Barrios 1994; Kiesling \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Mondragon Jacobo 2001; Las Casas et al. 2017).\u003c/p\u003e \u003cp\u003eThe high genetic diversity of Opuntias is conserved in active germplasm banks on all continents. In the Eastern Hemisphere, ex situ conservation has been carried out by Tunisia, Morocco, South Africa and Italy, while in the West, by Argentina, Brazil, Chile, the USA and Mexico (the largest centre of diversity). In Brazil, efforts have been directed towards the conservation of accessions for fodder, with 1,417 accessions, including accessions from various countries, varieties, hybrids and segregants from controlled crosses (Mondrag\u0026oacute;n \u0026amp; Chessa \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGermplasm characterisation is essential for providing information on the characters of each accession, promoting its classification by estimating genetic diversity within and between groups (Nefzaoui 2017).\u003c/p\u003e \u003cp\u003eSegundo Dennis et al., (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) At the molecular level, there are different molecular markers for assessing the genetic variability of species, characterising germplasm, conserving it, improving it genetically and/or carrying out population genetic analyses.\u003c/p\u003e \u003cp\u003eThere are several types of molecular markers based on the polymerase chain reaction (PCR), such as random amplified polymorphic DNA (RAPD) (Williams et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), amplified fragment length polymorphisms (AFLPs) (Vos et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Microsatellites (SSR), which are generally tandem regions of 6 bp (Lovin et al., 2009; Alencar, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and amplification of inter simple sequence repeats (ISSR) (Zietkiewicz et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ISSR marker is a microsatellite-based technique that is very useful for genome studies. The primers used in ISSR amplifications are based on SSR sequences (di, tri, tetra, or penta nucleotide repeats), anchored to genomic sequences that flank each side of the targeted simple sequence repeats (SSRs) (5\u0026lsquo; or 3\u0026rsquo;), using two to four arbitrary and often degenerate nucleotides. Unlike SSRs, this technique does not require prior sequence information and generates a large number of polymorphisms (Zietkiewicz et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eISSR markers are considered particularly useful for studying closely related individuals that require low levels of polymorphism (Zietkiewicz et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to ISSR, RAPD and ITS molecular markers have also proved to be efficient in accessing genetic diversity and discriminating between forage palm accessions (Bezerra et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this context, the aim of this study was to assess the genetic diversity of 40 Opuntia spp. genotypes from the Active Germplasm Bank of the National Semi-Arid Institute, using ISSR molecular markers.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eThe experiment was carried out in two stages, the first at the Molecular Biology Laboratory of the National Semi-Arid Institute (INSA - from Portuguese \" Instituto Nacional do Semi\u0026aacute;rido\" ) in the municipality of Campina Grande-PB, and the second at the Molecular Biology Laboratory of the Centre for Agrarian Sciences at Universidade Federal da Para\u0026iacute;ba. A total of 40 accessions from INSA's Active Germplasm Bank (BGA) were used, divided into 8 species of Opuntias.\u003c/p\u003e \u003cp\u003eThe collected cladodes were stored in a covered and ventilated area so that they could undergo a 15-day \u0026lsquo;curing\u0026rsquo; period, after which they were planted in high-density polypropylene pots with a capacity of 5 litres and kept under a 60% shade roof. 25 days after transplanting, the roots were collected using tweezers and stored in 2.0 ml Eppendorf tubes.\u003c/p\u003e \u003cp\u003eThe roots were washed in distilled water and then placed on paper towels to absorb the liquid in Petri dishes. After the roots were clean and dry, they were placed in brown paper BGAs containing 15 g of silica gel for 72 hours to dehydrate. After this period, the roots were placed in 2 ml Eppendorf tubes along with 3 ceramic beads for maceration in the Bead Ruptor 4 OMNI for 5 minutes. The beads were removed and the material was transferred to new 2 ml Eppendorf tubes labelled with the access number and stored in an upright freezer at -20\u0026ordm;C.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and purification Qiagen method\u003c/h2\u003e \u003cp\u003eDNA was extracted from the samples macerated in the Bead Ruptor using the DNeasy Plant Mini\u0026reg; Kit (Qiagen), following the manufacturer's recommendations: inserting 200 mg of the macerate into an Eppendorf tube, adding 400\u0026micro;l of AP1 buffer\u0026thinsp;+\u0026thinsp;4 \u0026micro;l of RNAse A, using the Vortex Na3600 North Scientific sample shaker to homogenise the solution and taken to a bain marie for 10 minutes at 65\u0026ordm;C, inverting the tube every 3 minutes; 130 \u0026micro;l of P3 buffer was then added, after which the samples were placed on ice for 5 minutes.\u003c/p\u003e \u003cp\u003eAfter this process, the samples were centrifuged using a Biocen 22R machine at 14,000 rpm for 5 minutes. After centrifugation, there was a separation between the liquid and solid parts, where the supernatant was collected and applied to the QIAshredder Mini spine (lilac colour) and the material was centrifuged at 14,000 rpm for 2 minutes.\u003c/p\u003e \u003cp\u003eThe filtered material was transferred to a new tube and 600 \u0026micro;l of AW1 buffer was added, mixing the liquids with the micropipette. 650 \u0026micro;l of the sample was transferred to another DNeasy Mini Spin column (white) and the material was centrifuged at 8,000 rpm for 1 minute. The material discarded in this process was the filtered liquid, and only the column filter remained. The previous operation was repeated using the rest of the sample.\u003c/p\u003e \u003cp\u003eAfter filtering the rest of the sample, the DNeasy Mini Spin column was inserted into a new 2 ml tube and 500 \u0026micro;l AW2 was added, the eluate was centrifuged for 1 minute at 8000 rpm, then the filtered material was discarded. 500 \u0026micro;l of AW2 buffer was added and the material was centrifuged again at 14,000 rpm for 2 minutes. The DNeasy Mini Spin column was inserted into a new 1.5 ml tube, adding 100 \u0026micro;l of TAE and left to rest for 5 minutes. The material was then centrifuged at 8000 rpm for 1 minute and the previous step was repeated. The process was completed when the resulting DNA pellet was resuspended in 200 \u0026micro;l of TAE.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA Quantification and Quality\u003c/h3\u003e\n\u003cp\u003eThe DNA samples extracted as above were subjected to 1% agarose gel electrophoresis using TBE 1X buffer (tris, boric acid and EDTA) to check the quantity and quality of the DNA, as shown in Fig.\u0026nbsp;1. For electrophoresis, 10\u0026micro;l of each DNA sample was taken and inserted into a 200\u0026micro;l tube, 5\u0026micro;l of loading buffer and 2\u0026micro;l of Sybr Green were added, the final concentration was 17\u0026micro;l, this mixture was applied to the wells of the agarose gel and this in turn was placed in the electrophoresis vat, which was connected to the KASVI K33-300V electrophoresis source at 120 Volts for 30 minutes.\u003c/p\u003e \u003cp\u003eAfter the run, the gel was transferred to the Gel Logic photodocumenter, Carestream MI SE and the image captured. A standard marker of known molecular weight (InvitrogenTM 1 Kb Plus DNA Ladder) was used (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigura 1\u003c/b\u003e Electrophoretic profile in 1% agarose gel of 19 forage palm accessions (Opuntia spp.), to check the quantity and quality of DNA, M \u0026minus;\u0026thinsp;100 bp molecular weight marker (InvitrogenTM 1 Kb Plus DNA Ladder).\u003c/p\u003e \u003cp\u003eSamples that showed a band pattern with a high quantity were diluted in a ratio of 5 \u0026micro;l of DNA to 45 \u0026micro;l of AE buffer; samples that showed no bands were extracted again.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOligonucleotide primers (ISSR).\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEleven ISSR oligonucleotide primers were used, the names and 5\u0026lsquo; \u0026minus;\u0026thinsp;3\u0026rsquo; sequences of which can be found in (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTabela 1\u003c/b\u003e ISSR (Inter Simple Sequence Repeats) primer oligonucleotides and their respective sequences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAGAGAGAGAGAGAGAGT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAGAGAGAGAGAGAGAGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAGAGAGAGAGAGAGAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC825\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eACACACACACACACACT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC827\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eACACACACACACACACG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAGAGAGAGAGAGAGAGYT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGAYT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGAYC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGAYG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePCR reactions using ISSR\u003c/h3\u003e\n\u003cp\u003eThe Polymerase Chain Reactions (PCR) were carried out using samples with a final volume of 25 \u0026micro;L, composed of 23 \u0026micro;L of a mixture (18.6 \u0026micro;L of Mili-Q water\u0026thinsp;+\u0026thinsp;1 \u0026micro;L of reaction buffer [200 mM Tris-HCl pH 8, 4, 500 mM KCl]\u0026thinsp;+\u0026thinsp;1.5 mM MgCl2, 100 \u0026micro;M of each of the four dNTPs\u0026thinsp;+\u0026thinsp;0.3 \u0026micro;M primer\u0026thinsp;+\u0026thinsp;0.6 unit of Taq polymerase (Invitrogen)) and 2 \u0026micro;L of DNA (10ng \u0026micro;L-1). A GeneAmp PCR System 9700 thermal cycler was used to carry out the amplification cycles, with the following temperatures and times: 5 minutes at 95\u0026deg;C, followed by 35 cycles of 45 seconds at 95\u0026deg;C (DNA denaturation); 35 seconds at 50\u0026deg;C (primer pairing to the DNA template) and 50 seconds at 72\u0026deg;C (primer extension), followed by 7 minutes at 72\u0026deg;C (final fragment extension) and the last cycle at 4\u0026ordm;C for 10 min.\u003c/p\u003e \u003cp\u003eAt the end of the PCR process, the amplified DNA samples received 5\u0026micro;l of loading buffer and 2\u0026micro;l of Sybr Green, increasing the final volume of the samples to 32\u0026micro;l and mixed by pipetting. 15\u0026micro;l of this mixture was applied to the wells of a 2.0% agarose gel, The gel was inserted into the electrophoresis machine and TBE 1X buffer (tris, boric acid and EDTA) was added until the gel was completely covered and the gel was subjected to an electric current of 85 V for one hour and twenty minutes, after which the gel was placed in the Gel Logic Carestream MI SE photodocumenter, where it was photographed. The molecular weights of the PCR products were estimated using a 100 bp DNA standard (InvitrogenTM 1 Kb Plus DNA Ladder) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor each primer, the number of amplified bands, number of polymorphic loci, number of monomorphic loci, polymorphic information content (PIC), marker index (MI), resolving power (Rp) and expected heterozygosity (He) values expressed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e were estimated. The PIC was calculated using the formula described by Serrote et al. (2019): PIC\u0026thinsp;=\u0026thinsp;1 - (p2\u0026thinsp;+\u0026thinsp;q2), where PIC is the polymorphic information content of the primer, p the frequency of bands present and q the frequency of bands absent. For dominant markers, the maximum value for PIC is 0.5 for p\u0026thinsp;=\u0026thinsp;0.5 (Serrote et al., 2019). MI and Rp were estimated as proposed by Gilbert et al. (1999), MI\u0026thinsp;=\u0026thinsp;PIC x polymorphic bands and Rp = \u0026sum;▒Ib, where Ib represents the band information, estimated as follows: Ib\u0026thinsp;=\u0026thinsp;1 - (2*|0.5 - p|), where p is the proportion of accessions containing band I. The percentage of polymorphic loci (PLP) was also estimated. The expected heterozygosity was estimated considering the Hardy-Weinberg equilibrium (2pq), where p is the frequency of bands present and q is the frequency of bands absent. The allelic diversity analyses were carried out using Microsoft\u0026reg; Excel for Mac software version 15.25.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMarkers and their sequences, number of amplified bands (NBA), number of polymorphic loci (NLP), number of monomorphic loci (NLM), percentage of polymorphic loci (PLP), polymorphic information content (PIC), resolving power (Rp), marker index (MI) and expected heterozygosity (He).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNBA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNLP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNLM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePLP (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePIC*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003csub\u003ep\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHe\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(AG)\u003csub\u003e8\u003c/sub\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e81,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,2419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3,4500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2,1774\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,2645\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(AG)\u003csub\u003e8\u003c/sub\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,3653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4,2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2,5572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,3872\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(AG)\u003csub\u003e8\u003c/sub\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,3007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6,3000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3,0071\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,3488\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(GA)\u003csub\u003e8\u003c/sub\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,1848\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1,2500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,7392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,1866\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(GA)\u003csub\u003e8\u003c/sub\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,3398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7,5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3,0581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,4297\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC825\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(AC)\u003csub\u003e8\u003c/sub\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,4150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3,0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,6600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,4688\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC827\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(AC)\u003csub\u003e8\u003c/sub\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,2479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,9500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,4958\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,2665\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(AG)\u003csub\u003e8\u003c/sub\u003eYT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,4478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2,9500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,7913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,4655\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(GA)\u003csub\u003e8\u003c/sub\u003eYT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,2347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2,2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,6428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,2372\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(GA)\u003csub\u003e8\u003c/sub\u003eYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e83,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,3849\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8,1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3,8490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,4485\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(GA)\u003csub\u003e8\u003c/sub\u003eYG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,1931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2,0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,1583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,1975\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e839\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e72\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAverage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e76,27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e6,55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1,73\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e80,60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0,31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3,81\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e2,01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0,34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*PIC equals observed heterozygosity when the molecular marker is dominant (Serrote et al., 2019).\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eThe DNA samples were subjected to PCR using 11 ISSR primers, resulting in 839 bands, distributed in 91 loci, of which 72 are polymorphic and 19 monomorphic, a polymorphism of 79.12%. Primers 825 and 834 showed 100% polymorphic loci.\u003c/p\u003e \u003cp\u003eThe PIC values for the 11 ISSR primers were moderately high, ranging from 0.1848 to 0.4478, with an average value of 0.31, indicating that the primers used in this study were effective and polymorphic (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The resolving power ranged from 0.9500 to 8.1500 with the highest value in UBC841 and the lowest in UBC827 and the average value was 3.81, the marker index ranged from 0.4958 to 3.8490 with the highest value in UBC841 and the lowest in UBC827 with an average of 2.01, while the highest expected heterozygosity was found in UBC825 0.4688 and the lowest was recorded in UBC810 0.1866 with an average value of 0.34.\u003c/p\u003e \u003cp\u003eBased on the data collected, it was possible to create the dendrogram (Fig.\u0026nbsp;3), using the method proposed by Ward, to analyse the genetic variability between and within species of the 40 Opuntia spp. accessions evaluated, which were allocated into nine different clusters according to genetic similarity and using the cut-off point (Mojena 1977).\u003c/p\u003e \u003cp\u003eCluster I was the largest of all, made up of seven accessions from different regions of Mexico, including 101, which is unidentified in the BGA and originates from the state of Guanajuato. Accessions 116, 119 and 120 originate from Zacatecas, 112 from the state of Coahuila, one of Mexico's 31 states, located in the north of the country, bordering the US state of Texas to the north and the Mexican states of Nuevo Le\u0026oacute;n to the east, San Luis Potos\u0026iacute; and Zacatecas to the south and Durango and Chihuahua to the west, accession 117, which also makes up this cluster, was collected in the city of Chapingo, located in the State of Mexico, accession 98, which originated in San Luis Potos\u0026iacute;, all these accessions belong to the O. megacantha species. Finally, accession 100, also unidentified in the BGA, is part of this group and was collected in the Chapingo region, a town near Texcoco in the State of Mexico. Accessions 100 and 101, which have not been identified in the BGA and which were grouped together with the O. megacantha accessions in this cluster, may also belong to this species, but more in-depth studies are needed to confirm this, especially cytogenetic analyses.\u003c/p\u003e \u003cp\u003eCluster II was made up of accessions 87, 88 and 86, all collected in the Zacatecas region of Mexico, 111, originating in Coahuila and 113 also collected in Zacatecas, belonging to the same species, O. megacantha, which is to be expected. Clusters I and II are made up of 13 accessions of the same species (O. megacantha), with genetic variability between them, which will allow them to be crossed and a segregating population to be obtained (Bor\u0026eacute;m \u0026amp; Miranda \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCluster III was made up of accessions 6 and 9 of the species O. cochenilifera, from the state of Michoac\u0026aacute;n and the city of Chapingo, respectively, and also 8 and 14, both of the species O. atropes, the first of which was collected in Chapingo and the second in Guanajuato.\u003c/p\u003e \u003cp\u003eThis grouping between the species O. cochenilifera and O. atropes is possibly related to the ploidy level of these species, which are diploid (2n\u0026thinsp;=\u0026thinsp;22). O. atropes, O. cochenilifera and O. undulata \u0026lsquo;African Elephant Ear\u0026rsquo; have 2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;22 chromosomes (Silva et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCluster IV includes accessions 19, 16, 17 and 18, all from the species O. ficus-indica, the first and last two from Chapingo, and the second collected in Coahuila.\u003c/p\u003e \u003cp\u003eCluster V grouped together the following accessions: 4 and 5, from the O. ficus-indica species, 94, from the O. robusta species and also accessions 1 and 2, which are O. ficus-indica collected in different regions of Mexico, the first three in Chapingo, the fourth in the city of Texcoco and the last in the state of Nuevo Le\u0026oacute;n. The grouping of these two distinct species may be related to the level of ploidy. According to Segura (2007), both O. ficus-indica and O. robusta are considered octoploids, with equal DNA content in 2C cells\u0026thinsp;=\u0026thinsp;4.98 pg, corroborating the results found in this study.\u003c/p\u003e \u003cp\u003eCluster VI is made up of accessions 123, 121, 122 and 125, all of the O. megacantha species, and accession 126, of the O. joconostle species, all from different regions of Mexico: the first originated in San Mart\u0026iacute;n de las Pir\u0026aacute;mides, a city in the State of Mexico, the second and third in Zacatecas, the fourth in Chapingo and the last in San Luis Potos\u0026iacute;. In flow cytometry analyses, O. megacantha Salm-Dyck has (2C\u0026thinsp;=\u0026thinsp;4.43 pg), while O. joconostle FAC Weber has (2C\u0026thinsp;=\u0026thinsp;5.01 pg), and they are octaploid species (Segura 2007).\u003c/p\u003e \u003cp\u003eO. jonocostle and O. megacantha are the most widespread, domesticated and economically important species (Valadez-Moctezuma, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and possibly the process of domestication of these species over the years has contributed to altering their physicochemical characteristics (Nava, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), for example, the firmness and permeability of cell membranes have increased with the level of domestication, making them more similar and therefore grouped in the same cluster.\u003c/p\u003e \u003cp\u003eNava (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) also reported a gradient of domestication, in which O. streptacantha is closest to the wild species, while O. hyptiacantha and O. megacantha are intermediate species and O. albicarpa and O. ficus-indica are the most domesticated.\u003c/p\u003e \u003cp\u003eCluster VII consists of accessions 63, 58 and 67, belonging to the species O. cochenilifera and coming from the IPA in Arcoverde-PE, Brazil. Accession 76, of the species O. atropes, was already in the area of the INSA farm in Campina Grande-PB, Brazil. This formation was possibly due to the fact that both species are diploid, as occurred in cluster III.\u003c/p\u003e \u003cp\u003eCluster VIII, formed only by accession 135 of the species O. dillenii, which originated in the municipality of Soledade-Pb, Brazil, is the most genetically distant species when compared to the other accessions evaluated in this study. The ploidy level of O. dillenii is 2n\u0026thinsp;=\u0026thinsp;44 (Castro \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLastly, cluster IX, formed by accessions 85, O. atropes originating from Guanajuato in Mexico and the other accessions 54, 55, 56, all of the species O. undulata which originate from the IPA in Arcoverde-PE, Brazil. They were possibly grouped together because all the individuals assessed also had the same ploidy level (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;22) (Silva 2022).\u003c/p\u003e "},{"header":"CONCLUSIONS","content":"\u003cp\u003eBased on the results obtained in this study, it was found that there is genetic variability between and within the eight species that make up the 40 accessions of INSA's active germplasm bank (BGA) evaluated in this study.\u003c/p\u003e \u003cp\u003eBased on the genetic diversity assessed, we suggest accessions 1, 86, 87, 88, 101, 117, 119 and 135 as potential genitors for the Forage Palm Genetic Improvement Programme of the INSA, UFPB, UFV, Empaer, Emparn and Embrapa Semi\u0026aacute;rido consortium.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe corresponding author Ms J.M.A., was directly responsible for the conception of the manuscript, and also carried out the entire research, drafting and publication process.The author M.M.R., contributed substantially to the production of the manuscript as advisor and reviewer.The author E.R.R., contributed as a reviewer in the analysis and interpretation of two data.The authors declare that there is no conflict of interest.The authors state that they have not received funding.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFinancing\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that they have not received funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eALENCAR, L. 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Genetic Resources and Crop Evolution, v. 70, n. 3, p. 951\u0026ndash;970, 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVOS, P.; HOGERS, R.; BLEEKER, M.; REIJANS, M.; LEE, T. V.; HORNES, M.; FRIJTERS, A.; POT, J.; PELEMAN, J.; KUIPER, M. \u0026amp; ZABEAU, M. AFLP: A new technique forDNA fingerprinting. Nucleic Acids Res. 23:4407\u0026ndash;4414, 1995.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWARD, J. H. Hierarchical Grouping to Optimize an Objective Function. Journal of the American Statistical Association v. 58, n. 301, p. 236\u0026ndash;244,, mar. 1963.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWILLIAMS, J. G. K.; KUBELIK, A. R.; LIVAK, K. J.; RAFALSKI, A. \u0026amp; TINGEY, S. V. DNA polymorphisms amplified by arbitrary primers are useful as genetic mark-ers. Nucleic Acids Res. 18:6531\u0026ndash;6535, 1990.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZIETKIEWICZ, E.; RAFALSKI, A. \u0026amp; LABUDA, D. Genome fingerprinting bysimple sequence repeat (SSR)-anchored polymerase chain reaction amplification.Genomics 20:176\u0026ndash;183, 1994.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"issr, genetic resources, genetic improvement, cactaceae","lastPublishedDoi":"10.21203/rs.3.rs-6567776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6567776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Opuntia spp. forage palm is a viable solution for animal feed in arid and semi-arid regions due to its easy adaptation to this type of environment, its physiological characteristics and its low production cost. Given its importance, it is essential to learn more about its genetic characteristics. Molecular markers are one of the most widely used techniques. Within this context, the aim of this study was to assess the genetic diversity of 40 genotypes of Opuntia spp. from the Active Germplasm Bank (AGB) of the National Semi-Arid Institute (INSA), using Inter Simple Sequence Repeats (ISSR) molecular markers.\u003c/p\u003e \u003cp\u003eDeoxyribonucleic acid (DNA) was extracted from the accessions using the DNeasy Plant Mini\u0026reg; Kit (Qiagen). Eleven ISSR oligonucleotide primers were used. This technique amplified 839 fragments, distributed over 91 loci. Nine groups were constructed from the genetic distances of the 40 accessions. It was found that there is high genetic diversity between and within the species evaluated, helping to broaden the genetic base of the segregating populations, suggesting intraspecific crossing between accessions 87 and 117 and interspecific crossing with accessions 1 x 119, 86 x ?(101), 86 x 135, 88 x 135, ?(101) x 135.\u003c/p\u003e","manuscriptTitle":"Genetic Diversity Between and Within Species of Opuntia Spp. Revealed by Issr Molecular Marker","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 06:00:57","doi":"10.21203/rs.3.rs-6567776/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c69b7530-68a5-4205-993c-06422e45a6c7","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-20T08:09:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-15 06:00:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6567776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6567776","identity":"rs-6567776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}