بررسی تنوع ژنتیکی درون گونه‌ای پنج رقم ارکیده فالانوپسیس شاخه بریده با استفاده از نشانگرهای مولکولی RAPD و IRAP

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه علوم باغبانی، دانشکده علوم کشاورزی، دانشگاه گیلان، رشت، ایران

2 گروه زیست فناوری، دانشکده کشاورزی، دانشگاه صنعتی اصفهان، اصفهان ، ایران

10.22059/ijhs.2023.343479.2030

چکیده

فالانوپسیس  یکی از شناخته‌شده‌ترین جنس‌های تیره ارکیده است که به دلیل قابلیت سازگاری بالا با شرایط محیطی مختلف از رشد نسبتاً مناسبی نیز برخوردار است. برنامه اصلاح فالانوپسیس و بررسی کامل  نتاج به‌طور معمول  نیاز به سه تا پنج سال زمان دارد که با استفاده از نشانگرهای مولکولی این زمان را می‌توان کاهش داد. در این تحقیق، نتاج حاصل از تلاقی 5 رقم مختلف، به‌منظور کوتاه کردن زمان جهت انتخاب ژنوتیپ‌های برتر با استفاده از نشانگرهای مولکولی رپید و آیرپ  مورد بررسی قرار گرفتند. از مجموع 299 نوار تولید شده در رپید، 86 درصد نوارها چند شکل بودند. میانگین تعداد نوارهای چند شکل 5/13 نوار برای هر آغازگر بود. میزان حداقل تشابه ژنتیکی با استفاده از نشانگر رپید و ضریب تشابه نی  بین هیبریدهای 'Sevilla'×'Sevilla' و 'Manila'×'Bombay' 43 درصد و حداکثر آن بین هیبریدهای ''Sevilla'×'Okayama و 'Okayama'×'Sevilla'  معادل 72 درصد به‌دست آمد. از 6 ترکیب آغازگر انتخابی آیرپ، در مجموع 83 نوار تولید شد که از این تعداد 72 نوار چند شکل بودند. بالاترین درصد چندشکلی را ترکیب آغازگرهای 3′LTR- LTR6150 و 3′LTR-3′LTR و پایین‌ترین را Sukkula -3′LTR تولید کردند. میزان حداکثر تشابه ژنتیکی با استفاده از نشانگر آیرپ بین هیبریدهای 'Sevilla'×'Okayama'  و 'Sevilla'×'Manila'  با مقدار 82 درصد و کمترین همسانی بین هیبریدهای 'Sevilla'×'Sevilla'  و ''Manila'×'Bombay در حد تشابه 32 درصد مشاهده شد که به ترتیب حاکی از میزان نزدیکی و دوری ژنتیکی این ژنوتیپ‌ها نسبت به یکدیگر می‌باشد. با استفاده از نتایج این پژوهش، ژنوتیپ‌های نوترکیب با الگوی باندی متفاوت از والدین می­توانند برای برنامه‌های به‌نژادی جهت تولید و معرفی ارقام جدید استفاده شوند.  

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

The evaluation of genetic diversity among five Phalaenopsis species using IRAP and RAPD molecular markers

نویسندگان [English]

  • Khosro Balilashaki 1
  • Hedayat Zakizadeh 1
  • Jamal-Ali Olfati 1
  • Aboozar Soorni 2
1 Department of Horticultural Sciences Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran
2 Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran
چکیده [English]

Phalaenopsis is one of the most well-known genera of the orchid family and has relatively good growth due to its high adaptability. The Phalaenopsis breeding program and full investigation of progenies takes three to five years, which can be reduced by using molecular markers. In this research, in order to decrease the process of selecting superior genotypes, progenies obtained from crosses between 5 different cultivars were examined using IRAP and RAPD markers. Among 299 bands produced in RAPD, 86% of the bands were polymorphic. The average number of polymorphic bands was 13.5 bands per primer and the minimum genetic similarity (43%) was obtained between 'Sevilla'×'Sevilla' and 'Manila'×'Bombay' hybrids, while the maximum similarity (72%) was found between 'Sevilla'× 'Okayama' and 'Okayama'×'Sevilla' hybrids based on Nei similarity coefficient. From 6 selected IRAP primer combinations, 83 bands were produced, among them 72 bands were considered polymorphic bands. The highest ratio of polymorphism was obtained by 3′LTR-LTR6150, 3′LTR-3′LTR primers combination and the lowest by Sukkula -3′LTR. The maximum genetic similarity, 82%, using IRAP marker was observed between 'Sevilla'×'Okayama' and 'Sevilla'×'Manila' hybrids and the lowest amount, 32%, was obtained between 'Sevilla'×'Sevilla' and 'Manila'×'Bombay' hybrids, indicating the genetic proximity and distance, respectively, of the studied genotypes. Recombined genotypes obtained in this research, which had different band patterns with their parents, can be used for breeding programs and introducing new cultivars.

کلیدواژه‌ها [English]

  • Molecular Markers
  • Retrotransposon
  • RAPD
  • Polymorphism

Extended Abstract

Introduction

Phalaenopsis orchid is very popular in the floricultural industry due to their unique traits like morphological diversity, color variation, and medicinal properties. Over 90 native species have been categorized into five subgenera, driving diverse cultivar development with vibrant colors and fragrances. Breeding programs, primarily utilizing native species like Phalaenopsis spp., aim to enhance traits such as color variation, flower size, and disease resistance.

Understanding genetic diversity is key to advancing breeding strategies. Molecular markers offer advantages in evaluating genetic variation and phylogenetic relationships due to their high variability and co-dominant inheritance. Techniques like RAPD and IRAP serve as cost-effective alternatives, with RAPD's simplicity and lack of radioactive materials being noteworthy. Studies have demonstrated significant genetic diversity among orchid populations, exemplified by research on Indonesian Sarcanthinae orchids. Retrotransposon-based markers, such as IRAP, further extend marker capabilities, utilizing retrotransposon insertion sites for genetic diversity assessment.

In this research, in order to decrease the process of selecting superior genotypes, progenies obtained from crosses between different cultivars. Current research focuses on extending markers for various orchid species and hybrids to assess polymorphism and infer phylogenetic relationships. These findings contribute to understanding orchid genetics and safeguarding breeders' intellectual property rights.

Materials and methods

The study utilized five Phalaenopsis orchid cultivars selected based on specific traits like flower longevity, resistance to fungus, and the production of the second flower stalk. Experimental crosses were conducted bidirectionally, with each flower both providing and receiving pollen grains. Capsules were counted five weeks’ post-pollination and harvested 120 days later, then sterilized and cultured on a growth medium. DNA extraction from plantlets involved grinding frozen leaves and subsequent extraction using a commercial kit.

Polymerase chain reaction (PCR) with random primers was performed for genetic marker analysis, selecting appropriate primers for diversity assessment. RAPD and IRAP products were visualized on agarose gels. Statistical analysis included estimating seed germination percentage and calculating genetic similarity matrices, similarity coefficients, dendrograms, resolving power, polymorphism information content (PIC), and marker index (MI).

Polymorphic bands in genetic profiles were scored, and genetic likeness matrices were computed using Free Tree software. Dendrograms were generated using iTOL software. The resolving power of primers and PIC were estimated using specific formulas. The MI was calculated based on PIC and an effective multiplex rate, derived from the total numbers of polymorphic gene locations and uniform bands.

 

Results and Discussion

RAPD Analysis: Of the 50 RAPD primers tested, 22 detected polymorphic bands, with fragment lengths ranging from 300 to 1500 bp. across all genotypes, a total of 299 bands were amplified, of which 257 were polymorphic (86%). The average number of polymorphic bands per primer was 13.5. Primers varied in their polymorphism percentage, with PPB ranging from 50% to 100%. The highest marker index (MI) was 6.54, attributed to the OPT-11 primer. The resolving index ranged from 21.12 to 0.508, with OPAI-13 exhibiting the highest polymorphism information content (PIC) of 0.444. The BB-20 primer showed the highest Shannon’s information index (I; 0.635) and the number of effective alleles (Ne; 1.812), indicating its effectiveness in investigating genetic diversity. RAPD analysis of Phalaenopsis aphrodite subsp. formosana and related species demonstrated the technique's utility in discovering relationships among Phalaenopsis species.

IRAP Analysis: Using IRAP markers, 83 bands were produced, of which 72 were polymorphic (86.74%). Fragment sizes ranged from 200 to 800 bp. The average number of amplified bands per pair of primers was 14.4, with Sukkula-Sukkula, 3′LTR-3′LTR, and LTR6150-LTR6150 showing the highest polymorphic fragments. PIC values ranged from 0.62 to 0.93, with five pairs of primers exceeding 0.80. MI ranged from 5.60 to 13.10, while the Rp index ranged from 4.3 to 8.40. BARE-1 and Sukkula fragments exhibited higher polymorphism, possibly due to their abundance in the genome. The polymorphism percentage varied from 78.57% to 93.33%, with the 3′LTR-3′LTR primer showing the highest MI and Rp values. Primer 3′LTR-LTR150 displayed the highest PIC, I, and Ne indices.

 

Conclusion

In conclusion, both RAPD and IRAP analyses demonstrated high levels of polymorphism, indicating their efficacy in assessing genetic diversity within Phalaenopsis orchid cultivars. The diverse set of primers utilized in this study revealed varying levels of informativeness, with certain primers exhibiting particularly high polymorphic potential. These findings underscore the value of molecular marker techniques in elucidating genetic relationships and informing breeding strategies within the Phalaenopsis genus

امیدی بخش، محمد. (2005). مطالعه تنوع ژنتیکی گندم دوروم با استفاده از نشانگر SSR. پایان نامه کارشناسی ارشد، دانشگاه تهران، تهران، ایران.
Bhattacharyya, P., Kumaria, S., Kumar, S. & Tandon, P. (2013). Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl., an endangered medicinal orchid species. Gene, 529, 21–26.
Bhattacharyya, P., Kumaria, S. & Tandon, P. (2015). Applicability of ISSR and DAMD markers for phyto-molecular characterization and association with some important biochemical traits of Dendrobium nobile, an endangered medicinal orchid. Phytochemistry, 117, 306–316.
Becker, J., Vos, P. & Kuiper, M. (1995). Combined mapping of AFLP and RFLP markers in barley. Molecular and General Genetics MGG, 249(1), 65-73.
Chen, J. & Chang, W.C. (2006). Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biologia Plantarum, 50(2), 169-173.
Chen, T.Y., Chen, J.T. & Chang, W.C. (2002). Multiple shoot formation and plant regeneration from stem nodal explants of Paphiopedilum orchids. In Vitro Cellular & Developmental Biology-Plant, 38(6), 595-597.
Chuang, H.T. (2002). Identification of some species in the genus Phalaenopsis to Taiwan and Philippine by using RAPD and ISSR molecular markers. Master Thesis, Graduate Institute of Agriculture, National Chiayi University, Chiayi, Taiwan. (Chinese with English abstract)
Chugh, S., Guha, S. & Rao, I.U. (2009). Micropropagation of orchids: a review on the potential of different explants. Scientia Horticulturae, 122(4), 507-520.
Goh, M.W.K., Kumar, P.P., Lim, S.H. & Tan, H.T.W. (2005). Random amplified polymorphic DNA analysis of the moth orchids, Phalaenopsis (Epidendroideae:Orchidaceae). Euphytica 141, 11–22.
Griesbach, R. (2002). Development of Phalaenopsis orchids for the mass-market. Trends in new crops and new uses. ASHS Press, Alexandria, VA, 458-465.
Kalendar, R., Grob, T. & Regina, M. (1999). IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theoretical and Applied Genetics, 98(5), 704-711.
Kalendar, R. & Schulman, A.H. (2006). IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nature Protocol, 1, 2478–2484
Khadivi-Khub A, Soorni A. (2014). Comprehensive genetic discrimination of Leonurus cardiaca populations by AFLP, ISSR, RAPD and IRAP molecular markers. Molecular Biology Reports. 41(6):4007-16.
Kilinc, F. M., Süzerer, V., Çiftçi, Y., Onay, A., Yıldırım, H., Uncuoğlu, A.A., Tilkat, E, Metin, O.K., Ibrahim, K. & Akdemir, O.F. (2015). Clonal micropropagation of Pistacia lentiscus L. and assessment of genetic stability using IRAP markers. Plant Growth Regulation, 75, 75–88
Kumar, R., Singh, M., Kumar, P., Singh, J. (2015). Crop Selection Method to Maximize Crop Yield Rate using Machine Learning Technique. International Conference on Smart Technologies and Management for Computing, Communication, Controls, Energy and Materials1. 0.1109/ICSTM.2015.7225403.
Liu, B. (2010). Uncertain risk analysis and uncertain reliability analysis. Journal of Uncertain Systems, 4(3), 163-170.
Niknejad, A., Kadir, M.A., Kadzimin, S.B., Abdullah, N.A.P. & Sorkheh, K. (2009). Molecular characterization and phylogenetic relationships among and within species of Phalaenopsis (Epidendroideae: Orchidaceae) based on RAPD analysis. African Journal of Biotechnology, 8, 5225-5240.
Omidbakhsh Fard, M, (2005). Study of genetic diversity in durum wheat using SSR marker', MSc Thesis. Tehran University. Tehran, Iran. (in Persian).
Powel,W., Morgante, M. & Andre, C. (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Molecular breeding, 2(3), 225-238.
Puchooa, D. (2004). A simple, rapid and efficient method for the extraction of genomic DNA from lychee (Litchi chinensis Sonn.). African Journal of Biotechnology, 3(4), 253-255.
Spooner, D.M., McLean, K., Ramsay, G., Waugh, R. & Bryan, G.J. (2005). Single domestication for potato based on multilocus amplified fragment length polymorphism genotyping. Proceedings of the National Academy of Sciences. U.S.A. 102, 14694–14699.
Stewart, S.L. & Kane, M.E. (2006) Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell, Tissue and Organ Culture, 86(2), 147-158.
Tsai, C.C., Shih, H.C., Wang, H.V., Lin, Y.S., Chang, C.H., Chiang, Y.C. & Chou, C.H. (2015). RNA-Seq SSRs of moth orchid and screening for molecular markers across genus Phalaenopsis (Orchidaceae). PLoS ONE, 10(11), e0141761.
Xiang, N., Hong, Y. & Lam-Chan, L.T. (2003). Genetic analysis of tropical orchid hybrids (Dendrobium) with fluorescence amplified fragment length polymorphism (AFLP). Journal of the American Society for Horticultural Science, 128, 731–735.
Varshney, R.K., Chabane, K. & Hendre, P.S. (2007). Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic diversity and conservation of genetic resources using wild, cultivated and elite barleys. Plant Science, 173(6), 638-649.
Wen, W.H., Liu, J.Y. & Qin, W.J. (2007). Targeted inhibition of HBV gene expression by single‐chain antibody mediated small interfering RNA delivery. Hepatology, 46(1), 84-94.