انگشت‎نگاری ژنتیکی بخشی از ژرم‎پلاسم خربزه ایران با استفاده از نشانگر‎های ریز‎ماهواره

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری اصلاح نباتات، گروه کشاورزی، دانشگاه آزاد اسلامی، واحد زاهدان، زاهدان، ایران

2 بخش تحقیقات زراعی باغی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی سیستان، سازمان تحقیقات، آموزش و ترویج کشاورزی، زابل، ایران

3 گروه کشاورزی، دانشگاه آزاد اسلامی، واحد زاهدان، زاهدان، ایران

4 گروه زیست شناسی، دانشکده علوم پایه، دانشگاه زابل، زابل، ایران

چکیده

آگاهی از میزان تنوع ژنتیکی بر اساس نشانگر‎های مولکولی روشی سریع است که نقش مهمی در برنامه‎های اصلاحی دارد. در این بررسی تنوع ژنتیکی 40 توده محلی خربزه از مناطق شرقی و مرکزی ایران با استفاده از 15 جفت آغازگر ریز‎ماهواره مورد ارزیابی قرار گرفت که 13 جفت از آغازگر‎های مورد استفاده، قادر به تکثیر مکان‎های ژنی ریز‎ماهواره شدند. تمام مکان‎های ژنی به جز آغازگر CMCT44، چند شکل بودند، در مجموع تعداد 25 آلل با میانگین 1/93 آلل به ازای هر جایگاه ژنی ریز‎ماهواره، مشاهده گردید. بالا‎ترین میزان مکان‎های چند شکلی (84/62 درصد) متعلق به توده KC-357009 بود. میزان بالای مکان‎های چند شکلی، تأیید‎کننده کارآیی نشانگر‎های مولکولی SSR در آنالیز ژنتیکی توده‎های بومی مورد بررسی است. میانگین محتوای اطلاعات چند شکلی و هتروزیگوتی مورد انتظار به ترتیب 0/24 و 0/23 به‎دست‎آمد. مقادیر بالای هموزیگوتی مشاهده شده با میانگین 0/84 نشان‎دهنده تنوع پایین درون توده‎های مورد بررسی است که دلیلی بر میزان پایین دگر‎گشنی بین توده‎ها و میزان بالای خویش‎آمیزی است. در بررسی ساختار ژنتیکی جمعیت‎ها، توده‎های مورد بررسی در دو گروه (زیر‎جمعیت) قرار گرفتند. نتایج حاصل از تجزیه ساختار جمعیت بر‎اساس روش بیزین و خوشه‎بندی UPGMA نشان داد که طبقه‎بندی ژنوتیپ‎های مورد مطالعه مستقل از منشاء جغرافیایی‎شان می‎باشد. با توجه به مقادیر حاصل از پارامتر‎های فوق از بین آغازگرهای مورد مطالعه، مکان‎های ژنی CMCCA145، CMGA172 و CMCT134b جهت تجزیه و تحلیل مجموعه‎های ژرم پلاسم خربزه در تحقیقات آتی توصیه می‎شود.

کلیدواژه‌ها


عنوان مقاله [English]

Genetic fingerprinting of a part of Iranian melon germplasm using microsatellite markers

نویسندگان [English]

  • Tahereh Shahraki 1
  • Mohammad Reza Naroui Rad 2
  • Hamid Reza Ganjali 3
  • Hossein Kamaladini 4
  • Hamid Reza Mobaser 3
1 Ph.D. Student in Plant Breeding, Department of Agriculture, Zahedan Branch, Islamic Azad University, Zahedan, Iran
2 Horticultural Research Department, Agricultural and Natural Resources Center of Sistan, Agricultural Research, Education and Extension Organization, Zabol, Iran
3 Department of Agriculture, Islamic Azad University, Zahedan Branch, Zahedan, Iran
4 Department of Biology, College of Basic Sciences, University of Zabol, Zabol, Iran
چکیده [English]

Introduction: Awareness of the extent of genetic diversity based on molecular markers is a rapid method that plays an important role in breeding programs. Cucumis melo L. is the scientific name for a melon that belongs to the Cucurbitaceae family (Kirkbride, 1993). This plant is a popular garden crop in warm and temperate climates across the globe, and Iran ranks fourth in the world in terms of output (FAOSTAT, 2019). Although several studies were conducted on the genetic structure of Iranian melons using various molecular markers including RAPD molecular markers (Feizian, 2004), ISSR (Maleki et al., 2018) and SSR microsatellite markers (Moayedi Nejad et al., 2010; Raghami et al., 2014) in recent years, due to the history of planting and the presence of huge genetic resources of melon, which makes Iran as one of the main centers of diversity of this crop in the world, research is inadequate.
Materials and Methods: Forty native melon stands used in this research were prepared from the melon germplasm of Iran Gene Bank. 4-5 leaves from each plant at the two- to three-leaf stage were collected and kept in a freezer at -80°C until DNA extraction. Their genomic DNA was extracted using the ZandBio plant kit. The markers used in this research were selected based on previous studies that were suggested by different researchers. Gradient PCR Vapo Protect thermocycler was used for polymerase chain reaction. PCR products were electrophoresed on 1.8% agarose gel in 1X TBE buffer and at 85 V for one hour. Observation and photographing of the gel was done with the help of UV-2100 geldoc device. Bands were scored as zero (not seeing a band) and one (observing a band). Evaluation of cluster analysis and genetic parameters such as observed heterozygosity, expected heterozygosity and number of effective alleles were obtained through Ntsys 2.1 and Popgene 1.32 software. To estimate the number of subpopulations (K) and distinguish pure and mixed genotypes, it was done using Structure software version 2.3 and using the Bayesian method (Pritchard et al., 2000).
Results and Discussion: All gene loci except CMCT44 primer were polymorphic. A total of 25 alleles with an average of 1.93 alleles per microsatellite gene locus were observed. The highest amount of polymorphic sites (84.62%) belonged to KC-357009 genotype.. The high amount of polymorphic sites confirms the effectiveness of SSR molecular markers in the genetic analysis of the investigated indigenous populations. The average content of polymorphism and expected heterozygosity information was obtained as 0.24 and 0.23, respectively. The observed high homozygosity values with an average of 0.84 indicate the low diversity within the investigated populations, which is a proof of the low level of variation between the populations and the high level of inbreeding. In the investigation of the genetic structure of the populations, the investigated populations were divided into two groups (sub-populations). The results of population structure analysis based on Bayesian method and UPGMA clustering showed that the classification of studied genotypes is independent of their geographical origin.
Conclusion: According to the values obtained from the above parameters, among the studied primers, gene locations CMCCA145, CMGA172 and CMCT134b are recommended for the analysis of melon germplasm collections in future research.
The results of this study can be used in hybridization programs. Crossing between the figures that have the least similarity will lead to the best results in achieving hybrids or achieving maximum separation in the generations after F1. Although genetic distance is not the only effective factor in identifying suitable parents for hybrid production and factors such as compatibility and genetic distance based on morphological traits should also be considered.

کلیدواژه‌ها [English]

  • Genetic structure
  • Heterozygosity
  • Local accessions
  • Molecular markers
Anderson, J.A., Churchill, G., Autrique, J., Tanksley, S. and Sorrells, M. 1993. Optimizing parental selection for genetic linkage maps. Genome, 36(1), PP.181-186. doi: 10.1139/g93-024
Bagheri, A.A., Darbandi, A. and Malbubi, M.A. 2007. Practical applications of plant molecular biology. Ferdwosi Mashhad Publications [In Persian].
Behbahanei, M. 2005. Genetic diversity among Iranian melons (Cucumis melo L.) via SSR markers. M. Sc. Thesis, Bu-Ali Sina University [In Persian].
Danin-Poleg, Y., Reis, N., Tzuri, G. and Katzir, N. 2001. Development and characterization of microsatellite markers in Cucumis. Theoretical and Applied Genetics, 102(1), PP.61-72. doi: 10.1007/s001220051618
Darvishzadeh, R. and Azizi, A. 2016. Molecular markers and their application in genetic diversity analysis. Urmia University Publishers [In Persian].
Ercisli, S., Ipek, A. and Barut, E. 2011. SSR marker-based DNA fingerprinting and cultivar identification of olives (Olea europaea). Biochemical Genetics, 49(9), PP.555-561. doi:10.1007/s10528-011-9430-z
FAOSTAT, 2019. FAO statistical database. https://www.fao.org/faostat/en/#data/QCL/visualize.
Feyzian, E., Javaran, M.J., Dehghani, H. and Zamyad, H. 2007. Analysis of the genetic diversity among some of Iranian melon (Cucumis melo L.) landraces using morphological and RAPD molecular markers. JWSS-Isfahan University Technology, 11, PP.151-163.
Filiz, E., Uras, M.E., Ozyigit, I.I., Sen, U. and Gungor, H. 2018. Genetic diversity and phylogenetic analyses of Turkish rice varieties revealed by ISSR markers and chloroplast trnl-F region. FEB-Fresenius Environmental Bulletin, 27(12), PP.835-8358.
Gonzalo, M., Oliver, M., Garcia-Mas, J., Monfort, A., Dolcet-Sanjuan, R., Katzir, N., Arus, P. and Monforte, A. 2005. Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.). Theoretical and Applied Genetics, 110(5), PP.802-811. doi: 10.1007/s00122-004-1814-6
Hocaoglu-Ozyigit, A., Ucar, B., Altay, V. and Ozyigit, I.I. 2020. Genetic diversity and phylogenetic analyses of Turkish cotton (Gossypium hirsutum L.) lines using ISSR markers and chloroplast trnL-F regions. Journal of Natural Fibers, 17(1), PP.1-14. doi: 10.1080/15440478.2020.1788493
Katzir, N., Danin-Poleg, Y., Tzuri, G., Karchi, Z., Lavi, U. and Cregan, P. 1996. Length polymorphism and homologies of microsatellites in several Cucurbitaceae species. Theoretical and Applied Genetics, 93(8), PP.1282-1290. doi: 10.1007/bf00223461
Kerje, T. and Grum, M. 2000. The origin of melon, Cucumis melo: a review of the literature. Acta Horticulture, 510, PP.37-44. doi: 10.17660/actahortic.2000.510.5
Kirkbride, J.H. 1993. Biosystematic monograph of the genus Cucumis (Cucurbitaceae): botanical identification of cucumbers and melons. Parkway Publishers, 26-35.
Laosatit, K., Amkul, K., Chankaew, S. and Somta, P. 2021. Molecular genetic diversity of winged bean gene pool in Thailand assessed by SSR markers. Horticultural Plant Journal, 8(1), PP.81-88. doi: 10.1016/j.hpj.2021.05.001
López-Sesé, A.I., Staub, J., Katzir, N. and Gómez-Guillamón, M.L. 2002. Estimation of between and within accession variation in selected Spanish melon germplasm using RAPD and SSR markers to assess strategies for large collection evaluation. Euphytica, 127(1), PP.41-51. doi: 10.1023/a:1019904224170
Maleki, M., Shojaeiyan, A. and Monfared, S.R. 2018. Population structure, morphological and genetic diversity within and among melon (Cucumis melo L.) landraces in Iran. Journal of Genetic Engineering and Biotechnology, 16(2), PP.599-606. doi: 10.1016/j.jgeb.2018.08.002
Matus, I. and Hayes, P. 2002. Genetic diversity in three groups of barley germplasm assessed by simple sequence repeats. Genome, 45(6), PP.1095-1106. doi:10.1139/g02-071
Moayedi Nejad, A., Irshadi, A. and Kohpayegani, J.A. 2012. Genetic diversity among Iranian cantaloupe landraces (Cucumis melo L.) using Microsatellite Markers. Agricultural Biotechnology, 1(1), PP.1-8.
Monforte, A., Garcia‐Mas, J. and Arus, P. 2003. Genetic variability in melon based on microsatellite variation. Plant Breeding, 122(2), PP.153-157. doi: 10.1046/j.1439-0523.2003.00848.x
Pritchard, J.K., Stephens, M. and Donnelly, P. 2000. Inference of population structure using multilocus genotype data. Genetics, 155(2), PP.945-959. doi: 10.1093/genetics/155.2.945
Raghami, M., López-Sesé, A.I., Hasandokht, M.R., Zamani, Z., Moghadam, M.R.F. and Kashi, A. 2014. Genetic diversity among melon accessions from Iran and their relationships with melon germplasm of diverse origins using microsatellite markers. Plant Systematics and Evolution, 300(1), PP.139-151. doi:10.1007/s00606-013-0866-y
Ritschel, P.S., de Lima Lins, T.C., Tristan, R.L., Buso, G.S.C., Buso, J.A. and Ferreira, M.E. 2004. Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.). BMC Plant Biology, 4(1), PP.1-14. doi: 10.1186/1471-2229-4-9
Saidi, A., Eghbalnegad, Y. and Hajibarat, Z. 2017. Study of genetic diversity in local rose varieties (Rosa spp.) using molecular markers. Banat's Journal of Biotechnology, 8(16), PP.148-157. doi:10.7904/2068-4738-viii(16)-148
Spataro, G., Tiranti, B., Arcaleni, P., Bellucci, E., Attene, G., Papa, R., Zeuli, P.S. and Negri, V. 2011. Genetic diversity and structure of a worldwide collection of Phaseolus coccineus L. Theoretical and Applied Genetics, 122(7), PP.1281-1291. doi: 10.1007/s00122-011-1530-y
Tamboli, A.S., Yadav, P.B., Gothe, A.A., Yadav, S.R. and Govindwar, S.P. 2018. Molecular phylogeny and genetic diversity of genus Capparis (Capparaceae) based on plastid DNA sequences and ISSR markers. Plant Systematics and Evolution, 304(2), PP.205-217. doi: 10.1007/s00606-017-1466-z
Tzitzikas, E.N., Monforte, A.J., Fatihi, A., Kypriotakis, Z., Iacovides, T.A., Ioannides, I.M. and Kalaitzis, P. 2009. Genetic diversity and population structure of traditional Greek and Cypriot melon cultigens (Cucumis melo L.) based on simple sequence repeat variability. HortScience, 44(7), PP.1820-1824. doi: 10.21273/hortsci.44.7.1820