ارزیابی تنوع ژنتیکی اکوتیپ‌های کلکسیون عناب ایران (Ziziphus spp.) با استفاده از نشانگر مولکولی ISSR

نوع مقاله : مقالات پژوهشی

نویسندگان

• هاجر شایسته ¹
• سعید ملک زاده شفارودی ²
• کمال غوث ³
• فرج اله شهریاری ⁴

¹ دانشگاه فردوسی مشهد

² بیرمینگهام انگلستان / دانشگاه فردوسی

³ جهاد کشاورزی استان خراسان جنوبی

⁴ فردوسی

https://doi.org/10.22067/jhorts4.v31i3.27523

چکیده

عناب (Zizyphus jujuba Mill.) به عنوان یک میوه ارزشمند و مقاوم به شرایط مختلف آب و هوایی در بسیاری از مناطق ایران گسترش دارد. از عناب علاوه بر صادرات بعنوان گیاه دارویی نیز استفاده می‌شود لذا ارزش اقتصادی قابل توجهی دارد. ارزیابی تنوع ژنتیکی در مواد گیاهی از اهمیت بالایی برخوردار بوده و گام اولیه برای شناسایی و نگهداری ذخایر ژنتیکی و از اصول اولیه اصلاح گیاهان محسوب می‌شود. استفاده از نشانگرهای DNA از بهترین روش‌های موجود برای بررسی تنوع ژنتیکی می‌باشند. در این بررسی از نشانگر ISSR به منظور بررسی تنوع ژنتیکی 31 اکوتیپ عناب جمع‌آوری شده از هشت استان عناب‌خیز کشور استفاده گردید. تعداد 13 آغازگر در واکنش PCR آزمایش شدند که شش آغازگر DNA‌ی الگو را بخوبی تکثیر و در بین نمونه‌ها چندشکلی را آشکار نمودند. از میان 84 مکان ژنی شناسایی شده، تعداد 70 مکان (83‌ درصد) چندشکلی نشان دادند. تجزیه خوشه‌ای اکوتیپ‌ها بر اساس ضریب تشابه دایس و به روش UPGMA انجام گرفت. در تجزیه خوشه‌ای، نمونه‌ها در سطح تشابه 85/0 در هفت گروه اصلی جای گرفتند. بیشترین تشابه ژنتیکی (95/0) بین نمونه‌های کلاله و درح و همچنین نوغاب و دوستیران و کمترین تشابه ژنتیکی (48/0) بین نمونه‌های برزادران و کنگان بدست آمد. آغازگرهای(AC)8YT و (GA)8Aمناسب‌ترین آغازگرها برای مطالعات بعدی تشخیص داده شد. نتایج این مطالعه نشان داد که تنوع کل نمونه‌های کشور تقریباً در تنوع ناحیه خراسان‌جنوبی خلاصه شده است. همچنین در اکوتیپ‌های متعلق به استان‌های اصفهان و مازندران، سطح قابل ملاحظه‌ای از تنوع مشاهده شد.

کلیدواژه‌ها

• تنوع ژنتیکی
• نشانگر مولکولی ISSR
• Zizyphus jujuba

20.1001.1.20084730.1396.31.3.17.3

عنوان مقاله [English]

Evaluation of Genetic Diversity in Collection from Iranian Jujube Ecotypes (Ziziphus spp.) using ISSR-molecular Marker

نویسندگان [English]

• Hajar Shayesteh ¹
• Saeid Malekzadeh Shafaroudi ²
• Kamal Ghous ³
• Farajollah Shahriari ⁴

¹ Ferdowsi University of Mashhad

² Ferdowsi University of Mashhad

³ Jahad-e-Keshavarzi Institute of Southern Khorasan

⁴ Ferdowsi university of Mashhad

چکیده [English]

Introduction Jujube (Zizyphus jujuba Mill.) as a valuable medicinal plant and adapted to different climatic conditions is widespread in many parts of Iran. Nowadays, beside the export of its fruit, jujube is also used as an herbal medicine to treat the diseases, so it has a high economic value. Study on genetic diversity is the first step to identify and preservation of germplasm. It is also considered as the basic principles of plant breeding. DNA markers seem to be the best way in determination of the genetic diversity. Inter simple sequence repeats (ISSR) markers are highly polymorphic and combine most benefits of Simple Sequence Repeats (SSRs) and amplified fragment length polymorphism (AFLP) to the generality of random amplified polymorphic DNA (RAPD).
Materials and Methods In order to study of the genetic diversity among 31 ecotypes collected from eight Jujube-rich provinces, including South Khorasan, Razavi Khorasan, Mazandaran, Golestan, Qom, Isfahan, Lorestan and Fars. Genomic DNA was extracted by CTAB method and polymerase chain reaction (PCR) was performed with 13 ISSR primers in which six most efficient primers were selected. Cluster analysis based on Dice similarity coefficient and Unweighed Pair Group Method with Arithmetic Mean (UPGMA) was carried out and POPGENe.3.2 software was used to determine the similarity of populations with each other.
Results and Discussion 84 loci were amplified and 70 of them (83%) revealed a proper polymorphism with the size between 200 and 2000 base pair. The average number of amplified and polymorphic bands per primer was 14 and 11.6 respectively. Primers with di-nucleotide repeats produced more polymorphic bands than ones with tri-nucleotide repeats. It seems that this is due to a higher frequency of sequences containing di-nucleotide repeats in intergenic regions and higher possibility of mutation revealed in more diversity in comparison to gene coding regions. Anchored primers with 1 or 2 nucleotides at the 5’ end make sure annealing only to the ends of SSRs in template DNA, so avoiding internal priming and smear formation. In addition, the anchor lets only a subset of the microsatellites to serve as priming sites. Primers (AC)8YT and (GA)8A with the higher percentage of polymorphism is recommended for further analysis. According to the cluster analysis, the ecotypes could be classified into seven main groups at the 0.85 level of genetic similarity. The most genetic similarity (0.95) was observed between ecotypes from Kalaleh and Doroh and also Noghab and Dustiran and the least genetic similarity (0.48) observed between Kangan and Borzaderan. POPGENe.3.2 software data indicated that populations of Isfahan and South Khorasan had the slightest difference while populations of Isfahan and Razavi Khorasan showed the most difference.
Conclusions In general results demonstrated that the total diversity of jujube ecotypes in Iran is summarized in the area of South Khorasan province. Given data showed that South Khorasan has been an original place of cultivation of this medicinal plant, this area could be considered as one of the important centers of jujube diversity. In addition, significant levels of diversity were observed among ecotypes belonging to Isfahan and Mazandaran provinces.

کلیدواژه‌ها [English]

• Genetic diversity
• ISSR marker
• Zizyphus jujuba

مراجع

1- Abbasi S., Malekzadeh Shafaroudi S., Ghooth K., and Shahriari F. 2012. Analysis of genetic diversity in Iranian Onnab ecotypes (Ziziphus spp.) using RAPD molecular Marker. Iranian Journal of Field Crops Research, 10(3):583-590. (in Persian)

2- Blair M.W., Panaud O., and McCouch S.R. 1999. Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L). Theoretical and Applied Genetics, 98: 780–792.

3- Fu J.X., Liu C.M., and Xie J.H. 2007. Identification and classification of ber cultivars based on ISSR and RAPD analysis. Acta horticulturae, 764:119-126.

4-Goulao L. and Oliveira C.M. 2001. Molecular characterisation of cultivars of apple (Malus x domestica Borkh.) using microsatellite (SSR and ISSR) markers. Euphytica, 122:81-89.

5- Ghouth K. 2010. Jujube Forgotten Fruit. Agricultural Organization of Southern Khorasan Province. (in Persian)

6- Ghouth K., and Malekzadeh Shafaroudi S. 2011. Identification Book of Iranian Collection of Jujube Ecotypes. Agricultural Organization of Southern Khorasan Province. (in Persian)

7- Khakdaman H., Pourmeidani A., and Adnani M. 2007. Study of genetic variation in Iranian Jujube (Zizyphus jujuba Mill.) ecotyps. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 14(4):202-214. (In Persian with English abstract)

8- Koohi dehkordi M., Rahimmalek M., Sayed Tabatabae B., Baninasab B., and Mobli M. 2006. Assessment of Genetic Relationship among some of the Iranian and Foreign Olive Cultivars using ISSR Markers. Journal of Horticultural Science and Technology, 7(2):93-102. (in Persian)

9- Kumar P., Gupta V.K., Misra A.K., Modi D. R., and Pandey B. K. 2009. Potential of Molecular Markers in Plant Biotechnology. Plant Omics Journal, 2:141-162.

10- Li J.D., Bi H.T., Li H.T., Li Z.S., and Fenga, J.C. 2009. Genetic Analysis of Ziziphus jujuba ‘Huizao’ Using ISSR Markers. Acta horticulturae, 840:135-142

11-Mondini L., Noorani A., and Mario Pagnotta A. 2009. Assessing plant genetic diversity by molecular tools. Diversity, 1:19-35.

12-Noroozi S., Baghizadeh A., and Javaran M. 2009. The genetic diversity of Iranian pistachio (Pistacia vera L.) cultivars revealed by ISSR markers. Biological Diversity and Conservation, 2(2):50-56

13-Nei M., and Li W.H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the Natural Academy of Science of the U.S.A, 76:5269-5273

14-Obeed R.S., Harhash A.L., and Abdel-Mawgood A.L. 2008. Fruit properties and genetic diversity of five ber (Ziziphus mauritiana Lam) cultivars. Pakistan Journal of Biological Science, 11:888-893

15- Okun D.O., Kenya E.U., Oballa P.O., Odee D.W., and Muluvi, G.M. 2008. Analysis of genetic diversity in Eucalyptus grandis (Hill ex Maiden) seed sources using inter simple sequence repeats (ISSR) molecular markers. African Journal of Biotechnolog, 7:2119–2123

16- Rao L.S., Usha-Rani P., Deshmukh P.S., Kumar P.A., and Panguluri S.K. 2007. RAPD and ISSR fingerprinting in cultivated chickpea (Cicer arietinum L.) and its wild progenitor Cicer reticulatum Ladizinsky. Genetic Resources and Crop Evolution, 54:1235–1244.

17- Reddy M.P., Sarla N., and Siddiq E.A.2002. Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica, 128:9–17.

18- Rohlf F.J. 1997. NTSYS-pc numerical taxonomy andmultivariate analysis system, version 2.0. Exeter Publications, N.Y.

19- Shahhoseini1 R., Babaei A., Kazemi M., and Omidbaigi R. 2012. A study on genetic variation in Iranian Jujube (Zizyphus jujuba Mill.) genotypes using molecular AFLP marker. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 20(1):55-68

20-Sing A.K., Devanshi L., Sharma P., Singh R., Singh B., Koundal K.R., and Singh N.K. 2007. Assessment of genetic diversity in Ziziphus mauritiana using inter-simple sequence repeat markers. Journal of Plant Biochemistry and Biotechnology, 16:35-40.

21-Singh A.K., Singh R., and Singh N.K. 2009. Comparative evaluation of genetic relationships among ber (Ziziphus sp.) genotypes using RAPD and ISSR markers. The Indian Journal of Genetics and Plant Breeding, 69(1): 106-118.

22-Yeh F.C., Yang R-C., Boyle T.B.J., Ye Z-H., and Mao J.X. 1999. POPGENE the User-Friendly Shareware for Population Genetic Analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada.

23- Zargari A. 1992. Medical Plants. First volume. University of Tehran Press. (in Persian)

24- Zeng J., Zou Y.P., Bai J.Y., and Zheng, H.S. 2002. Preparation of total DNA from recalcitrant plant taxa. Acta Botanica Sinica, 44:694-697.