Welcome to International Network for Natural Sciences | INNSpub

Rapid seed DNA extraction for species identification and diversity analysis of Pumpkin

Research Paper | January 1, 2017

| Download 4

Arindam Barman, Anshumali, Rituparna Mitra Barman, Chinky M Marak

Key Words:

J. Bio. Env. Sci.10( 1), 161-168, January 2017


JBES 2017 [Generate Certificate]


Cucurbita moschata an economically important species of the family Cucurbitaceae, shows high variability in fruit characteristics. A standardized DNA isolation protocol has been developed from dried pumpkin seeds for polymerase chain reaction for species identification and diversity analysis. Higher concentration of polysaccharides and polyphenols in pumpkin seeds interferes with DNA during its isolation resulting in no PCR products. Good quality DNA, with no coloured pigments and contaminants was isolated from sun dried pumpkin seeds with modified CTAB buffer protocol without using liquid nitrogen. The average DNA concentration obtained was 63.9μg/gm with purity ranging between 1.66 to 1.85. The isolated DNA was successfully amplified using barcoding (rbcL), RAPD and SSR primers. The quantity and quality of the DNA isolated by this method was high enough to perform more than 150 PCR reactions. The species identify for Cucurbita moschata was also confirmed through sequencing and NCBI BLASTn analysis of bracoding primer (rbcL) product using isolated DNA. On the basis of UPGMA analysis, 14 pumpkin genotypes were categorized into two broad clusters. Broad cluster I and II comprised of one genotype (NEHUP8) and 13 independent genotypes respectively. The major cluster-I comprised of two genotypes viz. NEHUP1 and NEHUP14 with a genetic similarity percent of 0.58 approximately. The major cluster-II was sub divided into three minor clusters. This modified DNA isolation protocol may be adequate for isolating high-molecular weight DNA from other cucurbitaceous species con­taining large amounts of secondary metabolites.


Copyright © 2017
By Authors and International Network for
Natural Sciences (INNSPUB)
This article is published under the terms of the Creative
Commons Attribution Liscense 4.0

Rapid seed DNA extraction for species identification and diversity analysis of Pumpkin

Csaikl UM, Bastian M, Brettschneider R, Gauch S, Meir A, Schauerte M, Scholz F, Sperisen C, Vornam B, Ziegenhagen B. 1998. Comparative analysis of different DNA extraction protocols: A fast universal maxi preparation of high quality plant DNA for genetic evaluation and phylogenetic studies. Plant Molecular Biology Reporter 16(1), 69-86. DOI: 10.1023/A:1007428009556.

Dawson MN, RaskoffK A, Jacobs DK. 1998.Field preservation of marine invertebrate tissue for DNA analyses. Molecular Marine Biology and Biotechnology 7, 145- 152.

Dellaporta SL, Wood J, Hicks JB. 1983. A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter 1, 19-21. DOI: 10.1007/BF02712670.

Deshmukh VP, Thakare PV, Chaudhari US, Gawande PA. 2007. A simple method for isolation of genomic DNA from fresh and dry leaves of Terminalia arjuna (Roxb.). Electronic Journal of Biotechnology 10, 468-472. DOI: 10.2225/vol10-issue3-fulltext-5.

Dillon D, Wu WI, Riedel B, Wissing JB, Dowhan W, Carman GM. 1996. The Escherichia coli pgp B gene encodes for a diacylglycerol pyrophosphate phosphatase activity. Journal of Biological Chemistry 271, 30548-30553. DOI: 10.1074/jbc.271.48.30548.

Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13-15.

Fang G, Hammer S, Grumet R. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques 13(1), 52-54.

Hebert PDCywinska ABall SL, De Waard JR. 2003. Biological identifications through DNA barcodes. Proceedings of Biological Sciences 270, 313-321. DOI: 10.1098/rspb.2002.2218.

Hills PN, Van Staden J. 2002. An improved DNA extraction procedure for plant tissues with a high phenolic content. South African Journalof Botany 68, 549-550. DOI: 10.1016/S0254-6299(15)30384-7

Katterman FR, Shattuck VI. 1983. An effective method of DNA isolation from the mature leaves of Gossypium species that contain large amounts of phenolic terpenoids and tannins. Preparative Biochemistry and Biotechnology 13, 347-359. DOI: 10.1080/00327488308068177

Keb Llanes M, Gonzalez G, Chimanzanero B, Infante D. 2002. A rapid and simple method for small scale DNA extraction in Agavaceae and other tropical plants. Plant Molecular Biology Reporter 20(3), 299a-299e. DOI: 10.1007/BF02782465.

Khanuja SPS, Shasany AK, Darokar MP, Kumar S. 1999. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oil. Plant Molecular Biology Reporter 17, 1-7. DOI: 10.1023/ A:1007528101452.

Kumar P, Gupta K, Misra K, Modi R, Pandey K. 2009. Potential of molecular markers in plant biotechnology. Plant Omics Journal 2(4), 141-162.

Loomis WD. 1974. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods in Enzymology 31, 528-545.

Moyo M, Amoo OS, Bairu MW, Finnie JF, Van Staden J. 2008. Optimizing DNA isolation for medicinal plants. South African Journal of Botany 74, 771-775. DOI: 10.1016/j.sajb.2008.07.001.

Mtambo J, Van Bortel W, Madder M, Roelants P, Backeljau T. 2006. Comparison of preservation methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for reliable DNA amplification by PCR. Experimental and Applied Acarology 38, 189-199. DOI: 10.1007/s10493-006-0004-4.

Salgado A, Vieiralves T, Flavia RM, Lamara, Leonardo LM, Assumpçao, Gomes D, Jascone L, Valadao AL, Albano RM, Lobo-Hajdu G. 2007. Field preservation and optimization of a DNA extraction method for Porifera. Porifera Research: Biodiversity, Innovation and Sustainability 22, 555-560.

Sarwat M, Negi MS, Lakshmikumaran M, Tyagi AK. 2006. A standardized protocol for genomic DNA isolation from Terminalia arjuna for genetic diversity analysis. Electronic Journal of Biotechnology 9, 86-91. DOI: 10.2225/vol9-issue1-fulltext-3.

Schander C, Halanych MK. 2003. DNA, PCR and formalin zed animal tissue a short review and protocols. Organisms Diversity and Evolution 3(3),195-205. DOI: 10.1078/1439-6092-00071

Schneerman MC, Mwangi J, Hobart B, Arbuckle J. 2002. The dried corncob as a source of DNA for PCR analysis. Plant Molecular Biology Reporter 20, 59-65.

ScottK D, Playford J. 1996. DNA extraction technique for PCR in rain forest plant species. Biotechniques 20(6), 974-978.

Shah MM, Yen Y, GillK S, Baenziger PS. 2000. Comparisons of RFLP and PCR-based markers to detect polymorphism between wheat cultivars. Euphytica 114,135-142. DOI: 10.1023/A:1003993930447.

Sharma AD, Gill PK, Singh P. 2002. DNA isolation from dry and fresh samples of polysaccharide-rich plants. Plant Molecular Biology Reporter 20(4), 415-415. DOI: 10.1007/BF02772129.

Suman PSK, AjitK S, Darokar MP, Kumar S. 1999. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Molecular Biology Reporter 17, 1-7. DOI: 10.1023/A:1007528101452.

Warude D, Chavan P, Joshi K, Patwardhan B. 2003. DNA isolation from fresh, dry plant samples with highly acidic tissue extracts. Plant Molecular Biology Reporter 21, 467. DOI: 10.1007/BF02772600.