獐ISSR-PCR反应体系的建立与优化及引物筛选

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Abstract

Five factors of amplification system,such as concentration of Mg²⁺,dNTPs,primers,Taq DNA polymerase and DNA template were investigated to optimize the ISSR-PCR reaction system of H.inermis through orthogonal tests.The data were analyzed by software DPS.As a result,a satisfactory ISSR reaction system for H.inermis with desirable repeatability and polymorphic bands was established.The optimized ISSR-PCR system of H.inermis included 2.5 mmol·L^-1Mg²⁺,1.25 U·25μL^-1 Taq DNA polymerase,0.3μmol·L^-1 primer,350 ng·25μL^-1 DNA template and 0.15 mmol·L^-1 dNTPs in 25μL PCR reaction system.Ten primers with stable amplification and rich polymorphism for ISSR were screened using this reaction system and its optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR.This optimized ISSR-PCR reaction system would provide the basis for the analysis of genetic diversity,phylogenetic analysis,physiological or pathological events and genetic variation of important traits of H.inermis.

Key words： Chinese water deer(H.inermis) orthogonal tests ISSR primer screening

Received: 25 August 2009

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	Q959.842

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	Articles by authors
	ZHANG Long-long
	BAO Yi-xin
	YU Hua-hui
	XU Jing
	SUN Bo

Cite this article:

ZHANG Long-long,BAO Yi-xin,YU Hua-hui, et al. Establishment and optimization of ISSR-PCR reaction system and primer screening for Chinese water deer(Hydropotes inermis)[J]. , 2009, 28(4): 335-341.

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http://www.ecolsci.com/EN/ OR http://www.ecolsci.com/EN/Y2009/V28/I4/335

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