DNA Extraction，Optimization of SSR-PCR Reaction System and Primer Screening of Persea americana

doi:10.13560/j.cnki.biotech.bull.1985.2016.04.019

Abstract

Abstract: This study is to establish a stable and reliable DNA extraction method，optimize the SSR-PCR reaction system，and screen the stable polymorphism primers for avocado（Persea americana Mill）SSR for providing the genetic foundation to conduct the SSR molecular marker of germplasm in avocados. Taking avocado’ leaves as the study material，3 avocado DNA extraction methods were compared，based on the L16（45）orthogonal experiment design，the SSR-PCR reaction system in avocados was optimized，and then by optimized reaction system the SSR primers were screened. To further test the stability of the optimized SSR-PCR system，the germplasms in 45 pieces of avocados were amplified by PCR using 5 pairs of polymorphism primers. The results showed that：among 3 DNA extraction methods of conventional 2×CTAB method，improved 2×CTAB method，and plant DNA kit method，the improved 2×CTAB method was the best regarding the extraction effect of avocado genomic DNA. The optimal SSR-PCR reaction system in avocados was：a total volume of 20 μL containing 40 ng of genomic DNA，1.5 mmol/L Mg2+，0.15 mmol/L dNTPs，0.5 U Taq DNA polymerase，0.5 μmol/L primer. Based on the above optimized reaction system，30 pairs of polymorphism primers with clear bands were screened from 73 SSR primers of avocados，indicating that the reaction system can be used for the further study of SSR markers in avocados. The bands of stability test were clear，showing that the optimized system was stable and reliable. Thus，improved 2×CTAB method can be used in DNA extraction of plentiful samples，and the optimized SSR-PCR reaction system and the 30 polymorphism primers can be utilized for the further study of SSR markers in avocados.

Key words: Persea americana Mill, DNA extraction, SSR marker, system optimization, primer screening

ZHOU Hai-lan, LI Shao-peng, LI Wei-liang, HE Jun-hu ,BAO Dong-hong, LI Mao-fu. DNA Extraction，Optimization of SSR-PCR Reaction System and Primer Screening of Persea americana[J]. Biotechnology Bulletin, 2016, 32(4): 143-150.

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