ISSR(Inter-simple Sequence Repeat)-PCR(Polymerase Chain Reaction) molecular marker method for macromitrium gymnostomum

Abstract

The invention relates to an ISSR(Inter-simple Sequence Repeat)-PCR(Polymerase Chain Reaction) molecular marker method for macromitrium gymnostomum. The ISSR-PCR molecular marker method comprises the following steps of firstly, extracting genome DNA (Deoxyribose Nucleic Acid) of the macromitrium gymnostomum; secondly, performing ISSR-PCR amplification, wherein a reaction system for performing the ISSR-PCR amplification in step (2) is as follows: 30mu L reaction system contains 3mu L of 10*LAPCR (Polymerase Chain Reaction ) buffer solution, 50ng of template DNA, 1.5mmol / L of Mg<2+>, 0.25mmol / L of dNTP (Deoxyribonucleoside Triphosphate), 1.5U of Taq enzyme and 0.5mu mol / L of primer. A primer UBC(University of British Columbia) 834 is taken as an example, ISSR amplification comprises conditions of: annealing at the temperature of 94DEG C for 4 minutes, then annealing at the temperature of 94DEG C for 45 seconds, at the temperature of 53DEG C for 45 seconds and at the temperature of 72DEG C for 1 minute for 45 circles, and finally, prolonging the annealing time at the temperature of 72DEG C by 10 minutes. Compared with the prior art, the ISSR-PCR molecular marker method disclosed by the invention has the advantages that a strip amplified by an established ISSR-PCR reaction system for the macromitrium gymnostomum has the characteristics of high stability, high definition and high polymorphism; the shortages in the research on the genetic diversity of the macromitrium gymnostomum are made up; the achievements of the invention can be used for researching the genetic relationship, a molecular marker and biogeography, and has high scientific value and application value.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an ISSR-PCR molecular marker method for Moss dentata. Background technique [0002] The genus Macromitrium Bridel in the family Orthotrichaceae is a group with pantropical distribution. China has a very rich variety of plants in the genus Macromitrium gymnostomum. Macromitrium gymnostomum is one of them. It is called Macromitrium gymnostomum because the capsule has no teeth. The main feature of this species is that the main stem grows prostrate and densely red Brown rhizomes; branches erect, single or with several small short branches, up to 5.0mm. The middle cells of the leaves are transparent, oblong, thick-walled, and smooth; the upper cells are slightly opaque, round, with inconspicuous warts. Branches and leaves curl when dry, linear when wet, linear-lanceolate or ovate-lanceolate, sporangia erect, elliptic-cylindrical, shrunken, and capsule teeth missing. Capsule hood-sh...

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Problems solved by technology

ISSR molecular marker technology has been widely used in the analysis of genetic diversity of many angiosperms, but there are not many studies on bryophytes, only see Skotnicki's research on Cucumber moss, Liu Li et al. For the research on the genetic diversity of the species, the ISSR marker method is rarely used. In China, it can be found in Wang Chenying et al.'s research on the genetic diversity of Physcomitr angustifolia and Physaceae and Wang Yingying's research on the genetic diversity of Physcomitrella spp.

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