Study On ISSR Identification Technology Of Species,Clones And Hybrid Families Of Larch

The thesis takes the larch which is the main plants for afforestation and timber of northern area of China as the study object,use the early needles of the clones and controlled pollination of Larix gmelinii,Larix Olgensis and Larix Kaempferi which are in the seed orchard,bank and progeny test plantation progenies of Heilongjiang province Qingshan forestry centre as the experiment material.It identifies different species,clones,families such as hybrid progenies and parents,and different hybrid kinds by means of ISSR molecular techniques.The specific bands of different species and hybrid are reclaimed and sequenced,and then transformed into SCAR primers.It provides a new molecular marker system to identify the larch species,and it would establish the foundation for developing the heterosis mechanism research of larch.1.49 ISSR primers are selected from 488 primers to amplify all samples.2 primers, ISSR12 and ISSR32,are obtained to identify Larix Kaempfer;1 primer ISSR18 is obtained to identify Larix gmelinii;2 primers,ISSR38 and ISSR41,are obtained to identify Larix Olgensis. There is a primer ISSR16 used for identifing L.gmelinii×L,olgensis;a primer ISSR19 can identify L.Kaempferi×L.olgensis.2.There are 22 clones that can be identified at one time through different bands amplified by 9 primers,and the others can be distinguished by discrepant bands amplified by several primers.All clones can be divided into 3 kinds,they are Larix Kaempferi,Larix Olgensis and Larix gmelinii.3.The research identifies larch hybrid families by means of distinguishing hybrid progenies and parents,positive and negative families,and different families of the same parent with ISSR molecular marker.The percentage of the band by which hybrid progenies are different from the parents is no less than 90%;positive and negative families can be identified in terms of the band appearance proportion,and the band in which the positive and negative families are different from each other accounts for no less than 80%;when a parent is given, the possibility of another can be calculated by means of increasing the appearing frequency of the given parent.All the families can be divided into 4 kinds of hybrid,L.kaempferi×L. gmelinii,L.kaempferi×L,olgensis,L.gmelinii×L,olgensis,L.gmelinii×L,kaempferi at 0.4 genetic distance.4.Specific bands amplified by 7 ISSR primers are reclaimed,cloned and then sequenced. According to the result,SCAR primers are designed based on the DNA sequences.After confirmation,there are 3 pairs of SCAR primers that can be applied to identification,and they are SC18s/SC18a,SC16s/SC16a,SC19s/SC19a.This research provides an effective and reliable method for identification of larch.