Study On Conservation Genetics Of Five Endangered Abies Species By Marker Technology Of Biochip

Abies including almost 50 species, the second biggest genus of Pinaceae, are main species in forming frigid-temperate zone coniferous forest vegetation and alpine and subalpine coniferous forest vegetation. Abies form unique alpine forest scenic spots, and it can conserve source of water and be planted as main woody trees in alpine zone. Because Abies have the specification of tolerating cold weather, shade and suiting cold-moist climate, it maily distributed at high latitudes and altitude, and be found very few in south of China before 1970s. However some firs were found since 1976, such as A.beshanzuensis was found in Zhejiang, A.yuanpaoshanensis and A.ziyuanensis were found in Guangxi, A.fanjinshanensis was found in Guizhou, A.dayuanensis was found in Hunan. This significant finding denied the opinion that there were no Abies species in alpine and subalpine mountain in subtropical zone. All of the five firs are listed in national protected plant at levelⅠbecause of very few in wild. Little research about these firs were conducted, and there are some debates in classification among these firs, and protecting strategy does not have good foundation, moreover some of them are very few, such as A.beshanzuensis only have 3 trees in wild. 3 samples are too few for analysizing its genetic diversity in normal experiment of electrophoresis on the gel. Looking for new analysing methods which can promote the number of the detecting loci at a large scale was a need for offsetting the deficiency of samples for such endangered trees. Biochip technology has the specification of high throughout and efficiency, and it is a good method for detecting the loci.The biochip technology was used in this paper for detecting the genetic diversity of these firs, analysing the relationship and assaying the genetic differentiation among five firs. The cause resulting in endangered situation were discussed and the trials how to propagate the materials by water media was conducted and analysed in this paper. The main conclusion followed as below.1 The biochip technology had rigorously restricted for the quality of DNA, however there are lots of proteins, polysaccharides, pigments and phenol constituents. A modified method based on CTAB was used for extracting the genomic DNA of firs in this experiment, and a good result was got by this method. The result show that the value of DNA D260 nm/D280 nm was 1.9, the DNA was white woolly precipitation and was not sticky after resolving. The detecting well were not light, and the main band were clear when the sample of DNA were electrophoresed on the gel.2 The complicacy of DNA of firs was analyzed through comparing with barely and spruce after using enzyme to cut DNA of firs. The results show the DNA of firs was more complicate than barely and spruce, and the quantity of fragment cutting by PstⅠand TaqⅠwere too much, that indicated more enzyme should be used in decreasing the complicacy; There were more C,G than A,T in the genomic DNA of firs, that indicated the enzyme containing more C,G base should be used.3 The method of decreasing complicacy of DNA were conducted that cutting by seven combined enzyme and then amplifying by primers, the result showed that the band of amplification after cutting by PstⅠ+TaqⅠ+MseⅠwere almost 750 bp, the bands over 1500bp were few, that indicated this combined enzyme was the best, the combination of PstⅠ+BstNⅠ, PstⅠ+TaqⅠwere not good as the former .4 The library was created by PstⅠ+TaqⅠ+MseⅠ, and the microarray was spotted by that library, the quality of microarray was detected after hybridization. The result showed the correlation among the parameters of based ratio, media ratio and average media were over 0.9, the sig-to-noise was 79.017-92.024 in reference channel, 20.786-32.827 in detecting channel, all of these parameters showed the chip was good for detecting the diversity.5 The method how to make microarray, how to chose the parameters, and the impacts of different library and treatment for the productivity were researched in this paper, the results showed the suitable parameters should be adopted such as the reproducibility at 100%,call rate at 85%, the productivity was almost 18%, and the library had universal property, the productive rates of effective marks had preferable property, the productive rates of effective marks was the most if library and the samples had the same way of treatment.6 The genetic diversity of five firs within population were analyzed by PstⅠ+TaqⅠ+MseⅠaccording to the parameters such as P, the Index of Nei gene, the Index of Shannon and Frequency of alleles, the result showed that the order of genetic diversity within five firs were A.ziyuanensis, A.yuanpaoshanensis, A.fanjinshanensis, A.dayuanensis, A.beshanzuensis, from high to low.7 The genetic diversity across five firs , the average genetic diversity within firs ,the differential coefficient and the gene flow were 0.2608±0.0004, 0.1653±0.0004, 0.3664, 0.8647,respectively.The relationship among A.ziyuanensis, A.yuanpaoshanensis and A.dayuanensis were closer, the differential time among these three firs were from 4.5-9.9 thousand years. The differential time was the longest between A.fanjinshanensis, and A.beshanzuensis, it was almost 54 thousand years.8 The influences of phytohormones (ABT and IAA) and nutrient solution on rooting of Abies by water cultured medium was conducted. The Abies were treated by water (CK), 10mg/L ABT+ water, 10mg/L IAA+ water, 10mg/L ABT+ hoagland solution, 10mg/L IAA+ hoagland solution, then the rooting process was observed and the formation rate of callus, rooting rate, number of rooting, and root length were investigated and analyzed. The results showed ABT and IAA had obvious influences on callus induction, rooting rate and the number of root of Abies by water culture, so they were suitable to be used in water propagation of Abies.The treatments of phytohormones had no regular influences on the longest root length and average root length. The nutrient solutions would not generate obvious influence on propagation of Abies at firstly stage, but they generated influence on root growth after rooting. The research provided new ideas for propagation of Abies ,which could make it out of endangered situation quickly.9 That the climate became warm after last glacier was the direct cause which made those firs endangered, cutting wood by human people and destroying environment also accelerated the endangered situation, emission of waste gas of industry also impacted the habitat of firs. Decreasing of the genetic diversity and inbreeding within population was the genetic cause. A powerful strategy should be made to protect these firs and the management should be payed more. The methods of on-site conservation and off-site preservation should be adopted, moreover, propagation and returning to wild habitat by artificial methods also may benefit the wild trees.