基于生物芯片标记技术的五种濒危冷杉保护遗传学研究
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摘要
冷杉属(Abies)是松科中的第二大属,全世界约有50种,是组成寒温性暗针叶林和亚高山暗针叶林的建群树种,它们组成了高山地区独特的森林景观,在涵养水源、保护河川上游水资源方面起着重要作用,是各分布区高山造林和森林更新的重要树种;由于该属树种耐寒、耐荫性强,喜气候凉润,主要分布于北半球的高海拔或高纬度地区,在我国的东南部很难见到。1976年以来相继在我国浙江、广西、湖南、贵州和江西五省区发现5种冷杉新种,百山祖冷杉(A.besganzuensis)(吴鸣翔定名发表)、元宝山冷杉(A.yuanpaoshanensis)(吕庸浚和傅立国定名发表)、资源冷杉(A.ziyuanensis)(傅立国和莫新礼定名发表)、梵净山冷杉(A.fanjinshanensis)(黄威廉定名发表)和大院冷杉(A.dayuanensis)(刘起衔定名发表)。这5种冷杉植物的发现否定了以往亚热带的低、中山无冷杉分布的观点,被认为是植物地理学和植物学上的一大发现。这几种冷杉野生群体数量稀少,全部被列为国家一级重点保护植物。目前这几种冷杉分类存在争议,保护策略缺乏科学依据,而且部分材料野生植株极其稀少,如百山祖冷杉野生群体只剩3株。在样本很少情况下,要开展物种分类和保护遗传学研究只能寻求能大幅度提高检测位点数的方法才能提高实验可信度。生物芯片技术具有高通量、高效率的特点,可以达到此目的。本论文利用生物芯片技术的手段,开展了遗传标记生物芯片的制作与检测研究,并将该技术应用于5种濒危冷杉的保护遗传学研究,分析了5种冷杉的分子亲源关系和群体遗传分化,探讨了它们的濒危原因和保护策略,并对冷杉的水培繁殖作了研究,主要结论如下:
1生物芯片技术对模板DNA的质量要求较高,而冷杉叶片中含有大量的多糖、蛋白质、多酚和色素类等次生代谢物质。本试验采用改良的CTAB法提取了冷杉基因组DNA,有效地去除了各种影响实验结果的杂质。检测结果表明,提取的DNA样品的D260 nm/D280 nm比值都为1.9左右,均为白色絮状沉淀,溶液清亮,无黏稠感,纯度较好。在芯片工作缓冲液中检测表明,提取的DNA样品孔均不发亮,且DNA主带清晰,无弥散带,能适应生物芯片的工作缓冲液环境,完全满足生物芯片技术对DNA质量的要求。
2实验以小麦、云杉、为对照,研究了冷杉基因组DNA复杂性,将三种材料基因组DNA酶切后,采用不同的选择性碱基的引物扩增。结果表明冷杉基因组DNA复杂性高于小麦和云杉,提示冷杉基因组DNA复杂性降低须采取更有效措施;采用PstⅠ和TaqⅠ两种酶切组合得到的片段数过多,提示应采用更多种的酶切组合或选择性碱基引物扩增可降低冷杉基因组DNA复杂性;冷杉基因组含C、G碱基数可能多于含A、T碱基数,提示应选用相对富含CG多的限制性内切酶可能有利于酶切效果。
3实验采用7种不同的限制性内切酶组合酶切冷杉基因组DNA后,采用有无选择性碱基的PstⅠ引物扩增酶切片段,探讨适合冷杉基因组DNA复杂性降低的方法。结果表明酶切组合PstⅠ+TaqⅠ+MseⅠ酶切片段在无选择性碱基引物扩增时片段分布在750bp左右,分子量比较大的片段相对较少,分布比较均匀集中,从该酶切组合的+3碱基引物扩增时显示没有明显的条带,说明该酶切组合效果最好;酶切组合PstⅠ+BstNⅠ、PstⅠ+TaqⅠ效果次之,其它几种酶切组合不甚理想。
4实验采用PstⅠ+TaqⅠ+MseⅠ酶切组合酶切冷杉基因组DNA样品构建基因组DNA文库,并用所得文库点制冷杉基因组DNA芯片,再用同样酶切方法进行样品处理后与芯片杂交,检测各芯片质量参数。检测结果表明芯片样点基本比率、中值比率和均数比率相关系数均大于0.9,参考通道信噪比为79.017-92.024,杂交后检测通道信噪比为20.786-32.827,说明采用该方法得到的芯片质量比较好,可以用于构建冷杉基因组DNA芯片。
5实验利用6种冷杉的基因组DNA为建库材料,研究了冷杉基因组DNA芯片的制作方法,芯片质量参数和有效标记参数的选取,以及不同文库构建方法和不同样品处理方法对有效标记得率的影响,研究结果表明:综合分析后认为实验应选取重复性100%,召回率大于85%标记点比较合适,此时得到的平均有效标记得率为18%左右,不同方法制作的芯片文库具有一定通用性,有效标记得率具有一定偏好性,芯片文库构建方法和样品处理方法相同时有效标记产率最高。
6实验采用PstⅠ、TaqⅠ和MseⅠ3种限制性内切酶混合酶切5种冷杉基因组DNA,并用酶切后的DNA片段制作遗传多样性检测的芯片检测并分析了5钟冷杉群体内遗传多样性,分析的遗传多样性参数有多态位点百分率、观察等位基因数、有效等位基因数、Nei基因杂合度、Shannon多样性指数等,并对结果做了差异显著性分析和多重比较,结果显示,5种冷杉的遗传多样性排序顺序为:资源冷杉﹥元宝山冷杉﹥梵净山冷杉﹥大院冷杉﹥百山祖冷杉5种冷杉,资源冷杉和元宝山冷杉差异不显著,大院冷杉和百山祖冷杉差异不显著,元宝山冷杉和梵净山冷杉差异不显著,其它两两间差异极显著。(具体参数值见第7章列表)
7实验采用PstⅠ、TaqⅠ和MseⅠ3种限制性内切酶混合酶切5种冷杉基因组DNA,并用酶切后的DNA片段制作遗传多样性检测的芯片检测并分析了5钟冷杉群体间遗传分化,并计算了群体间遗传距离,利用遗传距离估算了群体分化时间,结果显示,5种冷杉群体209个位点平均的总基因多样度Ht=0.2608±0.0004,平均的群体内基因多样度Hs=0.1653±0.0004;群体间基因分化系数Gst=0.3664;群体间基因流Nm=0.8647。5种冷杉中资源冷杉、元宝山冷杉、大院冷杉三种冷杉间亲源关系最接近,这三种冷杉和百山祖冷杉关系比较接近,梵净山冷杉与以上4种冷杉关系最远。资源冷杉、元宝山冷杉、大院冷杉相互间分化时间最短,在4.5千年-9.9千年之间。分化时间最长的是梵净山冷杉和百山祖冷杉之间,为54千年。
8试验采用了水培方式研究了在水培条件下生根调节剂和营养液对冷杉水培生根过程中愈伤诱导、生根率、最长根长、平均根长等参数的影响。结果表明生根调节剂(ABT和IAA)对诱导百山祖冷杉水培愈伤诱导、生根率、生根数有明显作用,两者都适合在百山祖冷杉水培繁殖中应用。但激素处理对最长根长和平均根长无规律性影响。营养处理对百山祖冷杉水培繁殖前期各项均无影响,但在出根后会影响根的生长。冷杉水培试验研究冷杉繁育提供了新的思路,有望使冷杉脱离濒危现状,使该物种作为水培盆景进入寻常百姓的生活,对于物种多样性保护,具有重要的意义。
9末次冰期以来气候变暖使南方冷杉分布区极度缩小是冷杉濒危的直接原因;人为砍伐和对冷杉生境的破坏是冷杉濒危的重要原因;工业废气对环境的污染间接影响冷杉生境;冷杉的遗传多样性降低,存在近亲交配,导致冷杉适应性降低是冷杉濒危的遗传学原因。必须要制定有力的保护政策,加强管理;可采取就地保护和迁地保存措施,并进行人工繁育,归化自然。
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.
1生物芯片技术对模板DNA的质量要求较高,而冷杉叶片中含有大量的多糖、蛋白质、多酚和色素类等次生代谢物质。本试验采用改良的CTAB法提取了冷杉基因组DNA,有效地去除了各种影响实验结果的杂质。检测结果表明,提取的DNA样品的D260 nm/D280 nm比值都为1.9左右,均为白色絮状沉淀,溶液清亮,无黏稠感,纯度较好。在芯片工作缓冲液中检测表明,提取的DNA样品孔均不发亮,且DNA主带清晰,无弥散带,能适应生物芯片的工作缓冲液环境,完全满足生物芯片技术对DNA质量的要求。
2实验以小麦、云杉、为对照,研究了冷杉基因组DNA复杂性,将三种材料基因组DNA酶切后,采用不同的选择性碱基的引物扩增。结果表明冷杉基因组DNA复杂性高于小麦和云杉,提示冷杉基因组DNA复杂性降低须采取更有效措施;采用PstⅠ和TaqⅠ两种酶切组合得到的片段数过多,提示应采用更多种的酶切组合或选择性碱基引物扩增可降低冷杉基因组DNA复杂性;冷杉基因组含C、G碱基数可能多于含A、T碱基数,提示应选用相对富含CG多的限制性内切酶可能有利于酶切效果。
3实验采用7种不同的限制性内切酶组合酶切冷杉基因组DNA后,采用有无选择性碱基的PstⅠ引物扩增酶切片段,探讨适合冷杉基因组DNA复杂性降低的方法。结果表明酶切组合PstⅠ+TaqⅠ+MseⅠ酶切片段在无选择性碱基引物扩增时片段分布在750bp左右,分子量比较大的片段相对较少,分布比较均匀集中,从该酶切组合的+3碱基引物扩增时显示没有明显的条带,说明该酶切组合效果最好;酶切组合PstⅠ+BstNⅠ、PstⅠ+TaqⅠ效果次之,其它几种酶切组合不甚理想。
4实验采用PstⅠ+TaqⅠ+MseⅠ酶切组合酶切冷杉基因组DNA样品构建基因组DNA文库,并用所得文库点制冷杉基因组DNA芯片,再用同样酶切方法进行样品处理后与芯片杂交,检测各芯片质量参数。检测结果表明芯片样点基本比率、中值比率和均数比率相关系数均大于0.9,参考通道信噪比为79.017-92.024,杂交后检测通道信噪比为20.786-32.827,说明采用该方法得到的芯片质量比较好,可以用于构建冷杉基因组DNA芯片。
5实验利用6种冷杉的基因组DNA为建库材料,研究了冷杉基因组DNA芯片的制作方法,芯片质量参数和有效标记参数的选取,以及不同文库构建方法和不同样品处理方法对有效标记得率的影响,研究结果表明:综合分析后认为实验应选取重复性100%,召回率大于85%标记点比较合适,此时得到的平均有效标记得率为18%左右,不同方法制作的芯片文库具有一定通用性,有效标记得率具有一定偏好性,芯片文库构建方法和样品处理方法相同时有效标记产率最高。
6实验采用PstⅠ、TaqⅠ和MseⅠ3种限制性内切酶混合酶切5种冷杉基因组DNA,并用酶切后的DNA片段制作遗传多样性检测的芯片检测并分析了5钟冷杉群体内遗传多样性,分析的遗传多样性参数有多态位点百分率、观察等位基因数、有效等位基因数、Nei基因杂合度、Shannon多样性指数等,并对结果做了差异显著性分析和多重比较,结果显示,5种冷杉的遗传多样性排序顺序为:资源冷杉﹥元宝山冷杉﹥梵净山冷杉﹥大院冷杉﹥百山祖冷杉5种冷杉,资源冷杉和元宝山冷杉差异不显著,大院冷杉和百山祖冷杉差异不显著,元宝山冷杉和梵净山冷杉差异不显著,其它两两间差异极显著。(具体参数值见第7章列表)
7实验采用PstⅠ、TaqⅠ和MseⅠ3种限制性内切酶混合酶切5种冷杉基因组DNA,并用酶切后的DNA片段制作遗传多样性检测的芯片检测并分析了5钟冷杉群体间遗传分化,并计算了群体间遗传距离,利用遗传距离估算了群体分化时间,结果显示,5种冷杉群体209个位点平均的总基因多样度Ht=0.2608±0.0004,平均的群体内基因多样度Hs=0.1653±0.0004;群体间基因分化系数Gst=0.3664;群体间基因流Nm=0.8647。5种冷杉中资源冷杉、元宝山冷杉、大院冷杉三种冷杉间亲源关系最接近,这三种冷杉和百山祖冷杉关系比较接近,梵净山冷杉与以上4种冷杉关系最远。资源冷杉、元宝山冷杉、大院冷杉相互间分化时间最短,在4.5千年-9.9千年之间。分化时间最长的是梵净山冷杉和百山祖冷杉之间,为54千年。
8试验采用了水培方式研究了在水培条件下生根调节剂和营养液对冷杉水培生根过程中愈伤诱导、生根率、最长根长、平均根长等参数的影响。结果表明生根调节剂(ABT和IAA)对诱导百山祖冷杉水培愈伤诱导、生根率、生根数有明显作用,两者都适合在百山祖冷杉水培繁殖中应用。但激素处理对最长根长和平均根长无规律性影响。营养处理对百山祖冷杉水培繁殖前期各项均无影响,但在出根后会影响根的生长。冷杉水培试验研究冷杉繁育提供了新的思路,有望使冷杉脱离濒危现状,使该物种作为水培盆景进入寻常百姓的生活,对于物种多样性保护,具有重要的意义。
9末次冰期以来气候变暖使南方冷杉分布区极度缩小是冷杉濒危的直接原因;人为砍伐和对冷杉生境的破坏是冷杉濒危的重要原因;工业废气对环境的污染间接影响冷杉生境;冷杉的遗传多样性降低,存在近亲交配,导致冷杉适应性降低是冷杉濒危的遗传学原因。必须要制定有力的保护政策,加强管理;可采取就地保护和迁地保存措施,并进行人工繁育,归化自然。
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.
引文
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