【作者】 哀建国；

【导师】 傅懋毅；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2010， 博士

【摘要】 本研究针对广泛分布于我国的蕨类植物里白(Hicriopteris glauca)的遗传资源、生理生态特性和繁殖技术开展一系列研究。采用trnG-S引物片段对分布于全国十个省区(安徽、福建、广东、广西、贵州、湖北、江西、四川、云南、浙江)毛竹(Phyllostachys edulis)林中12个野生群体的124个里白个体的cpDNA进行扩增,通过对变异位点数(P)、单倍型多态性(h)、核苷酸多态性(π)等遗传特性的分析,研究其遗传多样性状况。在此基础上,通过对浙江大溪里白群体生境为期1年的监测,揭示了里白生境中诸如温度、湿度、光照等生态因子的变化状况;并对大溪里白生境开展群落学调查和多样性研究;对其土壤和植株中所含水解性氮、速效钾、有效磷等大量元素以及微量元素的含量变化进行相关性分析,探讨了里白中矿质元素含量年动态变化规律;开展了里白在干旱胁迫条件下的光合作用响应规律研究;对在不同基质配比、不同光、温调控水平以及不同激素水平下里白孢子萌发、配子体增殖和孢子体诱导进行了研究。本研究旨在为蕨类植物里白的遗传资源评价、引种繁育以及在竹林复合经营中栽培利用等提供理论依据和科学支撑。通过以上研究,得到以下主要研究结果:(1)里白物种水平的单倍型多态性(h)为0.453,核苷酸多态性(Pi)为0.00067。西部群体的平均水平的核苷酸多态性(π= 0.000292)与单倍型多态性(h = 0.2)比中东部群体要高得多(π= 0.000145; h = 0.067)。群体内单倍型多样性(hS)和核酸多样性(πS)在里白群体间变化很大,群体内单倍型多样性(hS)平均值为0.127±0.231,核酸多样性πS (×10-3)的平均值为0.212±0.364。单倍型多样性(hS)和核酸多样性(πS)与海拔和经纬度没有相关性。AMOVA分析结果表明:在物种水平上不存在明显的谱系地理结构;地区间的变异为2.97%,群体间的变异为64.54%,群体内的差异为32.49%,地区间的差异不显著,变异主要出现在群体间;群体间的遗传距离和地理距离之间没有显著相关性。研究发现,里白具有四川龙溪(虹口)与湖北交接处、云南大围山以及黄山山脉一带3个遗传多样性分布中心,这3个分布中心涵盖了所测的所有3种单倍型,而其它地区的群体均仅含1种广布单倍型(Hap1)。(2)浙江大溪里白生境年平均气温20.5℃,年平均相对湿度65.5%,年平均光照强度5800lx,年平均土壤含水量19.4%,适宜植株生长。经调查,该里白生境毛竹林内共有维管植物75种,隶属43科,64属;其物种多样性指数较高,Simpson指数平均值为0.778,Shannon-wiener指数平均值为2.258,种间相遇机率平均值为0.783,群落均匀度平均值为0.783。灌木层属次生的常绿、落叶阔叶混交林类型。(3)浙江大溪里白生境土壤中氮、磷、钾、钙、镁、锰等大量营养元素含量在里白生长高峰期达到峰值;除锰元素外,里白中硫、钙、镁、铁、硼、锌、铜、钼、钠等元素含量均在里白生长旺季出现吸收高峰,随后下降,其变化规律基本相似。里白植株中氮和磷、氮和钾、磷和钾、钾和铁、钙和钼、钠和氮、钠和铁等矿质元素含量间相关性显著,氮和镁、氮和铁、镁和铁、钠和镁之间相关性极显著,且里白植株中氮、磷、钾等三种大量元素和土壤中变化规律一致。(4)不同土壤水分条件下,里白的Ci基本不变,表明干旱胁迫下气孔导度的降低不是引起Pn下降的主要原因;土壤相对含水量为80～60%时,不会引起Fv/Fm、Fv′/F′m、qP、ФPSⅡ和ETR的明显改变,在60%以下时,Fv/Fm、Fv′/F′m、qP、ФPSⅡ和ETR等参数明显下降,PSII的线性电子传递速率降低;干旱胁迫下用于PSII天线色素非光化学能量耗散的光能显著升高,对光合机构起到一定的保护作用;随着干旱胁迫的加重,发生膜脂过氧化现象。(5)里白孢子繁育的最佳光强为40μmol·m-2·s-1,配子体发育为孢子体的发育率达48.4%;基质以泥炭土-黄心土(1:1)效果最好,砂子最差;最适温度为20～25℃,低于或高于此温度,里白处于配子体阶段,不能形成孢子体;从孢子萌发到孢子体形成需要90d左右的时间,这和其它蕨类植物相比较缓慢。(6)无菌条件下,以配子体为外植体,配子体增殖适宜的培养周期为41～50d;配子体在培养基MS+6-BA0.5 +NAA0.5和MS+6-BA0.5 +NAA1.0上增殖效果最佳,但常用不同培养基间对配子体增殖倍率没有显著性差异;25℃为配子体培养的最适温度;孢子体诱导至90d时有大量孢子体产生,此时需要及时转接以利于后续培养。更多还原

【Abstract】 In the study, the genetic resource, ecophysiological character and propagation in Hicriopteris glauca plants were investigated. The chloroplast DNA (cpDNA) was amplified using trnG-S primer in 12 wild-population 124 individual H.glauca which distributed in 10 provinces of china (including Anhui, Fujian, Guangdong, Guangxi, Guizhou, Hubei, Jiangxi, Sichuan, Yunnan, Zhejiang). The genetic diversity was studied by the polymorphic sites (P), haplotype diversity (h), nucleotide diversity (π). Based on the above, the variation tendency of ecological factors, such as temperature, humidity, illumination, etc. were monitored in H.glauca population in Daxi, Zhejiang. The community and diversity in H.glauca habitat were also inverstigated. The content of macroelement in plant and in soil, such as hydrolysis nitrogen, potassium, phosphorus, and microelement were assayed to explore the change tendency of mineral element. To explore the physiological response of H.glauca to water stress, the characteristics of photosynthesis of H.glauca was also studied. In addition, we studied the sporegermination and gamete reproduction as well as sporophyte elicit in different matrix, temperature, water and different hormones level. This study is important to provide the theoretical basis and science supporting in genetic resource valuation, propagation, cultrue. The main study results as follow:(1) The mean of the haplotype diversity (h) is 0.4528, and the nucleotide diversity (π) is 0.00067 in H.glauca population. The haplotype diversity and the nucleotide diversity of H.glauca population in western China (π= 0.000292; h = 0.2) was higher than that in Eastern china (π= 0.000145; h = 0.067). The mean of haplotype diversity in population (hs) and the nucleic acid diversity (πS) among H.glauca population was largely different. The haplotype diversity in population (hs) and the nucleic acid diversity (πS) were not related with altitude, longitude and latitude. ANOVA analysis indicated that the genetic variation among different H.glauca population is 67.54%, which inclued 2.97% as genetic diversity among the geography region, which was not significant. The variation happened in the intergroup, there were not significant correlation between the genetic distance and geography distance in the whole population distribution. In this studiy, we found that there were three genetic distribution center of H.glauca, which located in the junction between Longxi, Sichuan and Hubei, the Dawei mountain in Yunnan and Huang Mountain. The three center contain all the three kinds of Haplotype, and the other population only contain one widely haplotype (Hap1).(2) The annual average temperature of H.glauca habitat in Daxi, Zhejiang is 20.5℃, the annual average relative humidity is 65.5%, the annual average illumination intensity is 5800lx, and the annual average relative soil water content is 19.4%. The environment is fit to grow for plant. There are 75 species vascular plant, belonged to 43 families, 64 genus. The results showed that the diversity index is high. the mean Simpson index is 0.778, the mean Shannon-wiener index is 2.258, the mean PIE is 0.783, and the mean the community evenness is 0.783. The shrub type is secondary evergreen and deciduous broad-leaved mixed forest.(3) The content of macroelement, such as Nitrogen(N), Phosphorus(P), Potassium(K), Calcium(Ca), Magnesium(Mg), Manganese(Mn), were on the peak in top growth stage. And the content of many elements, such as sulphur(S), calcium(Ca), Magnesium(Mg), iron(Fe), boron(B), zinc(Zn), copper(Cu), molybdenum(Mo), sodium(Na), increased in growth stage, and then decreased. The variation trend was similar. There were significantly correlation between these mineral elements: N and P, N and K, P and K, K and Fe, Ca and Mo Na and N, Na and Mg. And the variation trend of N, P, K in H.glauca plant were simlar to that in soil.(4) Under different soil water content, the intercellular CO2 content (Ci) had no change in H.glauca plants, which indicated that the decline of net photosynthetic rate (Pn) was not the main reason causing decline of stomatal conductance. The 80~60% of soil water content could not cause the obvious change in Fv/Fm, F′v/F′m, qP,ФPSⅡand ETR. However, when the soil water content was below 60%, the value of Fv/Fm、F′v/F′m、qP、ФPSⅡand ETR reduced obviously. These results suggested that the non-photochemistry energy dissipation in PSII antenna pigment is significantly rised, which have some protection effect on photosynthetic apparatus under the drought stress. With more seriously drought stress, the phenomenon of membrane lipid peroxidation happened.(5) The optimum illumination intensity of H.glauca spore propagation is 40μmol·m-2·s-1, and the developmental rate of gametophyte grow into sporophyte is 48.4%. The best matrix is peatsoil-subsoil, while worst effect is sand. The optimum temperature is 20～25℃. The gametophyte stage would keep below or over this temperature and could not grow into the sporophyte. The time from spore germination to sporophyte needed 90 days approximately, which was lower than the growth speed of other fern plant.(6) Under aseptic condition, the appropriate time of gametophyte propagation culture is about 41～50 day using gametophyte as explants. The optimal culture mediums were MS+6-BA0.5 +NAA0.5 and MS+6-BA0.5 +NAA1.0, and the optimal temperature is 25℃for gametophyte culture. However, there was no significant difference among gametophyte propagation using the different culture medium. When the large number of sporophyte were generated in sporophyte induction process, the successive transfer culture should be needed.更多还原