【作者】 刘卫东；

【导师】 刘友全；

【作者基本信息】 中南林业科技大学 ， 森林培育， 2010， 博士

【摘要】 从形态和ISSR分子标记两方面分别对湖南省的12个地区30份野生假俭草种质资源进行了研究,分析探讨它们之间的遗传多样性,结果表明：(1)30份假俭草种质资源在被测的12个形态学指标上出现较大的变异,其中根系深度、叶面积、叶毛、茎色和叶色这5个形态学指标的变异都达到20%以上；(2)对所测得的形态学指标进行相关分析表明,叶层高度与叶长、匍匐茎节直径、叶面积、叶毛、直立茎节间长、匍匐茎节间长、叶色这7个被测指标呈极显著正相关或负相关,r值范围在-0.791-0.803之间；叶色与叶长、直立茎节间长、匍匐茎节间长、匍匐茎节直径、叶面积、呈极显著正相关或负相关,r值范围在-0.889-0.793之间；(3)对形态指标结果聚类分析结果表明,在欧氏距离8.445处,30份种质资源可以分为三大形态类型,即普通型、高大型和矮生型；(4)综合考虑叶片形态和绿期,采集自岳阳市临湘县围城村的野生假俭草种质资源很适合在长沙地区作为坪用草坪草；(5)通过ISSR分子标记扩增反应体系的试验摸索,确立了适合于湖南地区野生假俭草的ISSR分子标记扩增反应体系；(6)从70个ISSR引物中选出了7个ISSR多态性引物用于30份野生假俭草种源遗传多样性分析,结果表明,湖南野生假俭草具有丰富的遗传变异。7个ISSR多态性引物共检测到126个条带,其中多态条带123个,总的多态位点百分率为97.62%；在单份材料水平上,I=0.1922,H=0.3155；群体水平上, I=0.0878,H=0.1302；(7)Nei’s遗传多样性分析表明,12群体间产生了一定程度的遗传分化(GST=0.5434)。最低遗传多样性水平出现在衡阳假俭草群体、娄底假俭草群体、邵阳假俭草群体,而最高遗传多样性水平出现在永州假俭草群体；(8)湖南野生假俭草群体间的Nei’s遗传距离(D)的变化范围为0.0274～0.1942,根据Nei’s遗传距离(D),利用UPGMA法构建的12个群体的遗传关系聚类图得出,在欧氏距离0.12处可将12个群体分为6个大类,划分类群与地理分布有较明显的关系,其中怀化假俭草群体和吉首假俭草群体的亲缘关系最近；邵阳假俭草群体和衡阳假俭草群体遗传距离最大；(9)利用ISSR标记获得的数据,使用NTSYS-pc2.10软件对30份湖南野生假俭草种质材料进行聚类分析结果表明,在相似系数0.780为阈值时,可以将材料分为八大类,根据ISSR遗传相似系数划分的类群与种质资源地理分布存在一定关系,这与主成分分析结果较一致；(10)形态和ISSR分子标记从不同层次上揭示了湖南省野生假俭草的遗传多样性。不能以一方法代替另一种方法,只有相互结合,综合分析,才能够较全面地揭示湖南省野生假俭草的遗传本质。生理学特性是植物在各种环境条件下的生命活动规律和机理的综合性表现,本研究以岳阳市临湘县E22假俭草(Eremochloa ophiuroides)为研究对象,选择长沙本地狗牙根(Cynodon dactylon)、山东青岛中华结缕草(Zoysia sinica)、美国百喜草(Paspalum notatum)三种暖季型草坪草作为对照,在各种环境条件下,研究草种的外部形态、生理活性以及光合特性,并对其抗旱性、抗盐性、抗寒性作出评价。结果表明：(1)在干旱胁迫下,假俭草叶片中具有较低的质膜透性,脯氨酸积累时间较长且积累量大,叶绿素含量降低较为缓慢且下降幅度小,水分饱和亏和丙二醛含量增加缓慢,且增加量较低。利用模糊隶属函数分析法,对生理指标进行综合分析,假俭草的抗旱性要比狗牙根、结缕草及百喜草弱；(2)随着盐分胁迫的加大,假俭草根、茎、叶干重出现下降趋势,随着处理时间的延长和盐浓度的增高,植株正常生长过程减短,萎蔫程度和死亡数量逐渐增加,同时叶色由绿到黄,甚至褐色,叶质由软到硬。五项生理指标中叶片的丙二醛含量和相对电导率逐渐上升,叶绿素总含量逐渐下降,脯氨酸含量和过氧化氢酶活性则是先上升后下降。耐盐指标与盐浓度之间的统计分析表明,耐盐指标与盐浓度之间均呈线形回归关系,在盐胁迫下其叶片内发生的一系列生理反应中,脯氨酸、相对电导率、丙二醛含量和过氧化氢酶活性四项指标是指示草坪草耐盐性的最佳生理指标；(3)在自然降温的过程中,假俭草的枯黄期为73d,细胞膜电解质渗透率为97%,超氧化物歧化酶活性的增加幅度要小于其它三种暖季型草坪草,可溶性糖含量为28.79%,脯氨酸含量在低温胁迫12h出现极端峰值,达到4.48mg/g。综合大田绿期观测、各个生理指标的分析,假俭草的抗寒性要比其它三种暖季型草坪草弱；(4)假俭草的光合速率(Pn)日变化类型属于典型的“双峰型”,上午和下午各有一次高峰,中午有“午休”现象,光合速率季节变化呈“单峰型”变化趋势,假俭草光合作用的光饱和点和光补偿点分别为1375和46.25μmol·m-2·s-1,CO2补偿点和饱和点分别为21.91 u 1·L-1和1810μ1·L-1,遮荫条件下,假俭草净光合速率日变化规律呈明显的“单峰型”,且在各种遮荫程度下,全年的平均光合速率均低于对照水平,但是全年的峰值与对照均出现在9月,说明遮荫会降低草坪草的生长速率,但不会改变其生长节律,chla/chlb值反映出假俭草具有较好的适应遮荫的能力。坡度对最大截留量有极为显著性的影响。在不同坡度下对四种暖季型草坪草进行综合评价分析,其茎叶截留效应为百喜草>狗牙根>假俭草>结缕草。假俭草根系最大抗拉力与根径均成线性正相关,抗拉强度与根径均成指数负相关。根系平均最大抗拉力分别为百喜草11.06 N>假俭草9.05 N>结缕草5.92 N>狗牙根5.06 N；平均抗拉强度为百喜草34.64 mPa>假俭草27.58 mPa>狗牙根26.31 mPa>结缕草22.82 mPa。假俭草土体抗剪强度要高于对照的结缕草和狗牙根,但低于百喜草。草种根系明显增加了土体最大剪切力,运用加权平均法进行综合评价分析,在自然状态下(垂直压力10kPa)土体抗剪强度依次为百喜草>假俭草>狗牙根>结缕草。更多还原

【Abstract】 This paper studied genetic diversity among 30 wild Eremochloa ophiuroides germplasm resources collected from 12 places in Hunan province by using Inter-Simple Sequence Repeat (ISSR) markers and analyzing morphological characteristics. The results got as follows:(1) Big variation of 30 Eremochloa ophiuroides took place on 12 morphological indexes, among which the variations of root depth, leaf area, length of leaves-hair, stem color and leaf color achieved above 20 percent. (2) The correlation analysis on morphological indexes results got indicated that the height of leaf layer has highly significant positive correlation or negative correlation with seven morphological indexes such as leaf length, prostrate stem diameter, leaf area, lamellar seta, stipites length of erect stem, stipites length of creeping stem and leaf color. The r value range was from -0.791 to 0.803. Leaf color has highly significant positive correlation or negative correlation with leaf length, stipites length of erect stem, stipites length of creeping stem, the diameter of creeping stem and leaf area. R value was between -0.889 and 0.793. (3)The cluster analysis on morphology indexes of 30 materials tested indicated that in the Euclidean distance of 8.445,30 germplasm resources tested were possible to be divided into three shape types, namely normal, tall and big, and dwarf form. (4) Considering both leaf form and green period, wild Eremochloa ophiuroides germplasm resources picked from Weicheng, a village in Linxiang county of Yueyang city, were quite suited to Changsha as lawn grass. (5)Basing on the previous studies, a set of simple, rapid and accurate techniques have been explored for Hunan wild Eremochloa ophiuroides DNA extraction and purification, and an ISSR-PCR Reaction System suit for Eremochloa ophiuroides has been established. (6)The study took a genetic diversity test on 30 Hunan wild Eremochloa ophiuroides germplasm resources by using seven polymorphic ISSR primers picked from 70 ISSR primers. The test showed that Hunan wild Eremochloa ophiuroides germplasm resources have a rich hereditary change. 126 bands may be analyzed were found, among which, polymorphic bands were 123. General polymorphic sites accounted for 97.62%. In single share material level, 1=0.0878, H=0.1302. In community level I=0.0878, H=0.1302. (7)Nei heredity multiplicity analysis indicated that a certain degree heredity differentiation (GST =0.5434) existed among 12 communities. The lowest heredity diversity levels appeared in Hengyang, Loudi, Shaoyang Eremochloa ophiuroides community. While the highest heredity diversity levels appeared in Yongzhou Eremochloa ophiuroides community. (8) The variation range of Nei’s heredity distance (D) among 30 Hunan wild Eremochloa ophiuroides communities was from 0.0274 to 0.1942. According to Nei’s heredity distance got, this study constructed heredity relations cluster charts of 12 communities by using UPGMA. At 0.12 of Euclidean distance,12 communities could be divided into 6 kinds. The division group has obvious relations with geographic distribution. Since Huaihua Eremochloa ophiuroides community was closely related to Jishou Eremochloa ophiuroides community, they were gathered together in the same place firstly. Shaoyang Eremochloa ophiuroides community and Hengyang Eremochloa ophiuroides community’s heredity was far away from each other. (9)The study took cluster analysis by NTSYS-pc2.10 software to 30 Hunan wild Eremochloa ophiuroides germplasm resources using data got from ISSR markers. The analysis results indicated the materials would be divided into 8 big types when similarity factor 0.780 was threshold value. Communities divided according to ISSR heredity similarity coefficient have certain relations with geographic distribution. This was consistent with the principal components analysis result. (10) Inter-Simple Sequence Repeat (ISSR) markers and morphological characteristics told us genetic diversity of Hunan wild Eremochloa ophiuroides from different levels. The two methods could not be substituted for another. Only the integration analysis of the two ways, could we comprehensively revealed the genetic nature of Hunan wild Eremochloa ophiuroides.Physiological characteristics refer to a comprehensive performance of plants’ life activity law and mechanism in a variety of environmental conditions. In this study, Linxiang E22 Eremochloa ophiuroides in Yueyang city was taken as research object. Three kinds of warm-season turfgrasses, local Changsha Cynodon dactylon, Qingdao Zoysia sinica in Shandong province and American Paspalum notatum were selected as contrast. External form, physical activity, photosynthetic characteristics, drought resistance, salt resistance and cold hardiness of turfgrasses were evaluated. The results indicated:(1) Under drought stress, the leaves of Eremochloa ophiuroides has a lower membrane permeability, proline accumulated long and accumulation was large, chlorophyll content decreased slowly and decline range was small, water deficit MDA increased slowly and small. A comprehensive analysis of physiological indicators of four warm-season turfgrasses was made by fuzzy subordination method. Centipedegrass drought resistance was weaker than bermudagrass, zoysiagrass, and Bahiagrass. (2) As salt stress to Eremochloa increased, the dry weights of roots, stem, and leaf were trending downward. With the extension of processing time and increasingly higher salt concentration, plants’normal growth was shortened, wilting degree and number of deaths gradually increased. While leaf color turned from green to yellow or brown and leaf quality from soft to hard. Among five physiological parameters, leaf MDA content and relative electrical conductivity gradually increased, total content of chlorophyll decreased, proline content and catalase activity was first increased and then decreased. Statistical analysis between salt tolerance index and salt concentration showed that linear regression relationship existed between salt tolerance index and salt concentration. A series of physiological reactions occurred in the leaves under salt stress showed that proline, relative conductivity and MDA content and catalase activity were four best physiological parameters to indicate salt tolerance. (3) In natural cooling process, the period of Eremochloa ophiuroides turning withered was 73 days. The membrane electrolyte leakage was 97%. Superoxide dismutase activity in the rate of increase was less than the other three warm-season turf grasses. Soluble sugar content was 28.79%. Proline content reached extreme peak 4.48 mg/g in 12-hour under cold stress. Basing on field green stage observation and various physiological analysis, Eremochloa ophiuroides’s cold hardiness was weak compare to the other three warm-season turf grasses. (4) Eremochloa ophiuroides’Diurnal variation of photosynthetic rate was typical "bimodal", that is to say it has a morning and afternoon peaks, and noon "nap" phenomenon. Seasonal change of photosynthetic rate was "single-peak-type" trend. Eremochloa ophiuroides’photosynthetic light saturation point (LSP) and light compensation point (LCP) were 1375 and 46.25μmol·m-2·s-1 respectively. CO2 compensation point was 21.91μl·L-1 and CO2 saturation point was 1810μl·L-1. Diurnal variation trend of net photosynthetic rate was obviously a "single peak" and in a variety of shading, the annual average photosynthetic rate was lower than the control level. However, the peak of Pn in one year appeared in September, which was the same with control. It indicated that the shade would reduce turfgrass growth rate, but it would not change its growth rhythm. Chla/chlb value reflected the Eremochloa ophiuroides has good ability to adapt to shade.The slope has a significant effect on lawn grass maximum interception amount, and through the synthetic analysis on the four warm-season lawn grass under the conditions of different slopes, the stems and leaves interception effect of the four lawn grass kind arranged in order as follows:Paspalum notatum> Cynodon dactylon> Eremochloa ophiuroides> Zoysia sinica.The maximum compressive stress of Eremochloa ophiuroides’root system was in direct linear proportional to the root diameter while the tensile strength was inversely proportional to the root diameter. The average maximum compressive stress of the four warm-season lawn grass’s root system lists in order as follows:Paspalum notatum 11.06 N> Eremochloa ophiuroides 9.05 N> Zoysia sinica 5.92 N> Cynodon dactylon 5.06 N, and the average tensile strength lists in order as follows:Paspalum notatum 34.64 mPa> Eremochloa ophiuroides 27.58 mPa> Cynodon dactylon 26.31 mPa> Zoysia sinica 22.82 mPa.Eremochloa ophiuroides’soil shear strength was higher than the control of Zoysia sinica and Cynodon dactylon, but less than Paspalum notatum. Turfgrass roots significantly increased the maximum shear strength of soil. The weighted average method to conduct a comprehensive evaluation and analysis was used. Results showed that in a natural state (vertical pressure 10kPa) shear strength of soil arranged in order as follows:Paspalum notatum> Eremochloa ophiuroides>Cynodon dactylon> Zoysia sinica.更多还原