全 文 ：第 16 卷 第 5 期
Vol. 16 No . 5
草 地 学 报
ACTA AGRESTIA SINICA
2008 年 9 月
Sep. 2008
# 博士论文摘要#
西藏几种嵩草种子萌发特性与分子系统发育研究
博士生 :郑红梅 导师:呼天明
(西北农林科技大学 动物科技学院 草业科学系, 陕西 杨凌 712100)
嵩草属(K obr esia Willd. )植物是多年生草本植物,草质
柔软、营养丰富, 家畜喜食。大多数嵩草植物是青藏高原高
寒草甸的主要建群种,其良好生长对维持青藏高原的生态平
衡起着重要作用。嵩草属植物主要分布在北半球, 共有 70
种,其中 59 种分布在中国 (主要分布在青藏高原东部)。由
于环境及人为影响,我国嵩草草地面临草地退化、载畜能力
下降等严峻问题,而且嵩草属植物大多是无性繁殖, 对环境
变化及不良环境适应性较弱。目前,对嵩草属植物的分子系
统发育关系及其适应性研究尚未见报道。本文探索其种内
遗传多样性、遗传结构、系统发育关系及种子特性,旨在为进
一步研究高寒草甸生态系统的结构、功能及演化提供依据。
主要结果如下:
1. 在实验室条件下, 分别对 7 种嵩草属植物的千粒
重、种子吸水曲线及不同溶液处理发芽率进行了比较, 结合
种子微形态结构、变温层积及生理指标测定等,探讨了嵩草
种子的萌发特性及解除休眠的技术。试验发现嵩草都具有
坚硬而厚的种皮, 40%氢氧化钠处理 3 h, 种皮的通透性有所
改善, 且在萌发初期, 大花嵩草( K . macrantha BÊ cklr . )、藏
北嵩草(K . littledalei C. B. Clarke)、矮生嵩草( K . humilis
( C. A . Mey) Ser g. )中, 可显著地提高种子中过氧化物酶、
过氧化氢酶和脱氢酶的活性,大多数嵩草的发芽率明显高于
对照(P< 0. 05)。嵩草种皮坚硬且与种子发芽力有关的几
种酶的活性较低,致使其发芽率较低。试验还发现其种皮保
护组织的厚度占果实横切面厚度的比例之大是少见的; 电镜
扫描微形态特征虽然种皮基本纹饰一致, 但从种子形态来
看,尚有一定差别。此外, 受材料数目限制,要通过电镜扫描
种皮微形态性状尚难于确定嵩草属种间的关系,本试验只能
作为一个关系划分的参考依据。
2. 对扩增长度多态性 ( AFLP ) 和随机扩增多态性
DNA( RAPD)几个关键参数进行优化,结果表明用于酶切的
嵩草基因组 DNA 模板以 600 ng 为宜, 连接最适反应时间为
10 h, 预扩增产物最适稀释倍数为 5 倍。采用优化好的体
系,以嵩草为材料进行 AFLP 标记, E-ACC + M-CAG、E-
ACA+ M-CAG、E-ACC+ M-CAA 和 E- ACA+ M-CTG 4 个
引物均获得多态性强且质量好的 AFLP 指纹图谱, 其中以
E-ACA+ M-CAG 引物组合效果最佳, 扩增出 41 条多态性
带, 比率为 99. 02%。用筛选出的 4 对 E + 3 和 M + 3 引物
及优化好的嵩草体系对 11 份嵩草基因组 DNA 进行 AFLP
扩增, 共得到 164 条清晰可辨条带, 多态性条带 154 条, 多
态性位点百分率为 93. 96% ,平均 Nei. s 基因多样性指数为
0. 2430, Shannon. s 多样性指数为 0. 4012, 表明嵩草种质间
存在丰富的遗传多样性。通过 UPGMA 聚类分析, 将 11 个
嵩草居群划分为 4 类。对 11 个嵩草群体的 RAPD 遗传多样
性进行分析表明, 10 个随机引物共扩增出 170 个清晰可辨
的条带, 扩增带数最多的引物为 22 条, 多态率达98. 16% ; 带
数最少的引物为 10 条, 多态性检出率最低为 45. 45%。当
遗传相似系数为 0. 66 时, 11 个嵩草居群被分为 4 个类群。
研究表明嵩草居群基于 RAPD ( H = 0. 2221 和 I = 0. 3592)
数据的遗传多样性相对低于 AFLP ( H = 0. 2430 和 I =
0. 4012)。基于 RAPD 和 AFLP 遗传相似系数数据的 Man-
tel检验表明两者有着显著的相关性( r = 0. 65528, P = 0.
002) ,说明 RAPD 和 AFLP 在嵩草遗传多样性的研究中都
是有效的。根据 AFLP 和 RAPD两种分子标记方法, 嵩草
居群的遗传相似系数与海拔之间没有相关性, 嵩草居群的生
境异质性影响遗传分化。
3. 序列分析为该属的系统分类及探讨其种间系统关
系提供了重要的科学依据。系统发育树不仅表明了各种间
的亲缘关系, 而且对探讨嵩草的起源与演化具有重要意义。
虽然 RAPD 和 AFLP分子标记是遗传多样性研究的有效方
法, 但其不能得到足够的信息与前人研究相比较。我们用测
序方法 nr ITS、cptr nT-L-F、cpndhF 及 trnK-RPS16 开展进
一步研究。大花嵩草( L )居群和其他嵩草居群有着较远的
亲缘关系。具有相似的生境或具有相似适应性的嵩草居群
倾向聚在一起。大多数的聚类分析表明基因在 11 个嵩草居
群中存在着基因变异, 可能是由于基因突变或居群间分布距
离较远、海拔较高导致的基因漂流引起的。
关键词: 西藏; 嵩草;系统发育; AFLP; RAPD; 种皮
中图分类号: S330. 2; Q943. 2 文献标识码: A 文章编号: 1007-0435( 2008) 05-0542-03
完成时间: 2008 年 5 月
收稿日期: 2008-09-01; 修回日期: 2008-09-10
基金项目:西藏科技厅重点科技计划( 2005011)、科技部国际合作计划( 2006DFA33630)
作者简介: 郑红梅( 1978-) ,女,内蒙古赤峰人,博士研究生,研究方向为牧草种质资源
导师简介: 呼天明 ( 1958-) ,男,内蒙古鄂尔多斯人,博士,教授,研究方向为草种繁育及草畜一体化, E-mail: hut ianming@ 126. com
第 5期 郑红梅:西藏几种嵩草种子萌发特性与分子系统发育研究
Study on Seed Germination Characteristics and Molecular Phylogeny of
Kobresia Willd. in Tibetan Plateau
Candidate: ZHENG Hong-mei A dviso r: HU Tian-ming
( Department of Grassland Science, Northw est A& F University, Yangling, Sh aanxi Provin ce 712100, C hina)
The genus K obr esia Willd. is the dominant perennial sedge on the T ibetan Plateau and has high nutr-i
t ional quality preferred by liv estock and impor tant ecolog ical functions such as w ater r esource conserva-
t ion, river system and reg ional climate r egulat ion, and regional biodiversity maintenance. K obr esia spp.
can be found in the alpine mountains of the northern hemisphere and include about 70 species, w ith 59 re-
ported throughout China w hich are mainly dist ributed in east of the Tibet Plateau. How ever, K obr esia
pastures ar e confronted w ith environmental degradat ion and decreased car rying capacity due to natural and
anthropogenic impacts, and seldom resear ch studied the K obr esia species o f the Tibetan Plateau on their
phylo genet ic r elat ionship and adaptat ion in details , in part because of the diff iculty in accessing this cold,
harsh, and remote region. Vegetative g row th is the dominant means of reproduct ion fo r the K obr esia spp.
and the substant ial vegetative gr ow th can reduce the genet ic div ersity and r ender populat ions with disad-
vantages to buf fer the chang ing and ex tr eme conditions. It is necessary to protect the genet ic diver sity of
Kobresia and under stand how they thrive in a har sh environment. In the present study, the genet ic div ers-i
ty , genet ic st ructure, and seed characterist ics of Kobresia species w er e studied in order to provide theory
and technical references for understanding the mechanisms of adaptat ion and evolutionary histo ry among
the K obr esia accessions collected f rom the T ibetan Plateau.
At f ir st , the important pro cess of K obr esia community maintaining and r enew ing, i. e. seed germina-
t ion mechanism , w as invest igated. Seed germ inat ion and the dormancy breaking techniques w ere studied
by the examinat ion o f 1000-seed weight , seed w ater abso rbing curve, and germinat ion rate under different
solut ion t reatments to gether w ith seed mo rph-dissect ion, seed strat ificat ion under dif ferent temperatures,
and phy siolog ical parameters, etc. The r esults show that seed coats of K obresia were hard and thick,
tr eatment of 40% NaOH w ith 3h improved the permeability of seed coat and signif icant ly increased the ac-
t ivit ies o f POD, SOD and dehydrogenase of K . macr antha BÊ cklr. , K . humil is ( C. A. M ey ) Serg . , and
K . pygmaea C. B. Clarke during early seed germ inat ion and the germ inat ion r ates w ere significant ly high-
er than those of non- treated seeds ( P< 0. 05) . T he hard seed coat and low act ivities o f some enzymes rela-
t ing to germinat ing capacity w ere the r eason for low germ inat ion rate. T her e w as a seldom higher percent-
age of protect ive t issue to the cross sect ion o f Kobresia seed. The seed coat ornamentat ion w as similar by
scanning electr on m icroscope, but there w ere some dif ferences in mor pholog ical t rait o f seed and it w as dif-
f icult to ident ify the relat ionship o f Kobresia species ow ing to the limit ing number of materials.
The genetic div ersity of Kobresia accessions w ere analyzed at the molecular level and several main fac-
to rs inf luencing the results of the AFLP and RAPD methods w ere opt im ized fo r the establishment of the
AFLP and RAPD react ion sy stem. The results show the appr opr iate DNA template was 600ng, the opt-i
mal ligat ion t ime w as 10 hours, and the 5 t imes dilut ion w as best for the pr oducts of pre-amplif icat ion.
With the opt imized system , the clear polymor phic AFLP f ingerprints o f K obr esia were obtained from four
primer pairs ( E-ACC+ M-CAG, E-ACA + M-CAG, E-ACC+ M-CAA, and E-ACA + M-CTG) , among
which E-ACA + M-CAG was the best in detect ion of K obr esia genet ic diversif icat ion. 41 polymorphic
bands w ere amplified and the po lymo rphic band rate w as 99. 02%. Genom ic DNA of K obr esia was ampl-i
f ied w ith four E+ 3 and M + 3 primer combinat ions w ith AFLP and 164 legible bands w ere obtained, a-
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草 地 学 报 第 16卷
mong them 154 ( 93. 96%) w er e polymorphic and the polymorphic lo cus rate w as 93. 96% . T he mean Nei.
s gene diversity index ( H ) w as 0. 2430 and the Shannon. s informat ion index ( I ) w as 0. 4012, indicat ing
the abundant g enet ic diversity o f K obr esia. Result of RAPD genet ic div ersity analysis of 11 K obr esia acces-
sion show that 170 legible polymor phic bands w ere amplif ied, the most band number amplified by a primer
reached to 22 w ith polymorphic detect ion rate of 98. 16% and least 10 w ith po lymorphic detect ion rate of
45. 45% . The 11 K obr esia accessions w ere classified into four groups by Cluster Analysis based on the UP-
GMA method when similarity is 0. 66. Our study revealed that the genetic diversity of Kobresia populat ion
based on RAPD ( H = 0. 2221 and I = 0. 3592) w as relat iv ely low er than AFLP ( H = 0. 2430 and I =
0. 4012) . T he M antel test show ed a significant co rrelation betw een the RAPD- and AFLP-based genetic
similarity ( r= 0. 65528, P= 0. 002) . In general, there w as abundant g enetic div ersity among K obr esia re-
sources, the genet ic coef f icient of Kobr esia populat ion w as unrelated to their g eographic latitude and natu-
r al habitat heterogeneity inf luenced the genet ic different iat ion o f Kobresia.
Nucleot ide sequence analysis pro vided impo rtant scient ific base fo r species classif icat ion and interspe-
cific relat ionship o f Kobresia. T he phylo genic dendro gram not only can indicate the kinship among K obr e-
sia species, but also is meaningful for the study of K obr esia o rigin and evo lvement . A lthough molecular
marker by RAPD and AFLP is an ef fect ive method for g enet ic div ersity research, w e canno t obtain enough
informat ion w hich can be used for comparison w ith results f rom fo rmer researches. We adopted the se-
quence analy sis of nrITS, cpt r nT-L-F, cpndhF and t rnK-RPS16 for further invest igat ion. T here w as an
ulterior kinship betw een K . macr antha population and o thers. T he populat ions w ith similar habitats o r
similar adaptat ion tended to gather. Results f rom most cluster analysis indicate that there w er e genetic var-
iat ions among the 11 Kobresia populat ions w hich could be caused by the mutat ion or g enetic drift due to re-
mote distr ibution or high elevat ion.
Key words: T ibet; K obr esia; Phylo geny ; AFLP; RAPD; Seed coat
(责任编辑 才 杰 李 扬)
5草业学报6征订启事
5草业学报6创刊于 1990 年,由中国草学会、兰州大学草地农业科技学院、甘肃草原生态研究所共同主办,是国内外公开发
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