【作者】 张海琴；

【导师】 周永红；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 猬草属Hystrix Moench是禾本科Poaceae小麦族Triticeae Dumortier中一个多年生小属。该属约有6种，中国有3种。Moench（1794）依据Linneus（1753）描述的ElymusHystrix L．具有强烈退化的颖或无颖的特点而独立为一个属，即：Hystrix Moench，其模式种为Hystrix patula。Dewey（1984）报道了H．patula具有StH染色体组，并根据染色体组分析方法，将Hystrix放在Elymus中，但他仅对Hystrix的模式种——H．patula进行了染色体组组成分析。Jensene et al．（1997）报道Elymus coreanus（H．coreana）和E．californicus（H．californica）具有与赖草属Leymus相同的NsXm染色体组，并把这两个物种组合到Leymus中，使得Hystrix的分类更为复杂。关于Hystrix属的界限和物种划分一直处在争议中，主要分歧是：将这些物种置于Hystrix，还是作为Elymus的一部分以及还是放在Leymus中。本研究（1）通过形态学特征比较、细胞学和繁育学资料，探讨三个Hystrix物种的染色体组组成及亲缘关系；（2）通过RAPD分析和ISSR分析，探讨Hystrix与Leymus、新麦草属Psathyrostachys的属间亲缘关系；（3）比较RAPD和ISSR两种DNA分子标记在Hystrix及近缘属系统分类研究中的应用。主要结果如下： 1．华山新麦草（P．huashanica Keng，2n=2x=14，Ns^h）与三个猬草属物种间的杂交结实率平均为15.66％，变幅为4.59％-34.62％：竖立鹅观草（R．ciliari svar．Japonensis（Honda）B．R．Lu，Yen et J．L．Yang，2n=4x=28，StY）与H．patula间的杂交结实率为5.00％。杂种种子发育良好，不需通过幼胚拯救技术即可萌发，获得杂种植株。杂种F₁完全不育，自交不结实，而亲本育性和结实正常。表明彼此之间存在不同程度的生殖隔离，是独立的生物学物种。 2．H．duthiei ssp．longearistata×P．huashanica的杂种F₁花粉母细胞减数分裂中期Ⅰ染色体配对数为：9.46Ⅰ+5.65Ⅱ+0.07Ⅲ；平均每细胞交叉数为7.08，C-值为0.51。在形态性状上，杂种F₁的许多性状介于两亲本之间。表明H．duthiei ssp．longearistata与P．huashanica之间有一个染色体组具有较高的同源性，即H．duthiei ssp．longearistata具有Psathyrostachys的Ns染色体组。 3．H．duthiei×P．huashanica的杂种F₁花粉母细胞减数分裂中期Ⅰ染色体配对为7.44个单价体，6.55个二价体，0.12个三价体和0.02个四价体。64％的细胞在中期Ⅰ形成7个二价体和7个单价体；平均每细胞交叉数为9.76，C-值为0.70。说明双亲有一组染色体具有较高的同源性，即H．duthiei含有Ns染色体组。 4．H．patula×P．huashanica的杂种F₁花粉母细胞减数分裂中期Ⅰ，染色体配对构型为：20．43＋0．29 11。平均每细胞交叉数为 0；29，C－值为 0刀2。R．ciliaris var．jponensisX H Patula杂种 F；花粉母细胞减数分裂中期 1染色体配对构型为：4．48＋11DZ 11＋0．27Ill＋0．23IV。平均每细胞交叉数为 17．83，C－值为 0石4。表明 H patula含有 St染色体组，而不含NS染色体组。 5．Hystrix、L聊us及 PsathytostachyS三个属 11个种 1变种共 13份材料的 l；oyD和 ISSR分析表明：属间存在较大的遗传多样性；L聊us和 PsathyffistachyS属内种间的遗传变异较小。4份 Hystrir材料的遗传多样性最丰富：（）H duthiei与 H duthiei SsP．long6aristata的相似系数较高，聚为一支，这与周永红等门 999)通过形态学比较和细胞学研究的结果相一致，支持将H long6aristata处理为H duthiei的一个变种。＊)Hcore。a与 L．cinereus聚为一支，表明 H coreana与 Lrym。具有较近的亲缘关系。臼)H pQtula与所有供试材料的遗传相似系数都较低，聚类图中位于最顶端，单独聚为一支。表明 H patula与 L啊us和 PsathyffistachyS的亲缘关系较远，与其它三份 Hystrix材料也存在着较大的遗传差异。 6．RAPD标记和ISSR标记的比较 门)多态性比较：KAPD标记选用 40个上海生工公司十聚体核酸随机引物进行 PCR扩增，20个引物扩增出142条带谱，其中135条为多态性带纹，平均每个引物扩增7．10条带谱。而ISSR标记选用35个ISSR引物进行PCR扩增，12个引物能扩增215条清晰并呈现多态性带，平均每个引物扩增17．92条多态性带。表明ISSR标记的多态性高于RAPD标记，这是与两者在进行基因组扩增时引物结合的位置不同有关。 （2）结果分析的比较：通过两种不同 DNA分于标记得出的伽tris、Lcymus和PsathmptachyS三个属间的 Jaccard遗传相似系数及遗传聚类图具有极显著的相关性，表明研究中所用的RAPD和ISSR标记数量可以检测到这些遗传变异。因此，RAPD和ISSR这两种 DNA分子标记可作为评价 Hytrix、L洲us和 PsathytostachyS的系统亲缘关系研究的可靠而有效的补充研究方法。更多还原

【Abstract】 Hystrix Moench is a small perennial genus of the tribe Triticeae (Dumortier). Originally the only known species was described as Elymus Hystrix by Linneus (1753),on account of the strongly reduced or lacking glumes,it was later segregated in its own genus as Hystrix patula (Moench 1794). Since then 8 deferent species have been included in Hystrix,three of them grow in China. Dewey (1984) reported H. patula has StH genomes and according to genomeanalysis method,included it in Elymus. But he only studied the type specie of Hystrix------H.patula. Jensen et al. (1997) reported Elymus coreanus (H. coreand) and E. californicus (H. californicd) have the same genome constitution of Leymus (NsXm),and included them in Leymus. Thus,the taxonomy of Hystrix becomes more complicated. Until now,the definition of Hystrix and its precise taxonomic rank are still under discussion. The main controversial is included the species of Hystrix either in Hystrix,or regard it as a part of Elymus or Leymus. The objective of the present paper was to investigate the genome constitution of three species of Hystrix and their taxonomic status from cytological and molecular data. By RAPD and ISSR assay,the present study was to evaluate phylogenetic relationships among Hystrix,Leymus and Psathyrostachys,and to compare the use of two molecular markers in the genus and species of Triticeae. The main results are as follows:1. The seed set of Psathyrostachys huashanica Keng (2n=2x=14,Nsh) crossed with three species of Hystrix averaged 15.66% and ranged 4.59%-34.62%. That ofRoegneria ciliaris var. japonensis (Honda) B. R. Lu,Yen et J. L. Yang (2n=4x=28,StY) crossed with H. patula was 5.00%. All of the crosses produced hybrid seeds that developed into adult plants even if without embryo rescue culture. The hybrids were completely sterile while the pollen fertilities of parental species were normal. This suggested there are reproductive between all of five parents and they were dependent biological species.2. The mean meiotic configuration of H. duthiei ssp. longearistata with P. huashanica was 9.46 I + 5.6511+ 0.07III,the chiasma frequency was 7.08 and the C-value was 0.51. There were obvious morphological differences between two parental species and the F,plants appeared intermediate between the two parents. Therefore,we could conclude that H. duthiei ssp. longearistata and P. huashanica share one Ns genome.3. The mean meiotic configuration of H. duthiei with P. huashanica was 7.44 I + 6.55 II + 0.12III+ 0.02IV,the chiasma frequency was 9.76 and the C-value was 0.70,which indicated that H. duthiei had one Ns genome.4. The mean meiotic configuration of H. patula with P. huashanica and R. ciliaris var. japonensis were as follows:H. patula X R huashanica 20.43 I + 0.29 II,with C-value of 0.02;R. ciliaris var. japonensis X H. patula 4.48 I + 11.0211+ 0.27III + 0.23IV,with C-value of 0.64. These suggested that H. patula has St genome and no Ns genome exists.5. To assess the phylogenetic relationships among Hystrix,Leymus and Psathyrostachys,two DNA molecular markers RAPD and ISSR were used. The results indicated that the level of genetic variation of three different genera is higher than species within the genus. 4 species of Hystrix showed great genetic diversity:(1) H. duthiei var. longearistata closely related to H. duthiei and were clustered together,which corroborated the results from morphological and cytological studies by Zhou Yong-Hong (1999). (2) H. coreana was clustered with Leymus cinereus,which indicated they had closer relationships. (3) There were distinct genetic differences between H. patula and the other materials used in this study. H. patula was separated as a single group in the UPGMA dendrograms. It may suggest that H. patula had different genomes from Leymus,Psathyrostachys and the other three species of Hystrix in this study.6. Comparison between the results from RAPD and those from ISSR,it is concluded that:(1) 40 random primers were screened for RAPD fragments,20 primers produced polymorphic RAPD更多还原