【作者】 于海清；

【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， 2007， 博士

【摘要】 1980年，(?)skell L(?)ve根据染色体组分析的结论建立了以St染色体组为特征的拟鹅观草属Pseudoroegneria (?)．L(?)ve。拟鹅观草属物种为多年生，簇生或丛生草本，长花药，异花授粉。全世界约15～20种，主要分布于中东、高加索、中亚、中国西部以及北美西部。拟鹅观草属是小麦族极为重要的染色体组供体属，由它的St染色体组组合成多个异源多倍体属，因此，拟鹅观草属在小麦族系统演化上具有重要地位。L(?)ve建立了拟鹅观草属后，基于染色体组分析的结果，一些分类群先后被不同学者划入或划出拟鹅观草属，还有一些拟鹅观草属分类群有待作进一步的处理。因此，拟鹅观草属物种及其相关属物种，特别是一些四倍体物种间还存在一定的争议，它们的系统地位、物种界限、种间(内)亲缘关系还有待进一步研究。这些争议主要表现在：(1)鹅观草属无芒类群Roegneria alashanica、R．elytrigioides、R．magnicaespes和R．grandis有可能是中国分布的拟鹅观草属物种；(2)具有E~eSt染色体组的Pseudoroegneria geniculata ssp．scythica、Elytrigia caespitosa、Et．caespitosa ssp．nodosa和Et．intermedia的系统关系有待进一步探讨，拟鹅观草属P．geniculata、P．geniculata ssp．scythica和P．geniculata ssp．pruinifera的系统关系还不清楚；(3)具有PSt染色体组物种已经被划出拟鹅观草属，归入杜威草属，但这些物种的属间关系、种间关系以及母本来源还不够清楚；(4)St染色体组在拟鹅观草属内及其相关属间存在一定程度的分化。本文通过C-带分析、基因组原位杂交分析、染色体组分析、ITS序列分析和trnL-F序列分析对拟鹅观草属四倍体物种进行了研究，目的在于：(1)探讨鹅观草属无芒类群物种的种间关系以及它们与拟鹅观草属物种的属间关系；(2)研究Pseudoroegneria物种的系统发育关系；(3)探讨Pseudoroegneria、Elytrigia和Douglasdeweya的属间关系；(4)揭示Pseudoroegneria物种St染色体组的分化。研究结果如下：1．对Pseudoroegneria的11个分类群、Roegneria的5个物种、Elytrigia的2个分类群和Douglasdeweya的2个物种进行了带型分析。结果表明：(1)具有St染色体组的Pseudoroegneria二倍体和四倍体物种都具有强的末端带，二倍体物种具有更强的末端带。(2)P．geniculata ssp．scythica、Et．caespitosa和Et．caespitosa ssp．nodosa的带型有相似性，但也存在较大差异；P．geniculata ssp．pruinifera的染色体具有多而强的中间带或着丝点带。(3)R．elytrigioides的染色体表现出强的末端带，与具有St染色体组的Pseudoroegneria二倍体和四倍体物种的带型相似；R．alashanica和R．magnicaespes的末端带少而较弱、着丝点带较弱、中间带多而较弱；R．grandis和R．ciliaris的带型有相似性，但R．grandis表现出多而强的末端带。(4)D．wangii和D．deweyi的带型表现出一定的差异，它们可能是两个独立的种。2．分别选用P．spicata(St)、P．libanotica(St)、P．stipifolia(St)、R．ciliaris(StY)、A．cristatum(P)、Lo．elongatum(E~e)和Lo．bessarabicum(E~b)作探针，P．strigosa(St)、鲑精DNA和Lo．elongatum(E~e)作封阻，用基因组原位杂交(GISH)技术来探讨R．alashanica、R．elytrigioides、R．magnicaespes、R．grandis、P．geniculata、P．geniculata ssp．scythica和P．geniculata ssp．pruinifera的染色体组组成。结果表明：(1)R．alashanica具有一组St染色体组，另一组未知，但不是E、P或Y染色体组；(2)R．magnicaespes具有一组St染色体组，另一组未知，但不是Y染色体组；(3)R．elytrigioides具有两组St染色体组；(4)R．grandis和P．geniculata都具有一组St染色体组，另一组染色体组与St都有一定程度的同源性；(5)P．geniculata ssp．scythica和P．geniculata ssp．pruinifera的染色体组组成分别为EEStSt和EEEEStSt。3．分别选用鹅观草属物种R．alashanica(St-)、R．elytrigioides(StSt)、R．magnicaespes(St-)和R．grandis(StY)，拟鹅观草属物种P．geniculata(StSt)、P．geniculata ssp．scythica(E~eSt)、P．geniculata ssp．pruinifera(—)、P．cognata(St)、P．libanotica(St)、P．spicata(St)、P．strigosa(StSt)和P．tauri(St)，杜威草属物种D．wangii(PSt)和D．deweyi(PSt)、披碱草属物种Elymus wawawaiensis(HSt)，花鳞草属物种Anthosachne scabra var．scabra(StWY)，猬草属物种Hystrix patula(HSt)进行属间和属内物种间杂种的创制。对11个杂交组合进行了染色体组分析，对花粉育性和结实率进行了调查。结果表明：(1)R．alashanica具有一组St染色体组，另一组染色体组与H染色体组没有同源性；(2)R．grandis具有一组St染色体组，其Y染色体组与St染色体组有同源性。(3)St染色体组在各物种间表现出一定程度的分化。4．对Pseudoroegneria的17份材料(St或StSt)、Roegneria的7份材料(StY或StSt)，Elytrigia的4份材料(E~eSt或E~bE~eSt)、Lophopyrum的4份材料(E~b、E~e或E~bE~eE~xStSt)、Douglasdeweya的3份材料(StP)、Agropyron的5份材料(P)、Australopyrum的2份材料(W)、Hordeum的2份材料(H)、Psathyrostachys的2份材料(Ns)进行系统发育关系研究。对其ITS序列进行了简约分析，构建了严格一致系统树。结果表明：(1)St染色体组在Pseudoroegneria二倍体物种间表现出分化，Pseudoroegneria多倍体与二倍体物种亲缘关系近；(2)R．alashanica、R．elytrigioides和R．magnicaespes与四倍体物种P．geniculata有近的亲缘关系；(3)StY染色体组与St染色体组有同源性，St染色体组与Y染色体组可能有相同的起源；(4)P．geniculata ssp．scythica和P．geniculata ssp．pruinifera处理为P．geniculata的亚种不合适，P．geniculata ssp．scythica、P．geniculata ssp．pruinifera、Et．caespitosa、Et．caespitosa ssp．nodosa、Et．intermedia和Lo．ponticum应该被组合到Trichopyrum中；(5)E~b、E~e与St染色体组之间有近的亲缘关系；(6)D．wangii和D．deweyi的P染色体组来自于Agropyron二倍体物种，将StP染色体组的物种从Pseudoroegneria划入Douglasdeweya是合理的。5．对Pseudoroegneria的16份材料(St或StSt)、Roegneria的7份材料(StY或StSt)，Elytrigia的3份材料(E~eSt或E~bE~est)、Lophopyrum的4份材料(E~b、E~e或E~eE~e)、Douglasdeweya的3份材料(PSt)、Agropyron的4份材料(P)、Australopyrum的1份材料(w)、Hordeum的2份材料(H)、Psathyrostachys的2份材料(Ns)进行系统发育关系研究。对其trnL-F序列进行了简约分析，构建了严格一致系统树。结果表明：(1)Pseudoroegneria、Roegneria、Elytrigia、Lophopyrum和Douglasdeweya物种聚成一支，它们的trnL-F序列同源性很高，具有较近的亲缘关系；(2)Pseudoroegneria是多倍体物种St染色体组的母本供体；(3)D．wangif和D．deweyi的母本来自于Pseudoroegneria二倍体物种，父本来自于Agropyron二倍体物种；(4)Agropyron和Australopyrum物种聚成一支，它们的trnL-F序列同源性很高，具有较近的亲缘关系。更多还原

【Abstract】 Pseudoroegneria is erected by (?)skell L(?)ve in 1980 based on genome analysis. Thegenome symbol of Pseudoroegneria species is designated St. The Pseudoroegneria grassesare caespitose, long-anthered, and cross-pollinating perennials. Pseudoroegneria consistsof about 15～20 species that distribute in the Middle East, Transcaucasia, Central Asia,Northern China or Westem North America. Pseudoroegneria is one of the most importantgenome donor genera in tribe Triticeae. The St genome is found in combination with othergenomes in many alloploids genera. Therefore, Pseudoroegneria is important inphylogenetic evolution of tribe Triticeae.Based on the results of genome analysis, some taxa are moved into or grouped out thePseudoroegneria since it was established, and some taxa require further treatment in thefuture. It is still in controversy in taxa treatment of partial species in Pseudoroegneria andrelated genera, especially in some tetraploid species. The phylogenetic relationships arestill obscure among them. The disputes are (1) Roegneria alashanica, R. elytrigioides, R.magnicaespes and R. grandis, which belong to the awnless group in Roegneria, are mostlikely the Pseudoroegneria species distributed in China, (2) Phylogenetic relationshipsamong Pseudoroegneria geniculata ssp. scythica, Elytrigia caespitosa, Et. caespitosa ssp.nodosa and Et. intermedia, which contain E~eSt genomes, need further investigation.Phylogenetic relationships of P. geniculata, P. geniculata ssp. scythica and P. geniculatassp. pruinifera are obscure, (3) The species with PSt genomes have been grouped intoDouglasdeweya from Pseudoroegneria, however, the phylogenetic relationships amongspecies in the two genera and the maternal donor of species in Douglasdeweya are stillunclear, (4) the St genome is differentiated in some degree in Pseudoroegneria and relatedgenera.In the present study, the disputes mentioned above were investigated via the C-banding,GISH, genome analysis, ITS sequences and trnL-F sequences. The purpose is to (1)investigate the phylogenetic relationships among Roegneria awnless species and Pseudoroegneria species, (2) research the phylogenetic relationships among species inPseudoroegneria, (3) study the phylogenetic relationships among Pseudoroegneria,Elytrigia and Douglasdeweya, (4) reveal the differentiation of St genome inPseudoroegneria. The results are as follows:1. The C-banding of 11 Pseudoroegneria taxa were analyzed together with those of fiveRoegneria species, two Elytrigia taxa and two Douglasdeweya species. The resultsindicated that: (1) all diploid species (St) and tetraploid species (StSt) in Pseudoroegneriahad large telomeric bands, the telomeric bands were larger in diploid Pseudoroegneriaspecies; (2) P. geniculata ssp. scythica, Et. caespitosa and Et. caespitosa ssp. nodosa hadsimilarity in their band karyotypes, however, there were great variations among them. P.geniculata ssp. pruinifera had more interstitial and centromeric bands; (3) R. elytrigioideshad distinct telomeric bands, which were similar to those of diploid Pseudoroegneriaspecies. R. alashanica and R. magnicaespes had little and small telomeric bands, smallcentromeric bands, and more and small interstitial bands. The band karyotypes of R.grandis and R. ciliaris were similar, however, R. grandis had larger telomeric bands; (4)There were variations between band karyotypes of D. wangii and D. deweyi, the twospecies were likely to be separated.2. The genomic constitutions of R. alashanica, R. elytrigioides, R. magnieaespes, R.grandis, P. geniculata, P. geniculata ssp. scythica and P. geniculata ssp. pruinifera weredetected with GISH. P. spicata (St), P. libanotica (St), P. stipifolia (St), R. ciliaris (StY), A.cristatum (P), Lo. elongatum (E~e) and Lo. bessarabicum (E~b) were used as probes, and P.strigosa (St), ssDNA and Lo. elongatum (E~e) were used as blocking, respectively. Theresults indicated that: (1) R. alashanica contained one set of St genome, the other set ofgenome was still unknown, however it was not E, P or Y genomes; (2) R. magnicaespescontained one set of St genome, the other set of genome was still unknown, however it wasnot Y genome; (3) R. elytrigioides contained two sets of St genomes; (4) R. grandis and P.geniculata contained one set of St genome respectively, the other set of genome wasclosely related to St genome; (5) the genomic constitutions of P. geniculata ssp. scythicaand P. geniculata ssp. pruinifera were EEStSt and EEEEStSt, respectively.3. The artificial hybrids were obtained with R. alashanica(St-), R. elytrigioides (StSt), R. magnicaespes (St-), R. grandis (STY), P. geniculata (StSt), P. geniculata ssp. scythica(E~eSt), P. geniculata ssp. pruinifera (---), P. cognata (St), P. libanotica (St), P. spicata(St),P. strigosa (StSt), P. tauri (St), R. grandis (StY), Elymus wawawaiensis (HSt),Anthosachne scabra var. scabra (StWY) or Hystrix patula (HSt) as parents respectively.11 combinations were studied with genome analysis. The pollen fertility and seed set wereinvestigated. The results indicated that: (1) R. alashanica contained one set of St genome,the other set of genome was not H; (2) R. grandis contained one set of St genome, the Ygenome in R. grandis was closely related to St genome; (3) the St genome wasdifferentiated in some degree among species used in the present study.4. The ITS sequences of 17 Pseudoroegneria accessions (St or StSt) were analyzedtogether with those of seven Roegneria species (StY or StSt), four Elytrigia accessions(E~eSt or E~bE~eSt), four Lophopyrum accessions (E~b, E~e or E~bE~eE~xStSt), threeDouglasdeweya accessions (PSt), five Agropyron accessions (P), two Australopyrumaccessions (W), two Hordeum accessions (H) and two Psathyrostachys species (Ns). Thestrict consensus tree was obtained with parsimonious analysis. The results indicated that: (1)the St genomes in diploid species were differentiated, the polyploid Pseudoroegneriaspecies were closely related to diploid Pseudoroegneria species; (2) R. alashanica, R.elytrigioides, R. magnicaespes and P. geniculata were closely related; (3) StY genomeswere closely related to St genome, St genome and Y genome might have the same origin;(4) it is unreasonable to treat P. geniculata ssp. scythica and P. geniculata ssp. pruiniferaas the subspecies of P. geniculata, P. geniculata ssp. scythica, P. geniculata ssp. pruinifera,Et. caespitosa, Et. caespitosa ssp. nodosa, Et. intermedia, Et. intermedia ssp. intermediaand Lo. ponticum should be grouped into Trichopyrum; (5) E~b, E~e and St genomes wereclosely related; (6) diploid Agropyron species were the donor of P genome in D. wangiiand D. deweyi, it is reasonable to group them into Douglasdeweya.5. The trnL-F sequences of 16 Pseudoroegneria accessions (St or StSt) were analyzedtogether with those of seven Roegneria species (StY or StSt), three Elytrigia accessions(E~eSt or E~bE~eSt), four Lophopyrum accessions (E~b, E~e or E~eE~e), three Douglasdeweyaaccessions (PSt), four Agropyron accessions (P), one Australopyrum accessions (W), twoHordeum accessions (H) and two Psathyrostachys species (Ns). The strict consensus tree was obtained with parsimonious analysis. The results indicated that: (1) species inPseudoroegneria, Roegneria, Elytrigia, Lophopyrum and Douglasdeweya were clusteredinto one clade, their sequences were homologous and they were closely related; (2)thematernal donor of the polyploid species with St genome were diploid Pseudoroegneriaspecies; (3) diploid Pseudoroegneria species was the maternal donor of D. wangii and D.deweyi, and paternal donor was from diploid Agropyron species; (4) species in Agropyronand Australopyrum were clustered into one clade, their sequences were homologous andthey were closely related.更多还原