| Elymus L.was established by Linnaeus(1753)and the type species is Elymus?sibiricus L.According to the genomic system of classification(Love 1984),Elymus is the largest genus in the Triticeae which consists of about 150 species.They are widely distributed in temperate and subtropic regions.The definition and taxonomic status is still under discussion since its establishment.There are 12 species and 1 variety recorded in Elymus in Flora of China(Kuo,1987).Most of the species are hexaploid and used as forage grass in China.Elymus dahuricus,E.excelsus,E.tangutorum,E.cylindricus,E.purpuraristatus and E.villifer were included in E.dahuricus complex because they share subtle morphological difference(Lu 1993a).Based on the morphological characters and molecular data,Baum et al.(2011)and Yen and Yang(2013)treated E.excelsus,E.tangutorum and E.cylindricus as varieties of E.dahuricus in Campeiostachys Drobov.In China,Elymus breviaristatus and E.sinosubmuticus have been listed as the nationally key protected wild plants since 1999.They are widely used as forage grass in China for its advantages of high yield,high adaptability and drought,cold resistance.However,their chromosome number is not clear.There is lack of other biosystematics data for E.purpuraristatus and E.atratus.In the study,karyotype analysis,genome analysis,GISH and multicolor GISH as well as phylogenetic analysis using 5S nrDNA sequences have been done to determine the genomic constitution of the Elymus species distributed in China,and to investigate the taxonomic status or the phylogenetic relationships of E.dahuricus complex,E.breviaristatus and E.sinosubmuticus.The main results are as follows:1.The karyotype analysis of nine species of Elymus suggested that(1)the chromosome number of all the nine species is 42 and all the species are hexaploid;(2)The karyotype formula of Elymus breviariatatus,E.cylindricus,E.dahuricus,E.purpuraristatus and E.tangutorum is 2n = 6x = 42 = 36m + 6sm,and their karyotype type is 2A;(3)The karyotype formula of E.atratus and E.nutans is 2n = 6x = 42 ?38m + 4sm,and their karyotype type is 2A;(4)The karyotype formula of E.excelsus is 2n = 6x = 42 = 34m + 8sm,and its karyotype type is 2A.The karyotype formula of E.sinosubmuticus is 2n = 6x = 42 = 38m + 4sm,and its karyotype type is 2B;(5)Satellites were observed in E.beviariatatus,E.atratus,E.nutans and E.sinosubmuticus.2.Chromosome pairing at meiotic metaphase I of interspecific or intergeneric hybrids between Elymus species and the related species showed that(1)10.48,11.12 and 20.92 bivalents per cell were observed in the hybrids between Elymus tangutorum and Elymus wawawaiensis(StH),Roegneria grandis(StY)and Elymus dahuricus(StYH)with a C-value of 0.58,0.60 and 0.96,respectively,suggesting that E.tangutorum contains StYH genomes;(2)There were 10.0,11.30 and 20.92 bivalents per cell in the hybrids of E.cylindricus x E.wawawaiensis,E.cylindricus x R.grandis E.cylindricus×Edahuricus with a C-value of 0.46,0.64 and 0.92,respectively,indicating that E.cylindricus constitutes StYH genomes;(3)Hybrids between E.excelsus and R.grandis,E.dahuricus had 10.13 and 20.86 bivalents per cell with a C-value of 0.60,0.97,respectively,suggesting that E.excelsus has StYH genomes;(4)Hybrids E.breviaristatus x E.dahuricus and E.breviaristatus x E.cylindricus contained 19.60 and 19.27 bivalents per cell with a C-value of 0.82 and 0.79,respectively,indicating that E.breviaristatus shares homologous genomes with E.cylindricus and the genomic constitution of E.breviaristatus is StYH;(5)6.46 bivalents and 0.14 trivalents per cell was observed in the hybrid of E.wawawaiensis x R.grandis,indicating that E.wawawaiensis and R.grandis share a homologous St genome and there is a certain degree of homoeology between the St and Y genomes.3.Hordeum bogdanii(H),Pseudoroegneria libanotica(St)and Roegneria ciliaris(StY)were used as genomic probes in the GISH or multicolor GISH to detect the genomic constitution of the hexaploid Elymus species.The results showed that(1)E.atratus,E.breviaristatus and E.sinosubmuticus have StYH genomes and there was no intergenomic rearrangement;(2)Species of the E.dahuricus complex(E.cylindricus,E.dahuricus,E.excelsus and E.tangutorum)share same basic StYH genomes and E.purpuraristatus has one set of H genome;(3)Intergenomic translocations were observed in the species of E.dahuricus complex and different kinds of intergenomic translocations were observed in different populations of E.excelsus and E.tangutorum from different geographic locations.These results were agreement with the results from meiotic pairing data,suggesting that GISH provides a good method to detect the genomic constitution of the species in Triticeae.4.The taxonomic status of the species of E.dahuricus complex is not clear because of their subtle morphological difference.To investigate the relationships between the species in the complex,16 hybridizations were made among the species.Chromosome pairing at meiotic metaphase I of the hybrids was analyzed and pollen fertility,seed set of the parents and hybrids were recorded.The results showed that(1)The meiotic pairing in the hybrids was quite regular with most of the cells had 21 bivalents(most of them are ring bivalents)and the C-value is above 0.90,showing that the species share homologous genomes;(2)The pollen fertility and seed set of the parents were normal.The pollen fertility of the hybrids ranged from 75.73%to 94.51%and the seed set of the hybrids ranged from 62.96%to 80.11%,indicating that there was no reproductive isolation among the species and they should be treated as the varieties of E.dahuricus.However,the species in E.dahuricus complex are widely distributed and there may be differentiation among the populations.5.Multicolor GISH was used to investigate the genomic alterations of species in E.dahuricus complex(E.dahuricus,E.cylindricus,E.excelsus)and six populations of E.tangutorum from Sichuan with different elevations.The results showed that(1)7H/1Y chromosomes were involved in reciprocal translocations for E.dahricus,E.cylindricus,E.excelsus and five populations of E.tangutorum.Species in E.dahuricus complex should be treated as the varieties of E.dahuricus for their closely relationship.We propose that the translocation is species-specific in the E.dahuricus complex,may be associated with allopolyploidy,and may help to restore fertility and nucleocytoplasmic compatibility in a newly formed allohexaploid and facilitate the successful establishment of E.dahuricus as a stable species;(2)No St/Y translocation was observed,suggesting that St genome may share some homology with Y genome and there is lower "genomic-stress" elicited by the union of diverged genomes upon allopolyploidy;(3)Eight chromosomes were involved in translocations in population Y 2228 of E.tangutorum from the highest altitude(4150 m).7H/1Y intergenomic translocations were observed.6H and 7St were also involved in intergenomic translocations in population Y 2228.The genomic changes may contribute to promote the genetic variability.More complicated translocations in population Y 2228 help to adapt to more changeable and harsher environmental conditions and facilitate its rapid and successful establishment in nature.6.To detect the genomic constitution and investigate the evolutionary relationships among the species,we obtained 271 5S nrDNA sequences from 27 accessions of the hexaploid Elymus.We identified Long H1,Short S1 and Long Y1 unit classes in nine species.The presence of the 3 unit classes was confirmed by the NJ tree of each unit class from PAUP and the phylogeny tree of three unit classes from MrBayes.The results suggested that these hexaploid Elymus species distributed in China comprise StYH genomes.The phylogeny tree showed that St unit class and Y unit class can be clearly separated.However,Y unit class sequences formed a sister clade to St unit class sequences,implying that St and Y genomes are distinct from one another,but they may have some affinity.The phylogeny tree indicated thatspecies in sect.Turczaninovia(E.cylindricus,E.dahuricus,E.tangutorum,E.purpuraristatus and E.dahuricus var.violeus)may share a more recent commonancestor,while species in sect.Elymus(E.nutans,E.breviaristatus,E.sinosubmuticus and E.atratus)share closely relationship.By identifying one type of unit class for each genome,we propose that 5S nrDNA sequences of species in Campeiostachys may have undergone genome-specific concerted evolution,while concerted evolution of each genome evolves independently.7.The genomic constitution of E.cylindricus,E.excelsus and E.tangutorum is StYH and they share a close relationship based on the chromosome pairing data at MI in hybrids,GISH analysis and 5S nrDNA sequences.According to the genomic system of classification,E.cylindricus,E.excelsus and E.tangutorum should be treated as Campeiostachys dahurica(Turcz.ex Griseb.)B.R.Baum,J.L.Yang&C.Yen var.cylindrica(Franch.)B.R.Baum,J.L.Yang&C.Yen,C.dahurica(Turcz.ex Griseb.)B.R.Baum,J.L.Yang&C.Yen var.excelsis(Turcz.ex Griseb.)B.R.Baum,J.L.Yang&C.Yen and C.dahurica(Turcz.ex Griseb.)B.R.Baum,J.L.Yang&C.Yen var.tangutorum(Nevski)B.R.Baum,J.L.Yang&C.Yen,respectively.E.breviaristatus contains StYH genomes,indicating that it should be included in Campeiostachys and treated as C.breviaristata(Keng)C.R.Yang,H.Q.Zhang&Y.H.Zhou. |
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