Phylogenetic Analysis Of Hystrix And Leymus In Triticeae

The tribe Triticeae, which includs about28genus and380species, is an enormous important group in Poaceae. Many of species in Triticeae are important cereal and forage grasses, which are precious germplasm resources in crop improvement and forage breeding. So, it is important to study the genetic and phylogenetic relationships among Triticeae species before we use the Triticeae germplasms.Hystrix Moench is a small perennial genus of the tribe Triticeae. According to the distinct morphological character of highly reduced glumes or long setaceous awn-shaped ones, if present, it was established by Moench (1794). The type species is Hystrix patula Moench. Dewey (1982,1984) reported that Hy. patula contains the StH genome, and combined the species of Hystrix into Elymus. Several studies have suggested that the genomes of the other species of Hystrix are different from the StH genome of Hy. patula (Jensen&Wang1997; Svitashev et al.1998; Muramatsu2001; Zhang et al.2002; Zhang and Zhou2006; Zhang et al.2006). Jensen&Wang (1997) proposed that Hy. coreana and Hy. californica had the same genome constitution NsXm as Leymus Hochst., and combined these two species into Leymus. The chromosome pairing and genomeinsitu hybridization (GISH) analyses indicated that Hy. coreana, Hy. duthiei and Hy. duthiei ssp. longearistata shared the NsXm genomes of Leymus, and should be treated as species of Leymus (Zhang et al.2006; Zhang&Zhou2006). Based on the Southen and GISH analysis, Ellneskog-Staam et al.(2007) advocated that Hy. komarovii most likely had a variant of the StH genome of Hy. patula, and should be transferred to the genus Elymus. It was also considered that Hy. coreana, Hy duthiei and Hy. duthiei ssp. longearistata had the Ns’Ns2genome constitutions (Ellneskog-Staam et al.2007). Therefore, the definition of Hystrix and its precise taxonomic status are still under discussion today. Some authors included the species in either Hystrix (Sakamoto1973; Kuo1987; Osada1993; Baden et al1997; Zhou et al.2000) or Asperella (Keng1959; Baum1983; Ohwi1984; Koyama1987), while others regarded it as a part of Elymus (Dewey1982; Love1984) or Leymus (Jensen&Wang1997; Zhang et al.2006). Now the disputes about Hystrix are:whether it is a valid genus? What are the genome constitutions of Hystix species? How about the phylogenotic relationships between Hystrix and its related species?In order to inspect the genomic constitution of Hystrix species and their precise taxonomic status, and relationship between Hystrix and Leymus, the leaf epidermal micromorphology, lamina anatomy, interspecific hybridization, chromosome pairing behavior at MI, genomic in situ hybridization (GISH), molecular phylogenetic analysis from chloroplast atpB-rbcL, nuclear RPB2and ITS sequences for Hystrix species and their related species were carried out in this study. The main results showed as follows:1. To reveal the phylogenetic relationships and maternal donor of species in Hystrix and Leymus, the chloroplast atpB-rbcL sequences were analyzed for Hystrix and its related species. The results indicated that:(1) Hy. patula was closely related to Elymus, and the maternal donor of Hy. patula was the St genome;(2) Hy. duthiei and Hy. duthiei ssp. longearistata were closely related to the Eurasia Leymus, and the maternal donor of them were the Ns genome from Psathyrostachys;(3) Hy. coreana and Hy. komarovii were closely related to the Leymus species in North America, and their maternal donors might be the Xm genome.2. Low copy nuclear gene RPB2was analyzed for Hystrix and its related species. The results showed:(1) Ns copy sequences of RPB2of Hystrix and Leymus have more diversity than that in Psathyrostachys, and Tajima’s and Fu and Li’s D values were all negative significant for RPB2gene on the Ns and Xm genome, indicated that population expansion and rapid genetic differentiation might have occurred among the species with NsXm genome in Leymus and Hystrix;(2) More than2copies of RPB2in Hy. coreana, Hy. duthiei, Hy. duthiei ssp. longearistata, L. karelinii, L. innovatus, L. paboauns, L. salinus, L. shanxiens and L. mollis were obtained, and7sequences from Hy. coreana, Hy. duthiei, L. salinus and L. karelinii were detected as recombination sequenses;(3) Hy. patula was closely related to Pseudoroegneria, Hordeum and Elymus;(4) Hy. duthiei, Hy. duthiei ssp. longearistata, Hy. coreana and Hy. komarovii contain NsXm genome, and were closely related to Psathyrostachys and Leymus;(5) H genome of Hordeum may involve in the composition of Xm genome by introgression;(6) L. mollis distributed in North America might have migrated from Central Asia, and have close relationship with L. multicaulis; L. mollis might have gene exchange with the species contained StH genome by some way.3. Sequence and phylogenitical analysis were carried out for ITS sequence from species of Hystrix and Leymus combined with30diploid species in Triticeae. The results were:(1) abundent ITS polymorphism were detected among Hystrix and Leymus, and the pattern of rDNA variation is associated with geographic distribution pattern;(2) Ns-genomic types of Hystrix and Leymus were originated from Psathyrostachys, P/F and St genomic types of Leymus were originated from AgropyronlEremopyrum, and Pseudoroegneria species, respectively;(3) the occurrence of a higher proportion of Hystrix and Leymus species with dominant uniparental rDNA type may associated with the segmental allopolyploid origin, and adaptive radiation in Hystrix and Leymus.4. A total of23interspecific or intergeneric cross combinations involving Hystrix and Leymus species were performed, and13hybrid F1plants were obtained. Meiosis analysis of3hybrids from species of Leymus were carried out. Meiosis analysis showed that:(1) in the tetraploid hybrids of L. multicaulis x L. crassiusculus, L. qinghaicus x L. multicaulis, and L. multicaulis x L. qinghaicus, an average of12.87,12.07and11.97bivalents per cell was observed at MI, respectively, suggesting that L. crassiusculus and L. qinghaicus share the same basic NsXm genome of L. multicaulis. Thus, it is reasonable that L. crassiusculus and L. qinghaicus were treated in Leymus.5. Genomic in situ hybridization (GISH) analysis was carried out in three Hystrix species (Hy. komarovii, Hy. coreana and Hy. duthiei ssp. longearistatd) and4Leymus species (L. flexus, L. mundus, L. racemosus and L. secalinus). GISH analysis indicated that:(1) Hy. komarovii has the NsXm genome instead of the StH genome;(2) Hy. komarovii, Hy. coreana and Hy. duthiei ssp. longearistata have the NsXm genomeof Leymus;(3) in species of Leymus, the Xm genome is closely related to Ns genome, and the Xm genome may be another Ns genome;(4) Ee genome is homology with the NsXm genome in a lower degree;(5) Xm genome did not origin from P genome.6. The leaf epidermal micromorphology and anatomical structure of Hystrix, Leymus and Elymus was examined under light microscope. The results showed that:(1) a number of variations of epidermal micromorphology and anatomical structure exist at the species level, and may be used for distinguishing different speicies, but not for different genera;(2) variations of epidermal features includes variation in morphology and wall thickness of long cells, morphology and distribution patterns of short cells and distribution of prickles;(3) the leaf anatomical features such as the presence or absence of keel, the outline of keel if it is present, arrangement of vascular bundles, have more distinguishing value between different species;(4) the variation of epidermal micromorphology and anatomical structure is related closely to environment.