A genome assembly for Orinus kokonorica provides insights into the origin, adaptive evolution and further diversification of two closely related grass genera.

Ancient whole-genome duplication (WGD) or polyploidization is prevalent in plants and has played a crucial role in plant adaptation. However, the underlying genomic basis of ecological adaptation and subsequent diversification after WGD are still poorly understood in most plants. Here, we report a chromosome-scale genome assembly for the genus Orinus (Orinus kokonorica as representative) and preform comparative genomics with its closely related genus Cleistogenes (Cleistogenes songorica as representative), both belonging to a newly named subtribe Orininae of the grass subfamily Chloridoideae. The two genera may share one paleo-allotetraploidy event before 10 million years ago, and the two subgenomes of O. kokonorica display neither fractionation bias nor global homoeolog expression dominance. We find substantial genome rearrangements and extensive structural variations (SVs) between the two species. With comparative transcriptomics, we demonstrate that functional innovations of orthologous genes may have played an important role in promoting adaptive evolution and diversification of the two genera after polyploidization. In addition, copy number variations and extensive SVs between orthologs of flower and rhizome related genes may contribute to the morphological differences between the two genera. Our results provide new insights into the adaptive evolution and subsequent diversification of the two genera after polyploidization.

Single-gene FISH maps and major chromosomal rearrangements in Elymus sibiricus and E. nutans.

BACKGROUND: Chromosomal variations have been revealed in both E. sibiricus and E. nutans, but chromosomal structural variations, such as intra-genome translocations and inversions, are still not recognized due to the cytological limitations of previous studies. Furthermore, the syntenic relationship between both species and wheat chromosomes remains unknown. RESULTS: Fifty-nine single-gene fluorescence in situ hybridization (FISH) probes, including 22 single-gene probes previously mapped on wheat chromosomes and other newly developed probes from the cDNA of Elymus species, were used to characterize the chromosome homoeologous relationship and collinearity of both E. sibiricus and E. nutans with those of wheat. Eight species-specific chromosomal rearrangements (CRs) were exclusively identified in E. sibiricus, including five pericentric inversions in 1H, 2H, 3H, 6H and 2St; one possible pericentric inversion in 5St; one paracentric inversion in 4St; and one reciprocal 4H/6H translocation. Five species-specific CRs were identified in E. nutans, including one possible pericentric inversion in 2Y, three possible pericentric multiple-inversions in 1H, 2H and 4Y, and one reciprocal 4Y/5Y translocation. Polymorphic CRs were detected in three of the six materials in E. sibiricus, which were mainly represented by inter-genomic translocations. More polymorphic CRs were identified in E. nutans, including duplication and insertion, deletion, pericentric inversion, paracentric inversion, and intra- or inter-genomic translocation in different chromosomes. CONCLUSIONS: The study first identified the cross-species homoeology and the syntenic relationship between E. sibiricus, E. nutans and wheat chromosomes. There are distinct different species-specific CRs between E. sibiricus and E. nutans, which may be due to their different polyploidy processes. The frequencies of intra-species polymorphic CRs in E. nutans were higher than that in E. sibiricus. To conclude, the results provide new insights into genome structure and evolution and will facilitate the utilization of germplasm diversity in both E. sibiricus and E. nutans.

Molecular cytogenetic study on the plants of Elymus nutans with varying fertility on the Qinghai-Tibet Plateau.

A molecular cytogenetic investigation was conducted on plants of the allohexaploid species Elymus nutans with varying fertility on the Qinghai-Tibet Plateau. Molecular karyotyping revealed that chromosome variants were distributed unevenly among genomes and among different homologue chromosomes in each genome. The plants with varying fertility exhibited significantly higher numbers of chromosome variants than did the normal fertility samples, although both kinds of plants showed the same pattern of high-to-low polymorphism from the Y to St and H genomes. Heterozygosis and karyotype heterozygosity in the plants with varying fertility were 3- and 13-fold higher than those in normal samples, respectively. Significant negative correlations were found not only between seed setting rates and total genome heterozygosity but also between seed setting rates and heterozygosity of each genome in the plants of varying fertility. Chromosome pairing analysis was performed using genomic in situ hybridization in selected plants of different fertility levels. The pairing of chromosomes at meiotic metaphase I was mostly bivalent, although univalent, trivalent, quadrivalent, and other polyvalents also occurred; in addition, chromosome configuration forms and frequencies varied among the studied samples. ANOVA results showed that the average number of ring bivalents in the Y genome was significantly higher than those in the St and H genomes. Significant positive correlations between pollen grain fertility and ring bivalent number were found in the St and H genomes but not in the Y genome. Furthermore, chromosome configuration parameters (total bivalents, numbers of ring and rod bivalents) were found to be significantly correlated with heterozygosity and seed setting rates in the St and H genomes, respectively, but not in the Y genome. It was inferred that the seed setting rate and pollen grain fertility in E. nutans are strongly influenced by the heterozygosity of each genome, but the Y genome differs from the St and H genomes due to chromosome pair alterations. The St and H genomes may contain more chromosome structural variations than the Y genome in E. nutans.

Meiotic Chromosomal Abnormality Detected in a Heterozygote of Elymus nutans.

Elymus nutans is an allopolyploid with a genome constitution of StStYYHH (2n = 6x = 42). Highly frequent intergenomic translocations and chromosomal variations with repeat amplification and deletions in E. nutans have been identified in the previous studies. However, more complicated structural variations such as chromosomal inversions or intra-genomic translocations are still unknown in this species, so does the reason for the origin of the chromosomal variations. Heterozygotes with rearranged chromosomes always present irregular meiosis behaviors, which subsequently cause the secondary chromosome rearrangements. Investigation on the meiosis of heterozygotes, especially on the individual chromosome level, may provide the important clues to identify the more complicated chromosome structural variations in the populations, and clarify the origin of the chromosome variations. In this study, meiotic analysis was conducted on a heterozygote plant of Elymus nutans, which showed high intra- and inter-genome chromosomal variations, by sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), with each chromosome clearly recognized. The results showed chromosomal abnormalities at every meiotic stage and abnormalities in frequency variations between different sub-genomes and different individual chromosomes. The abnormalities were revealed as univalent, fragment, rod, or Y shape bivalent in diakinesis; univalent and rod bivalent in metaphase I; lagged and segregated chromatid, bridge, fragment of the sister chromatid, fragment, bridge accompanied with fragment, and unequal segregated chromosome in anaphase I; bridge and lagged chromatid in ana-telophase II; and micronucleus at uninucleate stage. Generally, the St and H genomes harbor more abnormalities than the Y genome. Moreover, a paracentric inversion in 2St was exclusively determined, and another paracentric inversion in 6Y was tentatively identified. In addition, novel deletions were clearly detected in 3H, 4H, 1Y, and 3Y homologous chromosomes; in particular, de novo pericentric inversion in 3H was repeatedly identified in metaphase I. The study revealed the chromosomal inversions pre-existed in parents or populations, as well as de novo inversions and deletions originated in the meiosis of the heterozygote in E. nutans. Moreover, it indicated wide range of meiosis abnormalities on different stages and different chromosomes, and suggests that secondary rearrangements contribute much to the chromosome variations in E. nutans.

Molecular Karyotyping on Populus simonii × P. nigra and the Derived Doubled Haploid.

The molecular karyotype could represent the basic genetic make-up in a cell nucleus of an organism or species. A doubled haploid (DH) is a genotype formed from the chromosome doubling of haploid cells. In the present study, molecular karyotype analysis of the poplar hybrid Populus simonii × P. nigra (P. xiaohei) and the derived doubled haploids was carried out with labeled telomeres, rDNA, and two newly repetitive sequences as probes by fluorescence in situ hybridization (FISH). The tandem repeats, pPC349_XHY and pPD284_XHY, with high-sequence homology were used, and the results showed that they presented the colocalized distribution signal in chromosomes. For P. xiaohei, pPD284_XHY produced hybridizations in chromosomes 1, 5, 8, and 9 in the hybrid. The combination of pPD284_XHY, 45S rDNA, and 5S rDNA distinctly distinguished six pairs of chromosomes, and the three pairs of chromosomes showed a significant difference in the hybridization between homologous chromosomes. The repeat probes used produced similar FISH hybridizations in the DH; nevertheless, pPD284_XHY generated an additional hybridization site in the telomere region of chromosome 14. Moreover, two pairs of chromosomes showed differential hybridization distributions between homologous chromosomes. Comparisons of the distinguished chromosomes between hybrid and DH poplar showed that three pairs of chromosomes in the DH presented hybridization patterns that varied from those of the hybrid. The No. 8 chromosome in DH and one of the homologous chromosomes in P. xiaohei shared highly similar FISH patterns, which suggested the possibility of intact or mostly partial transfer of the chromosome between the hybrid and DH. Our study will contribute to understanding the genetic mechanism of chromosomal variation in P. xiaohei and derived DH plants.

The Kengyiliahirsuta karyotype polymorphisms as revealed by FISH with tandem repeats and single-gene probes.

Kengyiliahirsuta (Keng, 1959) J. L. Yang, C. Yen et B. R. Baum, 1992, a perennial hexaploidy species, is a wild relative species to wheat with great potential for wheat improvement and domestication. The genome structure and cross-species homoeology of K.hirsuta chromosomes with wheat were assayed using 14 single-gene probes covering all seven homoeologous groups, and four repetitive sequence probes 45S rDNA, 5S rDNA, pAs1, and (AAG)10 by FISH. Each chromosome of K.hirsuta was well characterized by homoeological determination and repeats distribution patterns. The synteny of chromosomes was strongly conserved in the St genome, whereas synteny of the Y and P genomes was more distorted. The collinearity of 1Y, 2Y, 3Y and 7Y might be interrupted in the Y genome. A new 5S rDNA site on 2Y might be translocated from 1Y. The short arm of 3Y might involve translocated segments from 7Y. The 7 Y was identified as involving a pericentric inversion. A reciprocal translocation between 2P and 4P, and tentative structural aberrations in the subtelomeric region of 1PL and 4PL, were observed in the P genome. Chromosome polymorphisms, which were mostly characterized by repeats amplification and deletion, varied between chromosomes, genomes, and different populations. However, two translocations involving a P genome segmental in 3YL and a non-Robertsonial reciprocal translocation between 4Y and 3P were identified in two independent populations. Moreover, the proportion of heterozygous karyotypes reached almost 35% in all materials, and almost 80% in the specific population. These results provide new insights into the genome organization of K.hirsuta and will facilitate genome dissection and germplasm utilization of this species.

Identification and characterization of satellite DNAs in Poa L.

BACKGROUND: Poa L. is a large genus of grass in Gramineae, among which P. pratensis is widely cultivated as turf and forage. Satellite DNA is the main components of the plant genome. Information of satellites will helpful for dissection the genome composition and definition of the phylogeny relationship of these species. However, the knowledge about the satellites in genus Poa is still limited. RESULTS: Four satellite DNAs were identified using the Repeat Explorer pipeline in HiSeq Illumina reads from diploid plants in P. malaca (2n = 26). Two satellites showed high similarity with the previously identified PpTr-1 and PpTr-3, whereas two others are newly identified with the monomer of 326 bp (Poa-326) and 353 bp (Poa-353) respectively. The clone DNAs of PpTr-1 and PpTr-3, and oligonucleotides designed representing satellites Poa-326 and Poa-353 were probed to test on chromosomes across 13 Poa speceis with different polyploidy level by fluorescent in situ hybridization (FISH). PpTr-1, PpTr-3, and Poa-362 were stably positioned in the subtelomeric regions in nearly all species with the variation of hybridization sites number. However, Poa-353 showed different FISH patterns of multiple regions with the variation of hybridization intensity and distribution sites across species. In addition, 5S rDNA and 45S rDNA were used to characterize the genome of the Poa species. Four rDNA FISH patterns were revealed in the tested species. CONCLUSION: Four identified satellite were high conservable across Poa species. Genome distribution of these satellites can be characterized by FISH. The variation of satellite DNAs and rDNA chromosomal distributions between species provide useful information for phylogenetic analysis in genus Poa.

A chromosome-scale genome assembly of a diploid alfalfa, the progenitor of autotetraploid alfalfa.

Alfalfa (Medicago sativa L.) is one of the most important and widely cultivated forage crops. It is commonly used as a vegetable and medicinal herb because of its excellent nutritional quality and significant economic value. Based on Illumina, Nanopore and Hi-C data, we assembled a chromosome-scale assembly of Medicago sativa spp. caerulea (voucher PI464715), the direct diploid progenitor of autotetraploid alfalfa. The assembled genome comprises 793.2 Mb of genomic sequence and 47,202 annotated protein-coding genes. The contig N50 length is 3.86 Mb. This genome is almost twofold larger and contains more annotated protein-coding genes than that of its close relative, Medicago truncatula (420 Mb and 44,623 genes). The more expanded gene families compared with those in M. truncatula and the expansion of repetitive elements rather than whole-genome duplication (i.e., the two species share the ancestral Papilionoideae whole-genome duplication event) may have contributed to the large genome size of M. sativa spp. caerulea. Comparative and evolutionary analyses revealed that M. sativa spp. caerulea diverged from M. truncatula ~5.2 million years ago, and the chromosomal fissions and fusions detected between the two genomes occurred during the divergence of the two species. In addition, we identified 489 resistance (R) genes and 82 and 85 candidate genes involved in the lignin and cellulose biosynthesis pathways, respectively. The near-complete and accurate diploid alfalfa reference genome obtained herein serves as an important complement to the recently assembled autotetraploid alfalfa genome and will provide valuable genomic resources for investigating the genomic architecture of autotetraploid alfalfa as well as for improving breeding strategies in alfalfa.

Molecular karyotyping of Siberian wild rye (Elymus sibiricus L.) with oligonucleotide fluorescence in situ hybridization (FISH) probes.

Siberian wild rye (Elymus sibiricus L.), an allotetraploid species, is a potentially high-quality perennial forage crop native to temperate regions. We used fluorescently conjugated oligonucleotides, representing ten repetitive sequences, including 6 microsatellite repeats, two satellite repeats, and two ribosomal DNAs, to characterize E. sibiricus chromosomes, using sequential fluorescence in situ hybridization and genomic in situ hybridization assays. Our results showed that microsatellite repeats (AAG)10 or (AGG)10, satellite repeats pAs1 and pSc119.2, and ribosomal 5S rDNA and 45S rDNA are specific markers for unique chromosomes. A referable karyotype ideogram was suggested, by further polymorphism screening, across different E. sibiricus cultivars with a probe mixture of (AAG)10, Oligo-pAs1, and Oligo-pSc119.2. Chromosomal polymorphisms vary between different genomes and between different individual chromosomes. In particular, two distinct forms of chromosome E in H genome were identified in intra- and inter-populations. Here, the significance of these results, for E. sibiricus genome research and breeding, and novel approaches to improve fluorescence in situ hybridization-based karyotyping are discussed.

Development and Application of Transposable Element-Based Chromosomal Markers for the St Genome in Triticeae.

The St genome, originating from Pseudoroegneria (Nevski) Á. Löve, plays an important role in Triticeae. In this study, the Pseudoroegneria stipifolia genome (2n = 2x = 14, StSt) was screened to identify sequences that could be used for FISH. A total of 163 effective clones were obtained from the genomic plasmid library which was constructed by DNase I digestion of P. stipifolia nuclear genomic DNA. Analysis of these clones identified 112 with characteristics of transposable elements (TEs), 13 with characteristics of tandem repetitive sequences, 8 with characteristics of mRNA sequences, and 30 unknown sequences. Fluorescent signals were detected for 11 of 41 TE sequences on P. stipifolia chromosomes after in situ hybridization and were divided into 4 types according to signal distribution patterns: over the whole St genome chromosomes, telomere to pericentromeric regions, centromere to pericentromeric regions, and terminal regions. The affinity between St and Y genomes was studied using the 11 TE probes in 3 StStYY species. Five TE probes showed no obvious difference between subgenomes, 2 probes displayed divergence only in 2 StStYY species, and 4 probes exhibited significant differences among 3 StStYY species. These results provide a preliminary understanding of the sequence composition of the St genome and enabled 11 novel TE probes to be developed and applied.

Diversification of the P genome among Agropyron Gaertn. (Poaceae) species detected by FISH.

The genomes of five Agropyron Gaertner, 1770 species were characterized using all potential di- or trinucleotide simple sequence repeat (SSR) motifs and four satellite DNA repeats as fluorescence in situ hybridization (FISH) probes. The sites of 5S and 45S rDNA were relatively conserved among the diploid and tetraploid species. A number of sites for the dinucleotide SSRs AC, AG, and pSc119.2 was detected in all investigated species except A. mongolicum Keng, 1938. Several different trinucleotide SSRs were identified in different tetraploid species. All Agropyron species were suggested to include the basic P genome, although genome differentiation was still observed. The P genome of A. mongolicum was distinct from that of the diploid A. cristatum (Linnaeus, 1753) Gaertner, 1770. and other tetraploid species, with no hybridizations for AC, AG, or pSc119.2 observed. This finding supports designation of the P genomes of A. cristatum and A. mongolicum as Pc and Pm, respectively. An exceptional 5S rDNA site revealed in one set of homoeologous chromosomes strongly supports the allopolyploid origin of A. desertorum (Fischer ex Link, 1821) Schultes, 1824. However, the diploid donors to A. desertorum need further investigation. Similarly, the unique FISH pattern of a pair of 5S rDNA-carrying chromosomes was indicative of a potential allopolyploid origin for A. fragile (Roth, 1800) Candargy, 1984. The conserved distribution of 5S and 45S rDNA suggests A. cristatum (4x) and A. michnoi Roshevitz, 1929 are closely related. Two forms of B chromosomes were identified among individuals A. mongolicum and A. desertorum plants.

Rapid Development and Characterization of Chromosome Specific Translocation Line of Thinopyrum elongatum with Improved Dough Strength.

The protein content and its type are principal factors affecting wheat (Triticum aestivum) end product quality. Among the wheat proteins, glutenin proteins, especially, high molecular weight glutenin subunits (HMW-GS) are major determinants of processing quality. Wheat and its primary gene pool have limited variation in terms of HMW-GS alleles. Wild relatives of wheat are an important source of genetic variation. For improvement of wheat processing quality its wild relative Thinopyrum elongatum with significant potential was utilized. An attempt was made to replace Th. elongatum chromosome long arm (1EL) carrying HMW-GS genes related to high dough strength with chromosome 1AL of wheat with least or negative effect on dough strength while retaining the chromosomes 1DL and 1BL with a positive effect on bread making quality. To create chromosome specific translocation line [1EL(1AS)], double monosomic of chromosomes 1E and 1A were created and further crossed with different cultivars and homoeologous pairing suppressor mutant line PhI . The primary selection was based upon glutenin and gliadin protein profiles, followed by sequential genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH). These steps significantly reduced time, efforts, and economic cost in the generation of translocation line. In order to assess the effect of translocation on wheat quality, background recovery was carried out by backcrossing with recurrent parent for several generations and then selfing while selecting in each generation. Good recovery of parent background indicated the development of almost near isogenic line (NIL). Morphologically also translocation line was similar to recipient cultivar N61 that was further confirmed by seed storage protein profiles, RP-HPLC and scanning electron microscopy. The processing quality characteristics of translocation line (BC4F6) indicated significant improvement in the gluten performance index (GPI), dough mixing properties, dough strength, and extensibility. Our work aims to address the challenge of limited genetic diversity especially at chromosome 1A HMW-GS locus. We report successful development of chromosome 1A specific translocation line of Th. elongatum in wheat with improved dough strength.

Characterization of genome in tetraploid StY species of Elymus (Triticeae: Poaceae) using sequential FISH and GISH.

Genomes of ten species of Elymus, either presumed or known as tetraploid StY, were characterized using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH). These tetraploid species could be grouped into three categories. Type I included StY genome reported species-Roegneria pendulina, R. nutans, R. glaberrima, R. ciliaris, and Elymus nevskii, and StY genome presumed species-R. sinica, R. breviglumis, and R. dura, whose genome could be separated into two sets based on different GISH intensities. Type I genome constitution was deemed as putative StY. The St genome were mainly characterized with intense hybridization with pAs1, fewer AAG sites, and linked distribution of 5S rDNA and 18S-26S rDNA, while the Y genome with less intense hybridization with pAs1, more varied AAG sites, and isolated distribution of 5S rDNA and 18S-26S rDNA. Nevertheless, further genomic variations were detected among the different StY species. Type II included E. alashanicus, whose genome could be easily separated based on GISH pattern. FISH and GISH patterns suggested that E. alashanicus comprised a modified St genome and an unknown genome. Type III included E. longearistatus, whose genome could not be separated by GISH and was designated as StlYl. Notably, a close relationship between Sl and Yl genomes was observed.

Karyotypic evolution of the Medicago complex: sativa-caerulea-falcata inferred from comparative cytogenetic analysis.

BACKGROUND: Polyploidy plays an important role in the adaptation and speciation of plants. The alteration of karyotype is a significant event during polyploidy formation. The Medicago sativa complex includes both diploid (2n = 2× = 16) and tetraploid (2n = 2× = 32) subspecies. The tetraploid M. ssp. sativa was regarded as having a simple autopolyploid origin from diploid ssp. caerulea, whereas the autopolyploid origin of tetraploid ssp. falcata from diploid form ssp. falcata is still in doubt. In this study, detailed comparative cytogenetic analysis between diploid to tetraploid species, as well as genomic affinity across different species in the M. sativa complex, were conducted based on comparative mapping of 11 repeated DNA sequences and two rDNA sequences by a fluorescence in situ hybridization (FISH) technique. RESULTS: FISH patterns of the repeats in diploid subspecies caerulea were highly similar to those in tetraploid subspecies sativa. Distinctly different FISH patterns were first observed in diploid ssp. falcata, with only centromeric hybridizations using centromeric and multiple region repeats and a few subtelomeric hybridizations using subtelomeric repeats. Tetraploid subspecies falcata was unexpectedly found to possess a highly variable karyotype, which agreed with neither diploid ssp. falcata nor ssp. sativa. Reconstruction of chromosome-doubling process of diploid ssp. caerulea showed that chromosome changes have occurred during polyploidization process. CONCLUSIONS: The comparative cytogenetic results provide reliable evidence that diploid subspecies caerulea is the direct progenitor of tetraploid subspecies sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. However, the tetraploid subspecies falcata is far from a simple autopolyploid from diploid subspecies falcata although no obvious morphological change was observed between these two subspecies.

Isolation and characterization of chromosomal markers in Poa pratensis.

BACKGROUND: Poa pratensis L. is a turf grass and forage crop used worldwide. Being a facultative apomictic species, P. pratensis has a highly variable chromosome number. Chromosomal markers constitute a powerful tool for chromosome identification and for various aspects of genomic research. However, currently, no chromosomal markers are available for P. pratensis. RESULTS: Four novel chromosome markers were isolated from a screen of Cot-1 DNA libraries, combined with fluorescence in situ hybridization (FISH) in Poa pratensis. Three tandemly repetitive sequences (PpTR-1, PpTR-2, and PpTR-3) were characterized as subtelomeric. Monomers of 318 bp, 189 bp and 189 bp were identified in PpTR-1, PpTR-2, and PpTR-3, respectively. One tandemly repetitive sequence (PpCR-1) was shown to be centromeric or pericentromeric, and it had a monomer of 27 bp. The distribution patterns of PpTR-1, PpTR-2, and PpTR-3 were highly conserved across different P. pratensis cultivars and in the distantly related Poa species, whereas PpCR-1 was conserved across different P. pratensis cultivars, but less conserved across Poa species. CONCLUSION: In this study, we report the identification and characterization of four novel chromosomal markers in P. pratensis. These chromosomal markers are powerful tools for accurate assessment of chromosome count, genomic and phylogenetic analyses, as well as studies of apomixis in P. pratensis.

High molecular karyotype variation revealed in indigenous Elymus nutans in the Qinghai Plateau.

The karyotypes of 27 individuals of Elymus nutans from eight wild populations in the Qinghai Plateau were analyzed using sequential FISH and GISH. High FISH pattern polymorphism and karyotype variation were detected within and among populations. The chromosome variations were mainly characterized as repeat deletions and amplifications along with inter-genomic translocations. The chromosomes of the St and Y genomes demonstrated higher polymorphism than those of the H genome. Six different inter-genomic translocations were identified in 33.3% of individuals; type I and II translocations were detected with higher frequency. Further analysis revealed that type I and II translocations were distributed in different geographic regions. The origin of high karyotype variation of E. nutans in the Qinghai plateau is further discussed.

Chromosomal organization of repetitive DNAs in Hordeum bogdanii and H. brevisubulatum (Poaceae).

Molecular karyotypes of Hordeum bogdanii Wilensky, 1918 (2n = 14), and Hordeum brevisubulatum Link, 1844 ssp. brevisubulatum (2n = 28), were characterized by physical mapping of several repetitive sequences. A total of 18 repeats, including all possible di- or trinucleotide SSR (simple sequence repeat) motifs and satellite DNAs, such as pAs1, 5S rDNA, 45S rDNA, and pSc119.2, were used as probes for fluorescence in situ hybridization on root-tip metaphase chromosomes. Except for the SSR motifs AG, AT and GC, all the repeats we examined produced detectable hybridization signals on chromosomes of both species. A detailed molecular karyotype of the I genome of Hordeum bogdanii is described for the first time, and each repetitive sequence is physically mapped. A high degree of chromosome variation, including aneuploidy and structural changes, was observed in Hordeum brevisubulatum. Although the distribution of repeats in the chromosomes of Hordeum brevisubulatum is different from that of Hordeum bogdanii, similar patterns between the two species imply that the autopolyploid origin of Hordeum brevisubulatum is from a Hordeum species with an I genome. A comparison of the I genome and the other Hordeum genomes, H, Xa and Xu, shows that colocalization of motifs AAC, ACT and CAT and colocalization of motifs AAG and AGG are characteristic of the I genome. In addition, we discuss the evolutionary significance of repeats in the genome during genome differentiation.

Genome analysis of seven species of Kengyilia (Triticeae: Poaceae) with FISH and GISH.

The genome compositions and genetic relationships of seven species of Kengyilia were assessed using a sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) technique. Five species, K. kokonorica, K. rigidula, K. hirsuta, K. grandiglumis, and K. thoroldiana, are native to Qinghai (China). The other two, K. alatavica and K. batalinii, are distributed in Xinjiang (China) and Kyrgyzstan, respectively. Each chromosome could be easily identified using chromosome markers (45S rDNA, 5S rDNA, pAs1, and AAG repeats) by FISH and allocated to the St, P, or Y genome by GISH. Molecular karyotype comparison indicated that K. alatavica and K. batalinii were distinct from the Qinghai species in all three genomes. These results support that the species of Kengyilia from Central Asia and the Qinghai-Tibetan plateau have independent origins. Genomic differentiation was still detected among the species of Kengyilia from Qinghai. Specifically, a common species-specific pericentric inversion was identified in both K. grandiglumis and K. thoroldiana, and an identical St-P non-Robertsonian translocation was frequently detected in K. hirsuta. The Qinghai species formed three genetic groups, K. kokonorica-K. rigidula, K. hirsuta, and K. grandiglumis-K. thoroldiana. The possible role of species-specific inversions and translocations in the evolution of StPY species is discussed.

Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.).

Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70-80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.

Characterization of alien chromosomes in backcross derivatives of Triticum aestivum × Elymus rectisetus hybrids by using molecular markers and sequential multicolor FISH/GISH.

Wild Triticeae grasses serve as important gene pools for forage and cereal crops. Based on DNA sequences of genome-specific RAPD markers, sequence-tagged site (STS) markers specific for W and Y genomes have been obtained. Coupling with the use of genomic in situ hybridization, these STS markers enabled the identification of the W- and Y-genome chromosomes in backcross derivatives from hybrids of bread wheat Triticum aestivum L. (2n=42; AABBDD) and Elymus rectisetus (Nees in Lehm.) Á. Löve & Connor (2n=42; StStWWYY). The detection of six different alien chromosomes in five of these derivatives was ascertained by quantitative PCR of STS markers, simple sequence repeat markers, rDNA genes, and (or) multicolor florescence in situ hybridization. Disomic addition line 4687 (2n=44) has the full complement of 42 wheat chromosomes and a pair of 1Y chromosomes that carry genes for resistance to tan spot (caused by Pyrenophora tritici-repentis (Died.) Drechs.) and Stagonospora nodorum blotch (caused by Stagonospora nodorum (Berk.) Castellani and Germano). The disomic addition line 4162 has a pair of 1St chromosomes and 21 pairs of wheat chromosomes. Lines 4319 and 5899 are two triple substitution lines (2n=42) having the same chromosome composition, with 2A, 4B, and 6D of wheat substituted by one pair of W- and two pairs of St-genome chromosomes. Line 4434 is a substitution-addition line (2n=44) that has the same W- and St-genome chromosomes substituting 2A, 4B, and 6D of wheat as in lines 4319 and 5899 but differs by having an additional pair of Y-genome chromosome, which is not the 1Y as in line 4687. The production and identification of these alien cytogenetic stocks may help locate and isolate genes for useful agronomic traits.