Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.     

Varying chromosome composition of 56-chromosome wheat x Thinopyrum intermedium partial amphiploids.

Thinopyrum intermedium (2n = 42) is a source of many potentially useful genes for wheat improvement. Many partial amphiploids have been produced between Th. intermedium and Triticum aestivum that are fertile and stable. These partial amphiploids all have 56 chromosomes, including seven pairs of chromosomes from Th. intermedium. To explore the genomic composition of these lines, meiotic analysis was conducted on 32 hybrid combinations between eight different partial amphiploids. All but two of the chosen parents were distinguishable on the basis of perenniality, head morphology, and reactions to leaf, stripe, and stem rusts and to barley yellow dwarf virus. Chromosome pairing in the hybrids clearly indicated that all but two of the partial amphiploids differed in their composition of Thinopyrum chromosomes. The differences varied from one to five chromosomes. This confirms molecular evidence that the extra genome of the octoploid partial amphiploids is a variable synthetic genome combining chromosomes of the three Thinopyrum genomes E, J, and X. Though the extra synthetic genomes vary widely between different octoploids, they are nevertheless stable once formed. It is argued that the failure to establish these octoploid amphiploids as a new crop is a consequence of their differing chromosome complements, which makes it impractical to interbreed them.     

普通小麦与长穗偃麦草杂种及其衍生材料的细胞遗传学特性

对十倍体长穗偃麦草(Thinopyrum ponticum)与普通小麦杂交F1及其与普通小麦回交BC1F1的形态学和细胞学特性进行了分析。结果表明,长穗偃麦草与普通小麦‘兰考矮早八’衍生F1(‘兰考小偃麦’)的根尖细胞染色体数为56条;花粉母细胞减数分裂中期Ⅰ染色体构型平均值为19.81Ⅰ+15.78Ⅱ+0.75Ⅲ+0.59Ⅳ;基因组荧光原位杂交(GISH)显示,兰考小偃麦中含有35条完整的长穗偃麦草和21条小麦染色体。‘兰考小偃麦’/‘科育818’和‘兰考小偃麦’/‘Cp02-3-5-5’杂交F1的根尖细胞染色体数及其所遗传的长穗偃麦草染色体数分别为50~52和16~22条,且存在染色体易位;花粉母细胞减数分裂中期Ⅰ平均染色体构型为14.54Ⅰ+17.40Ⅱ+0.55Ⅲ+0.14Ⅳ,平均49.4%的细胞出现多价体(三价体或四价体)。这些材料为创造小麦-长穗偃麦草新种质奠定了基础。     

The transfer and characterization of resistance to common root rot from Thinopyrum ponticum to wheat.

Common root rot, caused by Cochliobolus sativus (Ito and Kurib) Drechs. ex Dastur, is a major soil-borne disease of spring and winter wheat (Triticum aestivum L. em Thell.) on the Canadian prairies. Resistance to common root rot from Thinopyrum ponticum (Podp.) Liu and Wang was transferred into wheat via crossing with Agrotana, a resistant wheat - Th. ponticum partial amphiploid line. Evaluation of common root rot reactions showed that selected advanced lines with blue kernel color derived from a wheat x Agrotana cross expressed more resistance than the susceptible T. aestivum 'Chinese Spring' parent and other susceptible wheat check cultivars. Cytological examination revealed 41 to 44 chromosomes in the advanced lines. Genomic in situ hybridization, using total genomic DNA from Pseudoroegneria strigosa (M. Bieb) A. L?ve (St genome) as a probe, demonstrated that the blue kernel plants had two pairs of spontaneously translocated J-Js and Js-J chromosomes derived from the J and Js genome of Th. ponticum. The presence of these translocated chromosomes was associated with increased resistance of wheat to common root rot. The lines with blue aleurone color always had a subcentromeric Js-J translocated chromosome. The subtelocentric J-Js translocated chromosome was not responsible for the blue kernel color. The genomic in situ hybridization analysis on meiosis revealed that the two spontaneous translocations were not reciprocal translocations.     

St基因组中的CRW同源序列在偃麦草中的FISH分析

为了确定十倍体长穗偃麦草(Thinopyrum ponticum, Liu & Wang)和六倍体中间偃麦草(Th. intermedium, [Host] Barkworth & Dewey )的基因组组成, 根据野生一粒小麦(Triticum boeoticum)着丝粒自主型反转录转座子(CRW)序列设计特异引物, 以二倍体拟鹅观草(Pseudoroegneria spicata, Á Löve )基因组 DNA为模板进行PCR扩增, 筛选到一条St基因组着丝粒区相对特异反转录转座子的部分序列pStC1, 长度为1.755 kb (GenBank登录号: FJ952565), 其中有800 bp与小麦着丝粒反转录转座子(CRW)的LTR区高度同源, 另有小部分片段与其外壳蛋白编码基因(gag)部分同源, 并且包含一段富含AGCAAC碱基的重复序列。以pStC1为探针, 对十倍体长穗偃麦草的FISH检测结果显示其基因组组成为两个St组3个E组(St₁St₂E^eE^bE^x); pStC1与中间偃麦草杂交时, 不仅St基因组上有强烈的荧光信号, 而且E基因组一些染色体的近着丝粒区域也有杂交信号, 说明偃麦草属异源多倍体物种在其形成及进化过程中St与E基因组之间在着丝粒及近着丝粒相关区域可能存在协同进化。     

Molecular characterization of a wheat--Thinopyrum ponticum partial amphiploid and its derivatives for resistance to leaf rust.

Leaf rust (caused by Puccinia triticina Eriks.) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang has provided several leaf rust resistance genes to protect wheat from this fungal disease. Three chromosome substitution lines, Ji806, Ji807, and Ji859, and two chromosome addition lines, Ji791 and Ji924, with a winter growing habit were developed from crosses between wheat (Triticum aestivum L. em Thell.) and the wheat - Th. ponticum partial amphiploid line 693. These lines were resistant to leaf rust isolates from China. Sequence-tagged site (STS) analysis with the J09-STS marker, which is linked to the gene Lr24, revealed that the partial amphiploid line 693 and all of the substitution and addition lines carried gene Lr24. Genomic in situ hybridization (GISH) analysis was carried out on chromosome preparations using total genomic DNA from Pseudoroegneria strigosa (M. Bieb) A. L?ve (St genome, 2n = 14) as a probe in the presence of total genomic DNA from T. aestivum 'Chinese Spring' wheat (ABD genomes, 2n = 42). The GISH analysis demonstrated that these lines had a pair of chromosomes displaying the typical pattern of a Js genome chromosome. This indicates that the chromosome that carries gene Lr24 belonged to the Js genome of Th. ponticum. In addition to 40 wheat chromosomes, eight Js and eight J genome chromosomes were also differentiated by GISH in the partial amphiploid line 693. Since most sources of Lr24 have a red grain color, the white-colored seeds in all of these substitution and addition lines, together with high protein content in some of the lines, make them very useful as a donor source for winter wheat breeding programs.     

Identification of Blue-grained Wheat Translocation Lines Using Fluorescence in Situ Hybridization

The blue-grained wheat substitution line (blue 58) originated from wild hybridization between Triticum aestivum L. and Agropyron elongatum (Host) Beauv= Elytrigia elongatum (Host) Nevski= Thinopyrum ponticum (Host) Barkworth and Dewey (2n=10x=70) was irradiated and four translocation lines were screened by fluorescence in situ hybridization from the offsprings. The results obtained include the following: (1) both the two translocation lines, 9906 and 9902, have 42 chromosomes. The length of the translocated blue-grained segment was approximately one-third of the short-arm and one-half of the long-arm of the translocated wheat chromosome in 9906 and 9902, respectively, and the blue-grained translocated segment in 9902 was located on D genome; (2) both 9915 and 9904 have 44 chromosomes. One pair of chromosomes was translocated and two chromosomes from Th. ponticum were added in 9903, while two pairs of chromosomes were translocated in 9904 by blue-grained wheat segment. The location and application of blue-grained wheat translocation lines were discussed.     

蓝粒小麦易位系的荧光原位杂交鉴定

普通小麦（Triticum aestivum L.)和长穗偃麦草（Agropyron elongatum (Host)Beauv=Elytriga elongatum(Host)Nevski=Thinopyrum ponticum (Host)Barkworth and Dewey,2n=10x=70)杂交后选育出的蓝粒小麦异代换系（蓝５８）,２n=42其中９９０６中被易位蓝粒片段的相对长度约占易位小麦染色体短臂的１／３,而９９０２中被易位蓝粒片段的相对长度约占易位小麦染色体长臂的１／２,并将９９０２的蓝粒易位片段定位在小麦Ｄ组染色体上;（２）９９１５易位附加和９９０４易位－易位附加,其体细胞染色体数均为４４,其中９９１５的体细胞染色体只有一对发生了易位,另外队了两条长穗偃麦草染色体;而９９０４有两对染色体发生了易位,并易位系中控制蓝粒性状的长穗偃麦草染色体片段的定位和蓝粒小麦易位系的应用进行了讨论。     

Characterization of derivatives from wheat-Thinopyrum wide crosses

Partial amphiploids are lines that contain 42 (38-42) wheat and 14 (14-18) alien chromosomes. They are derived by backcrossing wheat onto hybrids between wheat and either Thinopyrum intermedium (6x) or Th. ponticum (10x). GISH analysis has shown that, with possibly one exception, the alien genomes (chromosome sets) in partial amphiploids are found to be hybrids i.e. composed of chromosomes from more than one alien genome. The individual partial amphiploids are meiotically stable and nearly perfectly fertile, but hybrids between different lines were characterized by varying numbers of unpaired chromosomes and consequently variable degrees of sterility. Translocated chromosomes involving different Thinopyrum genomes or Thinopyrum and wheat genomes were found in partial amphiploids and consequently in the addition lines derived from them. Partial amphiploids have proven to be an excellent tertiary gene pool for wheat improvement, containing resistance to biotic stresses not present in wheat itself. Resistance to Barley Yellow Dwarf Virus (BYDV) and Wheat Streak Mosaic Virus (WSMV) have been found in partial amphiploids and addition lines derived from both Th. intermedium and Th. ponticum. Excellent resistance to Fusarium head blight has been found on a Th. intermedium chromosome that had substituted for chromosome 2D in wheat. Genes for resistance to leaf rust and stem rust have already been incorporated into wheat and tagged with molecular markers.     

Identification of the parental chromosomes of the wheat-alien amphiploid Agrotana by genomic in situ hybridization.

Labelled total genomic DNA from four alien species, Thinopyrum ponticum (Host) Beauv. (2n = 70, genomes J1J1J1J2J2), Th. bessarabicum (Savul. &Rayss) Love (2n = 14, genome J), Th. elongatum (Host) Beauv. (2n = 14, genome E), and Haynaldia villosa (L.) Schur. (2n = 14, genome V), were used as probes in combination with blocking wheat DNA for in situ hybridization of the chromosomes of Agrotana, a wheat-alien hybrid (2n = 56) of unknown origin. The results showed that genomic DNA probes from Th. ponticum and Th. bessarabicum both clearly revealed 16 alien and 40 wheat chromosomes in Agrotana, indicating that the J genome present in these two species has a high degree of homology with the alien chromosomes in Agrotana. Biotinylated genomic DNA probe from Th. elongatum identified 10 chromosomes from Agrotana, while some regions of six other chromosomes yielded a weak or no signal. The probe from H. villosa produced no differential labelling of the chromosomes of Agrotana. The genomic formula of Agrotana was designated as AABBDDJJ. We suggest that the alien parent donor species of Agrotana is Th. ponticum rather than Th. bessarabicum. Genomic relationships of the three Thinopyrum species are discussed in relation to the distribution of GISH signals in the chromosomes of Agrotana.     

Decaploidy in Fragaria iturupensis (Rosaceae)

The strawberry genus, Fragaria (Rosaceae), has a base chromosome number of x = 7. Cultivated strawberries (F. ×ananassa nothosubsp. ananassa) are octoploid (2n = 8x = 56) and first hybridized from F. chiloensis subsp. chiloensis forma chiloensis × F. virginiana subsp. virginiana. Europe has no known native octoploid species, and only one Asian octoploid species has been reported: F. iturupensis, from Iturup Island. Our objective was to examine the chromosomes of F. iturupensis. Ploidy levels of wild strawberry species, include diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), pentaploid (2n = 5x = 35), hexaploid (2n = 6x = 42), octoploid (2n = 8x = 56), and nonaploid (2n = 9x = 63). Artificial triploid (2n = 3x = 21), tetraploid, pentaploid, octoploid, decaploid (2n = 10x = 70), 16-ploid, and 32-ploid plants have been constructed and cultivated. Surprisingly, chromosome counts and flow cytometry revealed that F. iturupensis includes natural decaploid genotypes with 2n = 10x = 70 chromosomes. This report is the first of a naturally occurring decaploid strawberry species. Further research on F. iturupensis and exploration on northern Pacific islands is warranted to ascertain the phylogeny and development of American octoploid species.     

Polymorphic nuclear gene sequences indicate a novel genome donor in the polyploid genus Thinopyrum

For decades, the wheatgrass genus Thinopyrum has been of interest to plant breeders as a source of genes that confer competitive traits. This genus has been a considerable challenge to plant systematists because of the impacts of polyploidization on the evolution of this group. This study was aimed to augment existing cytogenetic data with a sequence-based investigation of the genomes of these species. Sequences of the internal transcribed spacer 1 (ITS1), introns 9 through 11 of the granule-bound starch synthase (GBSSI) gene and intron III of the beta-amylase gene (Bmy1) were isolated from the genomes of polyploid Thinopyrum species by PCR, cloning and sequencing and the evolutionary distances between these species and putative diploid ancestors were estimated with Kimura's two-parameter method. Phylogenetic analysis of these sequences largely agrees with what has been established through cytogenetic means for the Th. caespitosum (Koch) Liu & Wang and Ps geniculata (Trin.) á. L?ve, and suggests a contribution of the St genome of Ps. spicata (Pursh) á. L?ve to the tetraploids Th. scirpeum (Presl) Dewey and Th. junceiforme (á. L?ve & D. L?ve) á. L?ve. A unique Bmy1 allele, divergent from other Triticeae but shared between Th. caespitosum, Th. intermedium (Host) Barkworth & Dewey, Th. junceum (L.) á. L?ve and Th. ponticum Barkworth & Dewey, implies a connection between these species. Distinct oligonucleotide polymorphisms and distance calculations based on the three loci implicate Crithopsis delileana (Schult.) Roshev. and Taeniatherum caput-medusae (L.) Nevski in the evolution of the hexaploid Th. intermedium and the decaploid Th. ponticum and also suggest a potential connection of these polyploids with Elytrigia repens (L.) Desv. ex Nevski. None of these species have been previously associated with the Thinopyrum genus. Allele-specific PCR was employed to detect the putative Crithopsis allele of ITS1 in a number of accessions. Real-time PCR indicates that two of six genomes of the hexaploid Th. intermedium have the Crithopsis-type ITS1 allele and that all ITS1 loci in the decaploid Th. ponticum are of this type. These results are supportive of the hypothesis that concerted evolution has homogenized the rDNA of Th. ponticum to the allele derived from the Crithopsis or Taeniatherum ancestor. Discovery of these novel alleles, with close homology to Ta. caput-medusae, may represent a fundamental change in the view of the evolution of Th. intermedium and Th. ponticum.     

簇毛麦基因组特异性PCR标记的建立和应用

以普通小麦中国春、簇毛麦、中国春－簇毛麦二体附加系和代换系为材料进行RAPD分析,筛选出一个簇毛麦基因组特异性RAPD片段OPFO2757,该片段分布于簇毛麦所有染色体上。在对OPFO2757进行克隆、测序的基础上,设计一对PCR引物,建立了簇毛麦基因组特异性PCR标记。用这对PCR引物对不同普通小麦品种、不同硬粒小麦品种、不同居群的簇毛麦、中国春－簇毛麦二体附加系、中国春－簇毛麦二体代换系、普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体等材料进行扩增,凡具有簇毛麦染色体的材料都能扩增出一条长为677bp的DNA片段,而不具簇毛麦染色体的材料包括大麦、黑麦、长穗偃麦草、中间偃麦草等不能扩增出该片段。所以,该特异性PCR标记可用于快速跟踪检测小麦背景中的簇毛麦染色体。     

Cytological and molecular characterization of a Triticum aestivum x Lophopyrum ponticum backcross derivative resistant to barley yellow dwarf.

Barley yellow dwarf is the most damaging virus-caused disease in bread wheat (Triticum aestivum L.). A resistant line, SW335.1.2-13-11-1-5 (2n = 47), derived from a cross of T. aestivum x Lophopyrum ponticum was characterized by meiotic chromosome pairing, by in situ DNA hybridization and by expression of molecular markers to determine its chromosome constitution. All progeny of this line had three pairs of L. ponticum chromosomes from homoeologous chromosome groups 3, 5, and 6 and the 2n = 47 progeny had an additional L. ponticum monosome. The pairs from groups 3 and 6 were in the added state, while the group 5 pair was substituted for wheat chromosome 5D. Several wheat-wheat translocations with respect to the parental wheat genotype occurred in this line, presumably owing to the promotion of homoeologous chromosome pairing by L. ponticum chromosomes. It was hypothesized that homoeologous recombination results in homoeologous duplication-deletions in wheat chromosomes. An aberrant 3:1 disjunction creates the potential at each meiosis for replacement of these wheat chromosomes by homoeologous L. ponticum chromosomes. Wheat chromosomes 3A and 6A appeared to be in intermediate stages of this substitution process.     

应用荧光原位杂交和染色体配对研究八倍体小冰麦中2的染色体组构成及染色体特征

通过染色体配对分析和荧光原位杂交（ＦＩＳＨ）技术对八倍体小冰麦中２的染色体组构成进行分析,结果表明：八倍体小冰麦中２含有的冰草染色体是来自天蓝冰草（Ａｇｒｏｐｙｒｏｎ　ｉｎｔｅｒｍｅｄｉｕｍ（Ｈｏｓｔ）Ｐ．Ｂ．＝Ｅｌｙｔｒｉｇｉａ　ｉｎｔｅｒｍｅｄｉａ（Ｈｏｓｔ）Ｎｅｖｓｋｉ＝Ｔｈｉｎｏｐｙｒｕｍ　ｉｎｔｅｒｍｅｄｉｕｍ　（Ｈｏｓｔ）Ｂａｒｋｗｏｒｔｈ　ａｎｄ　Ｄｅｗｅｙ）具同亲关系的染色体组,但冰草的这种同亲关系的染色体组不同于二倍体长穗偃麦草（Ｔｈｉｎｏｐｙｒｕｍ　ｅｌｏｕｇａｔｕｍ　２Ｘ）的Ｅ组染色体。中２含有１２条冰草染色体,且有一对染色体为小麦（Ｔｒｉｔｉｃｕｍ　ａｅｓｔｉｖｕｍ　Ｌ．）染色体和冰草染色体之间易位所形成的。     

Physical mapping of the blue-grained gene(s) from Thinopyrum ponticum by GISH and FISH in a set of translocation lines with different seed colors in wheat.

The original blue-grained wheat, Blue 58, was a substitution line derived from hybridization between common wheat (Triticum aestivum L., 2n=6x=42, ABD) and tall wheatgrass (Thinopyrum ponticum Liu & Wang=Agropyron elongatum, 2n=10x=70, StStEeEbEx), in which one pair of 4D chromosomes was replaced by a pair of alien 4Ag chromosomes (unknown group 4 chromosome from A. ponticum). Blue aleurone might be a useful cytological marker in chromosome engineering and wheat breeding. Cytogenetic analysis showed that blue aleurone was controlled by chromosome 4Ag. GISH analysis proved that the 4Ag was a recombination chromosome; its centromeric and pericentromeric regions were from an E-genome chromosome, but the distal regions of its two arms were from an St-genome chromosome. On its short arm, there was a major pAs1 hybridization band, which was very close to the centromere. GISH and FISH analysis in a set of translocation lines with different seed colors revealed that the gene(s) controlling the blue pigment was located on the long arm of 4Ag. It was physically mapped to the 0.71-0.80 regions (distance measured from the centromere of 4Ag). The blue color is a consequence of dosage of this small chromosome region derived from the St genome. We speculate that the blue-grained gene(s) could activate the anthocyanin biosynthetic pathway of wheat.     

Genome Analysis of a Natural Hybrid with 2n= 63 Chromosomes in the Genus Elytrigia Desv. (Poaceae) Using the GISH Technique

Abstract: Genomic in situ hybridization (GISH), using genomic DNA probes from Thinopyrum elongatum (E genome, 2 n = 14), Th. bessarabicum (J genome, 2 n = 14), Pseudoroegneria stipifolia (S genome, 2 n = 14), Agropyron cristatum (P genome, 2 n = 28) and Critesion californicum (H genome, 2 n = 14), was used to identify the genome constitution of a natural hybrid population morphologically close to Elytrigia pycnantha and with somatic chromosome number of 2 n = 63. The GISH results indicated the presence of a chromosomal set more or less closely related to the E, P, S and H genomes. In particular, two sets of 14 chromosomes each showed close affinity to the E genome of Th. elongatum and to the P genome of A. cristatum. However, they included 2 and 10 mosaic chromosomes, respectively, with S genome specific sequences at their centromeric regions. Two additional sets (28 chromosomes) appeared to be very closely related to the S genome of Ps. stipifolia. The last genome involved (7 chromosomes) is related to the H genome of C. californicum but includes one chromosome with S genome-specific sequences around the centromere and two other chromosomes with a short interstitial segment also containing S genome related sequences. On a basis of GISH analysis and literature data, it is hypothesized that the natural 9-ploid hybrid belongs to the genus Elytrigia and results from fertilization of an unreduced gamete (n = 42) of E. pycnantha and a reduced gamete (n = 21) of E. repens. The genomic formula SSSSP^SP^SE^SE^SH^S is proposed to describe its particular genomic and chromosomal composition.     

The genomic composition of Tricepiro, a synthetic forage crop.

Chromosome in situ hybridization (FISH and GISH) is a powerful tool for determining the chromosomal location of specific sequences and for analysing genome organization and evolution. Tricepiro (2n = 6x = 42) is a synthetic cereal obtained by G. Covas in Argentina (1972), which crosses hexaploid triticale (2n = 6x = 42) and octoploid Trigopiro (2n = 8x = 56). Several years of breeding produced a forage crop with valuable characteristics from Secale, Triticum, and Thinopyrum. The aim of this work is to analyse the real genomic constitution of this important synthetic crop. In situ hybridization using total DNA of Secale, Triticum, and Thinopyrum as a probe (GISH) labelled with biotin and (or) digoxigenin showed that tricepiro is composed of 14 rye chromosomes and 28 wheat chromosomes. Small zones of introgression of Thinopyrum on wheat chromosomes were detected. The FISH using the rye repetitive DNA probe pSc 119.2 labelled with biotin let us characterize the seven pairs of rye chromosomes. Moreover, several wheat chromosomes belonging to A and B genomes were distinguished. Therefore, tricepiro is a synthetic hexaploid (2n = 6x = 42) being AABBRR in its genomic composition, with zones of introgression of Thinopyrum in the A genome of wheat.     

Construction of a DNA Library Enriched with Mouse 4xChromosome of T(X;4)37H Translocation

Normal mouse chromosomes are routinely separated into only 5 peaks by the current flow cytometry. Since this limited resolution hindered isolation of the normal mouse X chromosome with an appropriate purity, we attempted to sort the mouse 4^x chromosome, a larger translocation chromosome of T(X;4)37H, consisting of nearly the entire chromosome 4 and chromosome X by flow cytometry. To obtain a large number of cells having 4^x chromosome for flow sorting, we isolated a somatic hybrid cell line MHH-1 formed between S194 myelome cell line and normal splenocytes from a male mouse carrying T(X;4)37H. Flow karyotyping of propidium iodide-stained chromosomes from MHH-1 cell line revealed an additional peak containing 4^x chromosomes at about 80%. DNA purified from sorted 4^x chromosomes was cloned into phage lambda gtWES after complete digestion with EcoRl restriction endonuclease. Thus a 4^x chromosome-enriched library of about 4.4 × 10⁴ recombinant phages was made and 13 single copy DNA clones specific to the X chromosome were isolated from the library so far.     

Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe--a landmark approach for Thinopyrum genome research

The introduction of alien genetic variation from the genus Thinopyrum through chromosome engineering into wheat is a valuable and proven technique for wheat improvement. A number of economically important traits have been transferred into wheat as single genes, chromosome arms or entire chromosomes. Successful transfers can be greatly assisted by the precise identification of alien chromatin in the recipient progenies. Chromosome identification and characterization are useful for genetic manipulation and transfer in wheat breeding following chromosome engineering. Genomic in situ hybridization (GISH) using an S genomic DNA probe from the diploid species Pseudoroegneria has proven to be a powerful diagnostic cytogenetic tool for monitoring the transfer of many promising agronomic traits from Thinopyrum. This specific S genomic probe not only allows the direct determination of the chromosome composition in wheat-Thinopyrum hybrids, but also can separate the Th. intermedium chromosomes into the J, J(S) and S genomes. The J(S) genome, which consists of a modified J genome chromosome distinguished by S genomic sequences of Pseudoroegneria near the centromere and telomere, carries many disease and mite resistance genes. Utilization of this S genomic probe leads to a better understanding of genomic affinities between Thinopyrum and wheat, and provides a molecular cytogenetic marker for monitoring the transfer of alien Thinopyrum agronomic traits into wheat recipient lines.