应用基因组原位杂交鉴定蓝粒小麦及其诱变后代

应用基因组原位杂交技术（GISH）对普通小麦（Triticum aestivumL.)和长穗偃麦草[Agropyron elongatum(Host)Beauv,2n=10x=70]杂交后选育出的蓝粒小麦蓝-58及其诱变后代的染色体组成进行了鉴定。结果表明,GISH可方便地检测到小麦遗传背景中的长穗偃麦草染色体或易位的片段。如前人报道,蓝-58（2n=42)是一个具有2条长穗偃麦草4E染色体的异代换系（4E/4D）。LW004可能是一个具有两对相互易位染色体的纯合系,其田间表现磷高效特性,LW43-3-4为41条染色体的蓝单体（40W 1’4E）,种子颜色为浅蓝色,通过此法还检测出一些染色体结构发生很大变异的材料如4E的单端体（40W 1‘4E),种子颜色为浅蓝色,通过此法还检测出一些染色结构发生很大变异的材料如4E的单端体（40W 1‘t4E)以及组型为39W 1‘4E 1‘t4E的个体,此项研究结果更为直观地表明控制蓝粒体状的基因的确在来自长穗偃麦草的染色体上。同时说明有效的突变方法与灵活方便的检测手段的有机结合在染色体工程材料的创制和染色体工程育种中起着至关重要的作用。     

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。     

Transfer of small chromosome fragments of Agropyron elongatum to wheat chromosome via asymmetric somatic hybridization

~~Transfer of small chromosome fragments of Agropyron elongatum to wheat chromosome via asymmetric somatic hybridization1 .Dong,Y.C,GenePools of common wheat,Journal of Triticeae CroPs(in Chinese),2000,20(3):78-81. 2 .Wei,Y.M.,Zheng,YL.Zhou,R.H., Detectlon of the rye chro- matin in multisPikelet wheat germplasm 10-A background using fluorescence in situ hybridization(FISH)and RFLP markers,Acta Bot.Sinica(in Chinese),1999,41(7):722-725. 3 .Xiang,E N.,Xia,G M.…     

Transfer of small chromosome fragments of<Emphasis Type="Italic">Agropyron elongatum</Emphasis> to wheat chromosome via asymmetric somatic hybridization

The chromosome constitution of hybrids and chromatin patterns of Agropyron elongatum (Host)Neviski in F₅ somatic hybrid lines -1–3 and I-1-9 between Triticum aestivum L. and A. elongatum were analyzed. Based on the statistic data of pollen mother cells, F₅ I-1-9 and-1-3 had 20–21 bivalents with a frequency of 84.66% and 85.28%, of which, 89.83% and 89.57% were ring bivalents. The result indicated that both hybrid lines were basically stable in the chromosome constitution and behavior. RAPD analysis showed that the two hybrids contained biparental and integrated DNA. GISH (Genome in situ hybridization) revealed that in the form of small chromosome segments, A. elongatum chromatin was scattered on 4–6 wheat chromosomes near by the region of centromere and telomere in the two hybrid lines. SSR analysis indicated that A. elongatum DNA segments were distributed on the 2A, 5B, 6B and 2D wheat chromosomes in the hybrids, which was in accordance with the GISH results that small-segments intercalated poly-site.     

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.     

Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers

Based on the cross (Triticum aestivum L. x Secale cereale L.) x T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat--rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)2, 2R(2D)3, 3R(3B), 6R(6A)2. The chromosome composition of lines IR(1A), 2R(W)1, 5R(W), 5R(5A), and 6R(W)1, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)1, 5R(5D), 5R(5A), and 6R(6A)1, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The "combined" long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising "secondary gene pools" for the purpose of plant breeding.     

Construction and molecular and cytogenetic analyses of euploid (2n = 42) and telocentric addition (2n = 42 + 2t) alloplasmic lines (Hordeum marinum subsp gussoneanum)-Triticum aestivum

Individual plants from the BC1F5 and BC1F6 backcross progenies of barley--wheat (= H. geniculatum All.) (2n = 28) x T. aestivum L. (2n = 42)] and the BC1F6 progeny of their amphiploids were used to obtain alloplasmic euploid (2n = 42) lines L-28, L-29, and L-49 and alloplasmic telocentric addition (2n = 42 + 2t) lines L-37, L-38, and L-50. The lines were examined by genomic in situ hybridization (GISH), microsatellite analysis, chromosome C-banding, and PCR analysis of the mitochondrial 18S/5S repeat. Lines L-29 and L-49 were characterized by substitution of wild barley chromosome 7H1 for common wheat chromosome 7D. In line L-49, common wheat chromosomes 1B, 5D, and 7D were substituted with homeologous barley chromosomes. Lines L-37, L-38, and L-50 each contained a pair of telocentric chromosomes, which corresponded to barley chromosome arm 7H'L. All lines displayed heteroplasmy for the mitochondrial 18S/5S locus; i.e., both barley and wheat sequences were found.     

VE161小麦的外源染色体鉴定

通过染色体原位杂交、ＲＦＬＰ分析和染色体重双端体分析,对在小麦遗传育种研究中具有重要理论和应用价值的ＶＥ１６１小麦不育异代换系及其附加系进行了染色体鉴定。结果表明,ＶＥ１６１小麦代换的或附加的外源染色体为来自长穗偃麦草的４Ｅ染色体,被代换的小麦染色体为４Ｂ。过去曾鉴定为７Ｂ,可能是由于ＶＥ１６１早代染色体易位较多所致。同时发现,ＶＥ１６１小麦在５Ｂ,７Ｂ和１Ｄ所在的３个部分同源群中仍有染色体相互易     

巨穗小麦种质中外源遗传物质的细胞遗传学和分子生物学鉴定

利用C分带、基因组原位杂交并结合分子生物学手段,对12份巨穗小麦种质材料中的外源遗传物质进行了检测.结果表明,12份材料染色体数均为42,其中5份材料均具有一对小麦-黑麦(Triticum aestivum-Secalecereal)1BL/1RS易位染色体和一对中间偃麦草(Agropyron intermedium Garten)染色体、3份材料只具有一对中间偃麦草染色体、3份材料只具一对1BL/1RS染色体、1份材料无1BL/1RS和中间偃麦草染色体.进一步细胞学分析表明,此中间偃麦草染色体代换了普通小麦(Triticum aestivum L.)中的2D染色体,因其良好的同源补偿性,表示为2Ai.同时对2Ai在巨穗小麦种质中存在的遗传学意义及小麦遗传改良中的应用进行了讨论.     

Characterisation of mildew resistant wheat-rye substitution lines and identification of an inverted chromosome by fluorescent in situ hybridisation

Seven different mildew resistant wheat lines derived from crosses between triticale and bread wheat were examined by molecular cytogenetics and chromosome C-banding in order to determine their chromosomal composition. Genomic in situ hybridisation (GISH) showed the presence of rye germplasm in all the lines and identified three substitution lines, three double substitution lines and one addition-substitution line. C-banding identified rye chromosomes 1R and 4R in the addition-substitution line, rye chromosomes 1R and 6R in two substitution lines and 1R and 2R in the third line, and rye chromosome 1R in the three substitution lines. Two of the latter lines (7-102 and 7-169) contained a modified form of the chromosome; fluorescent in situ hybridisation (FISH) using five different repetitive DNA-probes showed a pericentric inversion of 1R in both lines. The breakpoints of the 1R inversion were between (1) the 5S rDNA site and the NOR-region on the satellite of the short arm, and (2) between two AAC(5) sites close to the centromere on the long arm. The role of the rye chromosomes in the mildew resistance, the utilisation of the inverted 1R and the significance of the lines in wheat breeding are discussed.     

Characterization of Hordeum chilense chromosomes by C-banding and in situ hybridization using highly repeated DNA probes.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.     

Standard karyotype of Triticum longissimum and its cytogenetic relationship with T. aestivum.

C-banding polymorphism was analyzed in 17 accessions of Triticum longissimum from Israel and Jordan, and a generalized idiogram of this species was established. C-banding analysis was further used to identify two sets of disomic T. aestivum - T. longissimum chromosome addition lines and 13 ditelosomic addition lines and one monotelosomic (6S1L) addition line. C-banding was also used to identify T. aestivum - T. longissimum chromosome substitution and translocation lines. Two major nucleolus organizing regions (NORs) on 5S1 and 6S1 and one minor NOR on 1S1 were detected by in situ hybridization using a 18S-26S rDNA probe. Sporophytic and gametophytic compensation tests were used to determine the homoeologous relationships of T. longissimum chromosomes. The T. longissimum chromosomes compensate rather well and fertility was restored even in substitution lines involving wheat chromosomes 2A, 4B, and 6B that contain major fertility genes. Except for the deleterious gametocidal genes, T. longissimum can be considered as a suitable donor of useful genes for wheat improvement.     

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.     

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.     

Identification of alien chromatin specifying resistance to wheat streak mosaic and greenbug in wheat germ plasm by C-banding and in situ hybridization

Summary The chromosome constitutions of eight wheat streak mosaic virus (WSMV)-resistant lines, three of which are also greenbug resistant, derived from wheat/ Agropyron intermedium/Aegilops speltoides crosses were analyzed by C-banding and in situ hybridization. All lines could be traced back to CI15092 in which chromosome 4A is substituted for by an Ag. intermedium chromosome designated 4Ai-2, and the derived lines carry either 4Ai-2 or a part of it. Two (CI17881, CI17886) were 4Ai-2 addition lines. CI17882 and CI17885 were 4Ai-2-(4D) substitution lines. CI17883 was a translocation substitution line with a pair of 6AL.4Ai-2S and a pair of 6AS.4Ai-2L chromosomes substituting for chromosome pairs 4D and 6A of wheat. CI17884 carried a 4DL.4Ai-2S translocation which substituted for chromosome 4D. CI17766 carried a 4AL.4Ai-2S translocation substituting for chromosome 4A. The results show that the 4Ai-2 chromosome is related to homoeologous group 4 and that the resistance gene(s) against WSMV is located on the short arm of 4Ai-2. In addition, CI17882, CI17884, and CI17885 contained Ae. speltoides chromosome 7S substituting for chromosome 7A of wheat. The greenbug resistance gene Gb5 was located on chromosome 7S.Contribution No. 90-515-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kan., USA     

Molecular cytogenetic characterization of wheat--Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat Fusarium Head Blight

Thinopyrum elongatum(2n = 2x = 14,EE),a wild relative of wheat,has been suggested as a potentially novel source of resistance to several major wheat diseases including Fusarium Head Blight(FHB).In this study,a series of wheat(cv.Chinese Spring,CS) substitution and ditelosomic lines,including Th.elongatum additions,were assessed for TypeⅡresistance to FHB.Results indicated that the lines containing chromosome 7E of Th.elongatum gave a high level of resistance to FHB,wherein the infection did not spread beyond the inoculated floret.Furthermore,it was determined that the novel resistance gene(s) of 7E was located on the short-arm(7ES) based on sharp difference in FHB resistance between the two 7E ditelosomic lines for each arm.On the other hand,Th.elongatum chromosomes 5E and 6E likely contain gene(s) for susceptibility to FHB because the disease spreads rapidly within the inoculated spikes of these lines. Genomic in situ hybridization(GISH) analysis revealed that the alien chromosomes in the addition and substitution lines were intact,and the lines did not contain discernible genomic aberrations.GISH and multicolor-GISH analyses were further performed on three translocation lines that also showed high levels of resistance to FHB.Lines TA3499 and TA3695 were shown to contain one pair of wheat-Th. elongatum translocated chromosomes involving fragments of 7D plus a segment of the 7E,while line TA3493 was found to contain one pair of wheat-Th.elongatum translocated chromosomes involving the D- and A-genome chromosomes of wheat.Thus,this study has established that the short-arm of chromosome 7E of Th.elongatum harbors gene(s) highly resistant to the spreading of FHB,and chromatin of 7E introgressed into wheat chromosomes largely retained the resistance,implicating the feasibility of using these lines as novel material for breeding FHB-resistant wheat cultivars.     

Molecular Cytogenetic Identification of Triticum aestivum-Secale cereale Substitution and Addition Lines

Two substitution lines, designated as 930498 and 930483, and one addition line, designated as 930029, via Fo immature embryo culture of Triticum aestivum x octoploid triticale ( x Triti-cosecale Wittmack) were identified. Fluorescence in situ hybridization (FISH) using total genomic DNA of rye ( Secale cereale L. ) as probe corroborated the existence of rye chromosomes, further confirmed through chromosome paring at meiotic metaphase 1, C-banding and glutenin SDS- PAGE. The results demonstrated that the two substitution lines are ID/IR, and the addition line is also IR addition. Rye chromosomes that are distinct to the red-colored wheat chromosomes appear yellow-green at mitotic metaphase after FISH.     

Molecular cytogenetic characterization of Thinopyrum intermedium-derived wheat germplasm specifying resistance to wheat streak mosaic virus

Thinopyrum intermedium is a promising source of resistance to wheat streak mosaic virus (WSMV), a devastating disease of wheat. Three wheat germplasm lines possessing resistance to WSMV, derived from Triticum aestivum×Th. intermedium crosses, are analyzed by C-banding and genomic in situ hybridization (GISH) to determine the amount and location of alien chromatin in the transfer lines. Line CI15092 was confirmed as a disomic substitution line in which wheat chromosome 4A was replaced by Th. intermedium chromosome 4Ai?2. The other two lines, CI17766 and A29-13-3, carry an identical Robertsonian translocation chromosome in which the complete short arm of chromosome 4Ai?2 was transferred to the long arm of wheat chromosome 4A. Fluorescence in situ hybridization (FISH) using ABD genomic DNA from wheat as a probe and S genomic DNA from Pseudoroegneria stipifolia as the blocker, and vice versa, revealed that the entire short arm of the translocation was derived from the short arm of chromosome 4Ai?2 and the breakpoint was located at the centromere. Chromosomal arm ratios (L/S) of 2.12 in CI17766 and 2.15 in A29-13-3 showed that the translocated chromosome is submetacentric. This translocated chromosome is designated as T4AL?? 4Ai?2S as suggested by Friebe et al. (1991).     

Analysis off cereal chromosomes by atomic force microscopy.

Atomic force microscopy has been applied to the study of plant chromosomes from cereal grasses Triticum aestivum (bread wheat), Triticum tauschii, and Hordeum vulgare (barley). Using standard mitotic metaphase squashes, high resolution images have been obtained of untreated chromosomes and also of chromosomes after C-banding, N-banding, and in situ hybridization. The true 3-dimensional nature of the images permits detailed analysis of the surface structure and, on untreated uncoated chromosomes, surface features on a length scale consistent with nucleosome structures have been observed. C+ and N+ regions are manifest as areas of high relief on a slightly collapsed chromosome structure. In situ hybridization leads to a more severe degradation of the native structure, although it is still possible to correlate the optical signal with the topography of the hybridized chromosome. Key words : atomic force microscope, AFM, chromosomes, C-banding, in situ hybridization.