Generation of PCR-based markers for the detection of rye chromatin in a wheat background

Oligonucleotide primers were developed to detect the presence of four rye sequences using a PCR assay. These assays give a rye-specific signal from wheat DNA template which contains various rye chromosomes or chromosome segments. The sequences identified were associated with the nucleolar organiser region, the 5S-Rrna-R1 locus, the telomere, and a widely dispersed, rye-specific repetitive element Ris-1. The primers amplified from the well-established loci Nor-R1 and 5S-Rrna-R1 on rye chromosome arm 1RS, and also located a 5s-Rrna locus on chromosome 3R. The telomere-associated sequence was present on every rye chromosome, and was also present, at a low copy number, in both wheat and barley. These assays will be particularly useful for introgression programmes aimed at reducing the rye content of the 1BL.1RS wheat-rye translocation. When multiplexed, the primers will enable a rapid, simultaneous assay for a number of distinct rye loci, which can be derived from a small portion of mature endosperm tissue.     

Isolation and characterization of wheat-rye recombinants involving chromosome arm 1DS of wheat

Summary The introgression of genetic material from alien species is assuming increased importance in wheat breeding programs. One example is the translocation of the short arm of rye chromosome 1 (1RS) onto homoeologous wheat chromosomes, which confers disease resistance and increased yield on wheat. However, this translocation is also associated with dough quality defects. To break the linkage between the desirable agronomic traits and poor dough quality, recombination has been induced between 1RS and the homoeologous wheat arm IDS. Seven new recombinants were isolated, with five being similar to those reported earlier and two havina new type of structure. All available recombinantsw ere characterized with DNA probes for the loci Nor-R1, 5SDna-R1, and Tel-R1. Also, the amount of rye chromatin present was quantified with a dispersed rye-specific repetitive DNA sequence in quantitative dot blots. Furthermore, the wheat-rye recombinants were used as a mapping tool to assign two RFLP markers to specific regions on chromosome arms 1DS and 1RS of wheat and rye, respectively.     

应用荧光原位杂交和RFLP标记检测多小穗小麦新种质10-A中的黑麦染色质

利用ＡＰＡＧＥ、荧光原位杂交技术和ＲＦＬＰ标记,对导入黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）多小穗等性状创制的小麦新种质１０＿Ａ进行了分子标记检测。ＡＰＡＧＥ分析发现,１０＿Ａ与其他１ＲＳ／１ＢＬ易位系一样,含有１ＲＳ的醇溶蛋白标记位点Ｇｌｄ１Ｂ３。以黑麦基因组总ＤＮＡ作探针,用中国春（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍｃｖ．ＣｈｉｎｅｓｅＳｐｒｉｎｇ）基因组ＤＮＡ作封阻,与１０＿Ａ根尖细胞有丝分裂染色体进行荧光原位杂交。结果表明,黑麦的１ＲＳ易位到１０＿Ａ中。用２５个ＲＦＬＰ探针进行Ｓｏｕｔｈｅｒｎ分析,进一步发现１０＿Ａ的１ＢＳ特异限制性片段发生丢失,代之以黑麦１ＲＳ的特异限制性片段,而位于其他染色体上的特异限制性片段未发生缺失。据此认为,多小穗小麦新种质１０＿Ａ属于１ＲＳ／１ＢＬ易位系。同时还讨论了１０＿Ａ在小麦遗传改良中的利用情况。     

Localization of repetitive DNA in cereals byin situ hybridization: Cross hybridization among wheat,rye, barley,and oat

³H-RNA, complementary to repetitive DNA of wheat, rye, barley, and oat, was hybridizedin situ to root tip or pollen mother cells of the species mentioned. The cRNAs hybridized best with the DNA in cell nuclei of the species from which they were prepared. Cross hybridization with cells of the other related species resulted in a significant but diminished labelling. Wheat, rye, and barley hybridized better to each other than to oat, andvice versa, in agreement with the usual taxonomical classification. Over the interphase nuclei the label was distributed unevenly; not all regions of dense chromatin were labelled, and little label was found over the nucleoli. On chromosomes, the repetitive DNA was located somewhere along the chromosome arms or near the centromers in wheat, barley, and oat. Only in rye, most of the label was located near the telomers, probably over the large heterochromatin areas.     

Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye (Secale cereale L.).

A diminutive rye chromosome (midget) in wheat was used as a model system to isolate a highly reiterated centromeric sequence from a rye chromosome. Fluorescence in situ hybridization (FISH) shows this sequence localized within all rye centromeres and no signal was detected on wheat chromosomes. DNA sequencing of the repetitive element has revealed the presence of some catalytic domains and signature motifs typical of retrotransposon genes and has been called the Bilby family, representing a diverged family of retrotransposon-like elements. Extensive DNA database searching revealed some sequence similarity to centromeric retrotransposons from wheat, barley, and centromeric repetitive sequences from rice. Very low levels of signal were observed when Bilby was used as a probe against barley, and no signal was detected with rice DNA during Southern hybridization. The abundance of Bilby in rye indicates that this family may have diverged from other distantly related centromeric retrotransposons or incorporated in the centromere but rapidly evolved in rye during speciation. The isolation of a rye retrotransposon also allowed the analysis of centromeric breakpoints in wheat-rye translocation lines. A quantitative analysis shows that the breakpoint in IDS.1RL and 1DL.1RS and recombinant lines containing proximal rye chromatin have a portion of the rye centromere that may contribute to the normal function of the centromeric region.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

Rye chromosome translocations in hexaploid wheat: a re-evaluation of the loss of heterochromatin from rye chromosomes

Summary Using in situ hybridization techniques, we have been able to identify the translocated chromosomes resulting from whole arm interchanges between homoeologous chromosomes of wheat and rye. This was possible because radioactive probes are available which recognize specific sites of highly repeated sequence DNA in either rye or wheat chromosomes. The translocated chromosomes analysed in detail were found in plants from a breeding programme designed to substitute chromosome 2R of rye into commercial wheat cultivars. The distribution of rye highly repeated DNA sequences showed modified chromosomes in which (a) most of the telomeric heterochromatin of the short arm and (b) all of the telomeric heterochromatin of the long arm, had disappeared. Subsequent analyses of these chromosomes assaying for wheat highly repeated DNA sequences showed that in type (a), the entire short arm of 2R had been replaced by the short arm of wheat chromosome 2B and in (b), the long arm of 2R had been replaced by the long arm of 2B. The use of these probes has also allowed us to show that rye heterochromatin has little effect on the pairing of the translocated wheat arm to its wheat homologue during meiosis. We have also characterized the chromosomes resulting from a 1B-1R translocation event.From these results, we suggest that the observed loss of telomeric heterochromatin from rye chromosomes in wheat is commonly due to wheat-rye chromosome translocations.     

Development and application of EST-based markers specific for chromosome arms of rye (Secale cereale L.)

To develop a set of molecular markers specific for the chromosome arms of rye, a total of 1,098 and 93 primer pairs derived from the expressed sequence tag (EST) sequences distributed on all 21 wheat chromosomes and 7 rye chromosomes, respectively, were initially screened on common wheat 'Chinese Spring' and rye cultivar 'Imperial'. Four hundred and fourteen EST-based markers were specific for the rye genome. Seven disomic chromosome addition lines, 10 telosomic addition lines and 1 translocation line of 'Chinese Spring-Imperial' were confirmed by genomic in situ hybridization and fluorescencein situ hybridization, and used to screen the rye-specific markers. Thirty-one of the 414 markers produced stable specific amplicons in 'Imperial', as well as individual addition lines and were assigned to 13 chromosome arms of rye except for 6RS. Six rye cultivars, wheat cultivar 'Xiaoyan 6' and accessions of 4 wheat relatives were then used to test the specificity of the 31 EST-based markers. To confirm the specificity, 4 wheat-rye derivatives of 'Xiaoyan 6 × German White', with chromosomes 1RS, 2R and 4R, were amplified by some of the EST-based markers. The results indicated that they can effectively be used to detect corresponding rye chromosomes or chromosome arms introgressed into a wheat background, and hence to accelerate the utilization of rye genes in wheat breeding.     

Isolation and chromosomal localization of new MITE-like sequences from <Emphasis Type="Italic">Secale</Emphasis>

A species-specific DNA sequence (marker) that can detect the presence of Secale cereale chromatin in common wheat background was developed by using wheat microsatellite primer Xgwm614. One rye-specific DNA amplified fragment of 416bp (pSa614₄₁₆) was obtained from Secale africanum and a wheat — S. africanum amphiploid. The primer Xgwm614 also gave rise to specific bands in all Chinese Spring-Imperial rye addition lines 1R to 7R. Sequence analysis revealed that pSa614₄₁₆ was strongly homologous to a miniature inverted transposable element (MITE) stowaway-like element. Results of fluorescence in situ hybridization showed that the signal of pSa614₄₁₆ was distributed along all S. cereale. cv Jingzhou chromosomes, but the signal strengths were unbalanced on the seven rye genome chromosomes. This repetitive element may be useful as a molecular marker for the introgression of rye germplasm into the wheat genome.     

Structural organization of an alien Thinopyrum intermedium group 7 chromosome in U.S. soft red winter wheat (Triticum aestivum L.).

Barley yellow dwarf virus (BYDV) resistance in soft red winter wheat (SRWW) cultivars has been achieved by substituting a group 7 chromosome from Thinopyrum intermedium for chromosome 7D. To localize BYDV resistance, a detailed molecular genetic analysis was done on the alien group 7 Th. intermedium chromosome to determine its structural organization. Triticeae group 7 RFLP markers and rye specific repetitive sequences used in the analysis showed that the alien chromosome in the P29 substitution line has distinguishing features. The 350-480 bp rye telomeric sequence family was present on the long arm as determined by Southern and fluorescence in situ hybridization. However, further analysis using a rye dispersed repetitive sequence indicated that this alien chromosome does not contain introgressed segments from the rye genome. The alien chromosome is homoeologous to wheat chromosomes 7A and 7D as determined by RFLP analysis. Presence of the waxy gene on chromosomes 7A, 7B, and 7D but its absence on the alien chromosome in P29 suggests some internal structural differences on the short arm between Th. intermedium and wheat group 7 chromosomes. The identification of rye telomeric sequences on the alien Thinopyrum chromosome and the homoeology to wheat chromosomes 7A and 7D provide the necessary information and tools to analyze smaller segments of the Thinopyrum chromosome and to localize BYDV resistance in SRWW cultivars.     

Identification and chromosomal organization of two rye genome-specific RAPD products useful as introgression markers in wheat.

Two rye genome-specific random amplified polymorphic DNA (RAPD) markers were identified for detection of rye introgression in wheat. Both markers were amplified in all of the tested materials that contained rye chromatin such as rye, hexaploid triticale, wheat-rye addition lines, and wheat varieties with 1BL.1RS translocation. Two cloned markers, designated pSc10C and pSc20H, were 1012 bp and 1494 bp, respectively. Sequence analysis showed that both pSc10C and pSc20H fragments were related to retrotransposons, ubiquitously distributed in plant genomes. Using fluorescence in situ hybridization (FISH), probe pSc10C was shown to hybridize predominantly to the pericentromeric regions of all rye chromosomes, whereas probe pSc20H was dispersed throughout the rye genome except at telomeric regions and nucleolar organizing regions. The FISH patterns showed that the two markers should be useful to select or track all wheat-rye translocation lines derived from the whole arms of rye chromosomes, as well as to characterize the positions of the translocation breakpoints generated in the proximal and distal regions of rye arms.     

Visualization of Secale cereale DNA in wheat germ plasm by fluorescent in situ hybridization

Homozygous wheat/rye (1BL/1RS or 1AS/ 1RL) translocation lines have significantly contributed to wheat production, and several other wheat/rye translocation lines show a potential promise against biotic and abiotic stresses. Detecting the presence of rye at the chromosome level is feasible by C-banding and isozyme protocols, but the diagnostic strength of genomic in situ hybridization for eventually analyzing smaller DNA introgressions has greater significance. As a first step we have applied the genomic in situ hybridization technique to detect rye chromosomes in a wheat background using germ plasm of agricultural significance. By this method rye contributions to the translocations 1BL/1RS, 1AL/1RS, 5AS/5RL and 6BS/6RL could be identified. Differential labelling has further enabled the detection of rye and Thinopyrum bessarabicum chromosomes in a trigeneric hybrid of Triticum aestivum/Th. bessarabicum//Secale cereale.     

In situ hybridization as a rapid means to assess meiotic pairing and detection of alien DNA transfers in interphase cells of wide crosses involving wheat and rye

Summary The objectives of this study were to determine if biotin-labelled total genomic DNA of rye (Secale cereale L.) could be used to (i) preferentially label rye meiotic chromosomes in triticale and (ii) detect translocation stocks at interphase and/or early prophase by in situ hybridization. Welsh triticale, a wheat-rye segmental amphiploid, and Kavkaz wheat, a wheat-rye translocation were used. The results indicated that labelled chromosomes of rye and unlabelled chromosomes of wheat could be observed throughout all meiotic stages in the triticale. For Kavkaz wheat, the presence of the translocated 1RS chromosome arm of rye was detected at the interphase or very early prophase stage. Rapid assessment of feasibility of gene transfers and detection of alien DNA in somatic cells at the interphase stage by in situ hybridization allows for rapid decision-making and saves time and expense in plant breeding programs.Plant Research Centre Contribution No. 1276     

K型小麦细胞质雄性不育保持系T911289中外源遗传物质的初步鉴定

利用荧光基因组原位杂交(GISH)、生化标记和DNA分子标记技术对普通小麦(triticum aestivum L.)K型细胞质雄性不育保持系T911289的染色体组成进行了鉴定与分析。GISH鉴定和黑麦特异散布重复序列的检测结果表明,T911289的外源遗传物质来源于黑麦,黑麦1RS上的微卫星引物SCM9扩增结果和醇溶蛋白酸性聚丙烯酰胺凝胶电泳(A—PAGE)分析、低分子量谷蛋白的sDS_PAGE分析均表明,T911289所含的黑麦遗传物质来源于1RS;A-PAGE和SDS-PAGE分析及小麦1BS上的微卫星引物的扩增结果则表明,‘1911289缺少1BS染色体臂或1BS末端片段。GISH鉴定结果还表明,‘1911289中有罗泊逊易位和小片段易位两种类型的杂交信号,说明T911289是一个异质群体,但其罗泊逊易位又不同于生产上大面积应用的1BL／1RS易位,它可能是一种新的复杂易位形式。虽然T911289的小片段易位未能打破优异农艺性状与劣质蛋白基因的连锁,但这种小片段易位的获得将有利于小麦和黑麦的遗传研究,这种种质材料在育种上的应用价值也应优于罗泊逊易位。     

Transfer to wheat (Triticum aestivum) of small chromosome segments from rye (Secale cereale) carrying disease resistance genes

One hundred wheat lines, derived from monosomic additions of chromosome 1R of rye inbred line R12 (Chinese rye), were detected by PCR amplification using rye-specific primer pairs. Only 5 wheat lines, 1R296, 1R330, 1R314, 1R725, and 1R734, were determined to contain rye chromatin. While 1R296 and 1R330 were highly susceptible to stripe rust and powdery mildew, 1R314, 1R725 and 1R734 were highly resistant to both diseases. Acid-polyacrylamide gel electrophoresis showed that the ω-secalin bands were absent in 1R314, but present in the other 4 wheat lines. Genomicin situ hybridization indicated that 1R296, 1R330, and 1R725 contained translocations involving the whole short arm of chromosome 1R. However, 1R314 and 1R734 contained a pair of wheat chromosomes with small, terminal, rye-derived chromosome segments. The results suggest that the translocation breakpoint of 1RS in 1R314 was located between theSec-1 locus and the disease-resistance loci, while in line 1R734, the breakpoint was located between theSec-1 locus and the centromere. Taking account of the improved disease resistance of 1R725, 1R314 and 1R734, the chromosome arm 1RS of R12 may represent new and valuable disease resistance resources for wheat improvement.     

Isolation and chromosomal distribution of a novel Ty1-copia-like sequence fromSecale, which enables identification of wheat—Secale africanum introgression lines

A repetitive sequence of 411 bp, named pSaO5₄₁₁, was identified in theSecale africanum genome (R^a) by random amplified polymorphic DNA (RAPD) analysis of wheat and wheat—S. africanum amphiploids. GenBank BLAST search revealed that the sequence of pSaO5₄₁₁ was highly homologous to a part of a Ty1-copia retrotransposon. Fluorescence in situ hybridization (FISH) analyses indicated that pSaO5₄₁₁ was significantly hybridized toS. africanum chromosomes of a wheat—S. africanum amphiploid, and it was dispersed along theSecale chromosome arms except the terminal regions. Basing on the sequence of pSaO5₄₁₁, a pair of sequence-characterized amplified region (SCAR) primers were designed, and the resultant SCAR marker was able to target both cultivated rye and the wildSecale species, which also enabled to identify effectively theS. africanum chromatin introduced into the wheat genome.     

Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers

The wheat line H960642 is a homozygous wheat-Thinopyrum intermedium translocation line with resistance to BYDV by genomicin situ hybridization (GISH) and RFLP analysis. The genomic DNA ofTh. intermedium was used as a probe, and common wheat genomic DNA as a blocking in GISH experiment. The results showed that the chromosome segments ofTh. intermedium were transferred to the distal end of a pair of wheat chromosomes. RFLP analysis indicated that the translocation line H960642 is a T7DS-7DL-7XL translocation by using 8 probes mapped on the homoeologous group 7 in wheat. The translocation breakpoint is located between Xpsr680 and Xpsr965 about 90–99 cM from the centromere. The RFLP markers psr680 and psr687 were closely linked with the BYDV resistance gene. The gene is located on the distal end of 7XL around Xpsr680 and Xpsr687. Project supported by the 863 program and the National Natural Science Foundation of China (Grant No. 39680027).     

Centromere structure and function analysis in wheat–rye translocation lines

1RS.1BL translocations are centric translocations formed by misdivision and have been used extensively in wheat breeding. However, the role that the centromere plays in the formation of 1RS.1BL translocations is still unclear. Fluorescence in situ hybridization (FISH) was applied to detect the fine structures of the centromeres in 130 1RS.1BL translocation cultivars. Immuno‐FISH, chromatin immunoprecipitation (ChIP)‐qPCR and RT‐PCR were used to investigate the functions of the hybrid centromeres in 1RS.1BL translocations. New 1R translocations with different centromere structures were created by misdivision and pollen irradiation to elucidate the role that the centromere plays in the formation of 1RS.1BL translocations. We found that all of the 1RS.1BL translocations detected contained hybrid centromeres and that wheat‐derived CENH3 bound to both the wheat and rye centromeres in the 1RS.1BL translocation chromosomes. Moreover, a rye centromere‐specific retrotransposon was actively transcribed in 1RS.1BL translocations. The frequencies of new 1RS hybrid centromere translocations and group‐1 chromosome translocations were higher during 1R misdivision. Our study demonstrates the hybrid nature of the centromere in 1RS.1BL translocations. New 1R translocations with different centromere structures were created to help understand the fusion centromere used for wheat breeding and for use as breeding material for the improvement of wheat.     

Origin and molecular structure of a midget chromosome in a common wheat carrying rye cytoplasm

The origin and molecular structure of the midget chromosome that is retained in a common wheat with rye cytoplasm, were studied by using fluorescent in situ hybridization (FISH). FISH with biotinylated rye genomic DNA as a probe clearly showed that the midget chromosome had originated from certain part(s) of rye chromosome(s). The midget chromosome did not possess sequences similar to wheat rDNA nor to a rye telomeric sequence with a 350 bp repeat unit. However, another repetitive sequence (120 bp family) of rye was found to occur at one end of the midget chromosome. The telomeric repeat sequences from Arabidopsis thaliana cross-hybridized to both ends of the midget chromosome as well as to wheat chromosomes. From the results obtained in this and previous studies, it is assumed that the midget chromosome originated from part of a rye chromosome, most likely the centromeric region of chromosome 1R, and that the telomeric sequences were synthesized de novo.by R. Appels     

A specific oligonucleotide of the 5S rDNA spacer and species-specific elements identify symmetric somatic hybrids between Solanum tuberosum and S. pinnatisectum

A genetic map of rye, Secale cereale L., chromosome 1R covering 247 cM was constructed utilizing 27 RFLP and four C-band markers, including terminal C-bands. Genetic mapping of C-bands and the centromere, and in situ hybridization of three RFLP clones, allowed for the integration of the genetic and cytological maps. Eight contact points between the genetic and cytological maps revealed variation in the recombination distance to cytological distance ratio ranging between 0.25 and 1.95, a 7.8-fold difference. Recombination was found to be highest in the satellite region of 1RS and lowest in the most distal region of 1RL.