Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat-rye chromosome pairing

 Fluorescence in situ hybridization (FISH) with multiple probes has been applied to meiotic chromosome spreads derived from ph1b common wheat x rye hybrid plants. The probes used included pSc74 and pSc 119.2 from rye (the latter also hybridizes on wheat, mainly B genome chromosomes), the Ae. squarrosa pAs1 probe, which hybridizes almost exclusively on D genome chromosomes, and wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH with a two-by-two combination of these probes allowed unequivocal identification of all of the rye (R) and most of the wheat (W) chromosomes, either unpaired or involved in pairing. Thus not only could wheat-wheat and wheat-rye associations be easily discriminated, which was already feasible by the sole use of the rye-specific pSc74 probe, but the individual pairing partners could also be identified. Of the wheat-rye pairing observed, which averaged from about 7% to 11% of the total pairing detected in six hybrid plants of the same cross combination, most involved B genome chromosomes (about 70%), and to a much lesser degree, those of the D (almost 17%) and A (14%) genomes. Rye arms 1RL and 5RL showed the highest pairing frequency (over 30%), followed by 2RL (11%) and 4RL (about 8%), with much lower values for all the other arms. 2RS and 5RS were never observed to pair in the sample analysed. Chromosome arms 1RL, 1RS, 2RL, 3RS, 4RS and 6RS were observed to be exclusively bound to wheat chromosomes of the same homoeologous group. The opposite was true for 4RL (paired with 6BS and 7BS) and 6RL (paired with 7BL). 5RL, on the other hand, paired with 4WL arms or segments of them in more than 80% of the cases and with 5WL in the remaining ones. Additional cases of pairing involving wheat chromosomes belonging to more than one homoeologous group occurred with 3RL, 7RS and 7RL. These results, while adding support to previous evidence about the existence of several translocations in the rye genome relative to that of wheat, show that FISH with multiple probes is an efficient method by which to study fundamental aspects of chromosome behaviour at meiosis, such as interspecific pairing. The type of knowledge attainable from this approach is expected to have a significant impact on both theoretical and applied research concerning wheat and related Triticeae. Received: 21 February 1996 / Accepted: 12 July 1996     

Homoeology of rye chromosome arms to wheat

Summary Cytological markers such as diagnostic C-bands, telocentrics, and translocations were used to identify the arms of rye chromosomes associated with wheat chromosomes at metaphase I in ph1b mutant wheat × rye hybrids. Arm homoeologies of rye chromosomes to wheat were established from the results of metaphase I pairing combined with available data on the chromosomal location of homoeoloci series in wheat and rye. Only arms 1RS, 1RL, 2RL, 3RS, and 5RS showed normal homoeologous relationships to wheat. The remaining arms of rye appeared to be involved in chromosome rearrangements that occurred during the evolution of the genus Secale. We conclude that a pericentric inversion in chromosome 4R, a reciprocal translocation between 3RL and 6RL, and a multiple translocation involving 4RL, 5RL, 6RS, and 7RS are present in rye relative to wheat.     

Nonhomoeologous translocations between group 4, 5 and 7 chromosomes within wheat and rye

Summary Genetic maps of wheat chromosome 4A and rye chromosome arm 5RL, and the chromosomal locations of 70 sets of isozyme and molecular homoeoloci have been used to further define the structure of wheat chromosomes 4A, 5A and 7B, and rye chromosomes 4R, 5R and 7R. We provide evidence, for the first time, which is consistent with the presence of an interstitial segment on 4AL originating from 5AL, and of a segment originally from 5RL on 7RS. The evolutionary origins of the present chromosomes are discussed.     

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.     

New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance

Powdery mildew is one of the serious diseases of wheat (Triticum aestivum L., 2n = 6 × = 42, genomes AABBDD). Rye (Secale cereale L., 2n = 2 × = 14, genome RR) offers a rich reservoir of powdery mildew resistant genes for wheat breeding program. However, extensive use of these resistant genes may render them susceptible to new pathogen races because of co-evolution of host and pathogen. Therefore, the continuous exploration of new powdery mildew resistant genes is important to wheat breeding program. In the present study, we identified several wheat-rye addition lines from the progeny of T. aestivum L. Mianyang11 × S. cereale L. Kustro, i.e., monosomic addition lines of the rye chromosomes 4R and 6R; a disomic addition line of 6R; and monotelosomic or ditelosomic addition lines of the long arms of rye chromosomes 4R (4RL) and 6R (6RL). All these lines displayed immunity to powdery mildew. Thus, we concluded that both the 4RL and 6RL arms of Kustro contain powdery mildew resistant genes. It is the first time to discover that 4RL arm carries powdery mildew resistant gene. Additionally, wheat lines containing new wheat-rye translocation chromosomes were also obtained: these lines retained a short arm of wheat chromosome 5D (5DS) on which rye chromosome 4R was fused through the short arm 4RS (designated 5DS-4RS·4RL; 4RL stands for the long arm of rye chromosome 4R); or they had an extra short arm of rye chromosome 4R (4RS) that was attached to the short arm of wheat chromosome 5D (5DS) (designated 4RS-5DS·5DL; 5DL stands for the long arm of wheat chromosome 5D). These two translocation chromosomes could be transmitted to next generation stably, and the wheat lines containing 5DS-4RS·4RL chromosome also displayed immunity to powdery mildew. The materials obtained in this study can be used for wheat powdery mildew resistant breeding program.     

Pairing and recombination between individual chromosomes of wheat and rye in hybrids carrying the ph1b mutation

Wheat-rye chromosome associations at metaphase I studied by Naranjo and Fernández-Rueda (1991) in ph1b ABDR hybrids have been reanalysed to establish the frequency of pairing between individual chromosomes of wheat and rye. Wheat chromosomes, except for 2A and 2D, and their arms were identified by C-banding. Diagnostic C-bands and other cytological markers such as telocentrics or translocations were used to identify each one of the rye chromosomes and their arms. Both the amount of telomeric C-heterochromatin and the structure of the rye chromosomes relative to wheat affected the level of wheatrye pairing. The degree to which rye chromosomes paired with their wheat homoeologues varied with each of the three wheat genomes; in most groups, the B-R association was more frequent than the A-R or D-R associations. Recombination between arms 1RL and 2RL and their homoeologues of wheat possessing a different telomeric C-banding pattern was detected and quantified at anaphase I. The frequency of recombinant chromosomes obtained supports the premise that recombination between wheat and rye chromosomes may be estimated from wheat-rye pairing.     

Relationship between QTLs for preharvest sprouting and alpha-amylase activity in rye grain

Preharvest sprouting (PHS) and high alpha-amylase activity (AA) negatively affect quality of rye grain. The objective of this study was to reveal genetic relationship between PHS and AA by developing a consensus map of QTLs controlling each trait. A method of composite interval mapping (CIM) was used to search for QTLs within the 541 × Ot1-3 and DS2 × RXL10 F₂ mapping populations representing wide variation range of both traits. Sixteen QTLs for AA were detected on chromosomes 1R (3), 2R (2), 3R (2), 4R (3), 5R (3), 6R (2) and 7R (1). Their distribution was not random showing a tendency of QTL location in distal regions of chromosomes. Nine QTLs for AA located on chromosome arms 1RS, 2RL, 3RS, 4RL, 5RS, 5RL, 6RS, 6RL and 7RS coincided with QTLs for PHS. Seven QTLs for AA independent from PHS were detected on chromosome arms 1RL (2), 2RS, 3RL, 4RS, 4RL and 5RL. Four QTLs for PHS not associated with those for AA were identified on chromosomes 1RL, 2RL, 5RL and 7RL. Partial overlapping of the genetic systems controlling AA and PHS suggests that alpha-amylase found in sound grain of rye could be produced through at least three independent mechanisms i.e. PHS at its initial stage, late maturity alpha-amylase (LMA) and/or retained pericarp alpha-amylase (RPAA). Six QTLs co-located on both maps were found on chromosome arms 1RS, 2RS, 5RS, 5RL, 6RS and 6RL. Valuable features of line Ot1-3 i.e. resistance to preharvest sprouting and low alpha-amylase production in ripening grain can be attributed to seven major QTLs from chromosomes 1RL, 2RL, 5RL (2), 6RL and 7R (2). This set of QTLs, identified in line Ot1-3, might be useful in breeding sprouting resistant cultivars of rye.     

Condensation of rye chromatin in somatic interphase nuclei of Ph1 and ph1b wheat

The Ph1 locus in hexaploid wheat (Triticum aestivum L.) enforces diploid-like behavior in the first metaphase of meiosis. To test the hypothesis that this chromosome pairing control is exercised by affecting the degree of chromatin condensation, the dispersion of rye chromatin in interphase nuclei in somatic tissues of wheat-rye chromosome translocations 1RS.1BL, 2RS.2BL, 2BS.2RL, 3RS.3DL and 5RS.5BL was compared in Ph1 and ph1b isogenic backgrounds. No significant differences in rye chromatin condensation that could be attributed to the Ph1 locus were detected. Regardless of the Ph1 status, each rye chromosome arm tested conformed to the general Rabl's orientation and occupied portions of the nuclei proportional to their length. Earlier observations that indicated the involvement of Ph1 locus in rye chromatin condensation in wheat could have been due either to specific loci on the studied 5RL rye arm that control the chromosome condensation process or to damage to the genetic system controlling chromatin condensation in the existing ph1b stocks of wheat. That damage might have been caused by homoeologous recombination and uneven disjunction of chromosomes from multivalents.     

Genetic control of 6-phosphogluconate dehydrogenase (6-PGD) isozymes in cultivated wheat and rye

Summary The 6-phosphogluconate dehydrogenase (6-PGD) zymogram phenotypes of wheat, rye and their aneuploid derivatives were determined. Two genes involved in the production of 6-PGD isozymes were located on chromosome arms CRL (4 RL) and FRL (6 RL) of Imperial rye. On the basis of differential interactions between wheat and rye chromosomes, evidence was obtained that genes located on chromosomes 6 A, 6 BL and 7 BL control 6-PGD isozyme activities in Chinese Spring wheat. The wheat and rye 6-PGD zymogram phenotypes were indicative of homoeologous relationships between rye chromosome 6 RL to wheat chromosomes of group 6, and rye chromosome 4 RL to wheat chromosomes of group 7.     

Isolation of lambda and YAC clones from defined regions of the rye genome

A dispersed, rye-specific element has been used to isolate clones of rye origin from wheat plants containing only a single rye chromosome arm or segment. In this way a set of 23 YAC clones has been isolated from the short arm of rye chromosome 1 (1RS). This technique was extended to isolate clones from a small region of 1RS that contains a large number of agronomically important genes. The targeted cloning method allowed the isolation of 26 classes of lambda clones representing about 5% of the region. Ten of the lambda clones could be mapped to segments within this region. A third example of the application of this technique involved the isolation of clones from a very small but fully functional rye chromosome, the midget chromosome. These clones have allowed the confirmation of the origin of the midget from 1RL, and may provide a tool for the isolation of structural elements of cereal chromosomes. This technique allows the identification of clone libraries for any rye chromosome or chromosome arm, since substitution, addition and translocation lines are available for all rye chromosomes. Furthermore, the technique allows isolation of clones derived from segments of the rye genome recombined into wheat. The method is technically simple and both lambda and YAC libraries can be constructed. Synteny between the genomes of the cereals allows region-specific libraries from rye to be used to target regions of the wheat and barley genomes.     

小麦遗传背景对黑麦抗叶锈基因Lr26的抗性表达的影响

利用1套从小麦纯系和黑麦自交系培育出的1R附加系、代换系和易位系,研究了1RS上的抗叶锈基因Lr26在小麦中的表达。结果发现,1R二体附加系和纯合1RS/1BL易位系高抗小麦叶锈病;而其小麦亲本、1R(1B)代换系和1BS/1RL易位系重感叶锈病。这一结果指出了黑麦染色体臂1RS上的抗小麦叶锈病基因Lr26在小麦中的表达受小麦染色体臂1BL上的基因的强烈影响,指出了外源基因在小麦中的表达可受染色体臂或基因水平上的相互作用的制约。文中讨论了外源基因与小麦遗传背景相互作用在小麦育种中的意义。     

Isolation of lambda and YAC clones from defined regions of the rye genome

A dispersed, rye-specific element has been used to isolate clones of rye origin from wheat plants containing only a single rye chromosome arm or segment. In this way a set of 23 YAC clones has been isolated from the short arm of rye chromosome 1 (1RS). This technique was extended to isolate clones from a small region of 1RS that contains a large number of agronomically important genes. The targeted cloning method allowed the isolation of 26 classes of lambda clones representing about 5% of the region. Ten of the lambda clones could be mapped to segments within this region. A third example of the application of this technique involved the isolation of clones from a very small but fully functional rye chromosome, the midget chromosome. These clones have allowed the confirmation of the origin of the midget from 1RL, and may provide a tool for the isolation of structural elements of cereal chromosomes. This technique allows the identification of clone libraries for any rye chromosome or chromosome arm, since substitution, addition and translocation lines are available for all rye chromosomes. Furthermore, the technique allows isolation of clones derived from segments of the rye genome recombined into wheat. The method is technically simple and both lambda and YAC libraries can be constructed. Synteny between the genomes of the cereals allows region-specific libraries from rye to be used to target regions of the wheat and barley genomes. Received: 25 June 1997 / Accepted: 11 November 1997     

Preferential elimination of chromosome 5R of rye in the progeny of 5R5D dimonosomics

Transmission of chromosome 5R of rye (Secale cereale L.) and chromosome 5D of common wheat (Triticum aestivum L.) through gametes of 5R5D dimonosomics (2n = 42, 20W″ + 5R′ + 5D′) was studied. Chromosome 5R was found to have lower competitiveness as compared to 5D. Gametes with the rye chromosome were two times less often involved in the formation of a progeny. The combined frequency of the karyotypes of wheat (5D5D) and wheat monosomics (5D) was 11.6-fold higher than the frequency of the karyotypes of substitution lines (5R5R) and monosomics for the rye chromosome (5R). The karyotypes of 10.38% of hybrid plants had aberrant 5R chromosomes with different translocations formed as a result of breakages in the centromere and in the proximal region of the long arm. Telocentrics for the short arm t5RS, i5RS isochromosomes, and chromosomes with a terminal deletion T5RS.5RL-del were identified. The absence of amplification of SSR markers mapped on 5RS and the detection of PCR products for a number of 5RL markers (including the genome-specific rye marker Xrms115) permitted nine plants carrying only the long arm of chromosome 5R to be revealed. Since t5RL telocentrics were not detected by the cytological analysis, the results obtained allow us to suggest the presence of small intercalary translocations of the long arm of chromosome 5R in chromosome 5D or in other wheat chromosomes.     

The centromere structure in Robertsonian wheat-rye translocation chromosomes indicates that centric breakage-fusion can occur at different positions within the primary constriction

Univalent chromosomes at meiotic metaphase I have a tendency to misdivide at the centromeres. Fusion of the misdivision products may produce Robertsonian translocations. The fine structure of the centromeres in Robertsonian wheat-rye translocation chromosomes was analyzed by fluorescence in situ hybridization (FISH) using two centromere-specific DNA clones: pRCS1, derived from rice, and pAWRC1, derived from rye. Clone pRCS1 hybridizes to the centromeres of all grasses including wheat and rye, whereas clone pAWRC1 is rye specific and hybridizes only to the centromeres of rye. Four of the six wheat-rye translocations derived from a single centric misdivision event (1st generation translocations) had hybrid centromeres, with approximately half of the centromere derived from rye and half from wheat. In the two other 1st generation translocations, the entire centromere was derived from rye. Among eight reconstructed wheat and rye chromosomes that originated from two consecutive centric misdivision-fusion events (2nd generation translocations), T1BS.1BL (derived from T1BS.1RL and T1RS.1BL) and one of three T2BS.2BL (derived from T2RS.2BL and T2BS.2RL) had hybrid centromeres. T1RS.1RL (derived from T1BS.1RL and T1RS.1BL), two of three T2BS.2BL, and all three T2RS.2RL (derived from T2RS.2BL and T2BS.2RL) had rye centromeres. All three 3rd generation translocations had hybrid centromeres with approximately half of the centromere derived from rye. There were no indications that the composite structure of the centromere in these chromosomes affected their behavior in mitosis or meiosis. These observations support the notion of a compound structure of the centromere in higher organisms, and indicate that during the centric breakage-fusion event, centromere breakage may occur in different positions along the segment of the chromosome that interacts with the spindle fibers. Normal behavior of the 1st, 2nd, and 3rd generation centric translocations in mitosis and meiosis indicates that, at least in wheat and rye, centromeres are not chromosome specific.     

Anthocyanin biosynthesis genes location and expression in wheat–rye hybrids

Studies into gene expression in a foreign background contribute toward understanding of how genes derived from different species or genera manages to co-exist in a common nucleus, on the one hand, and help to estimate possible effectiveness of wide hybridization for cultivated plant improvement, on the other hand. The aim of this study was to investigate conservation of wheat and rye expression networks, using the anthocyanin biosynthesis pathway (ABP) genes as a model system. We isolated and analyzed ABP genes encoding enzymes acting at different steps of the pathway: chalcone-flavanone isomerase (CHI), flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), and anthocyanidin-3-glucoside rhamnosyltransferase (3RT). The rye ABP genes locations we determined (Chi on chromosome 5RL, F3h on 2RL, Ans on 6RL, 3Rt on 5RL, the regulatory Rc—red coleoptile—gene on 4RL) were in agreement with the rearrangements established between rye and wheat chromosomes. Expression of the ABP structural genes was studied in wheat–rye chromosome addition and substitution lines. F3h activation by the Rc gene was found to be critical for the red coleoptile trait formation. It was shown that the rye regulatory Rc gene can activate the wheat target gene F3h and vice versa wheat Rc induces expression of rye F3h. However, lower level of expression of rye F3h in comparison with that of the two wheat orthologues in the wheat–rye chromosome substitution line 2R(2D) was observed. Thus, although work of the wheat and rye ABP gene systems following the formation of wheat–rye hybrids is finely coordinated, some divergence exists between rye and wheat ABP genes, affecting level of gene expression.     

Chromosome pairing in tetraploid rye with monosomic-substitution wheat chromosomes

In tetraploid rye with single-substitution wheat chromosomes - 1A, 2A, 5A, 6A, 7A, 3B, 5B, 7B - chromosome pairing was analysed at metaphase I in PMCs with the C-banding method. The frequency of univalents of chromosome 1A was considerably higher than that of the other four wheat chromosomes of genome A (6A, 5A, 7A and 2A). Among chromosomes of genome B, the lowest mean frequency of univalents was observed for chromosome 5B. In monosomic lines, wheat chromosomes 1A, 2A, 5A, 6A, 7A and 5B paired with rye homoeologues most often in rod bivalents and in chain quadrivalents (also including 3B). The 47% pairing of 5B-5R chromosomes indicate that the rye genomes block the suppressor Ph1 gene activity. In monosomic plants with chromosomes 5A, 2A, 6A, 7A and 5B, a low frequency of rye univalents was observed. It was also found that the wheat chromosomes influenced the pairing of rye genome chromosomes, as well as the frequency of ring and rod bivalents and tri- and quadrivalents. However, the highest number of terminal chiasmata per chromosome occurred in the presence of chromosomes 5A and 2A, and the lowest - in the presence of chromosomes 3B and 7B. In the presence of chromosome 5B, the highest frequency of bivalents was observed. The results of the present study show that the rye genome is closer related to the wheat genome A of than to genome B. The high pairing of wheat-rye chromosomes, which occurs in tetraploid rye with substitution wheat chromosomes, indicates that there is a high probability of incorporating wheat chromosome segments into rye chromosomes.     

Genetic mapping of Dn7, a rye gene conferring resistance to the Russian wheat aphid in wheat

The Russian wheat aphid is a significant pest problem in wheat and barley in North America. Genetic resistance in wheat is the most effective and economical means to control the damage caused by the aphid. Dn7 is a rye gene located on chromosome 1RS that confers resistance to the Russian wheat aphid. The gene was previously transferred from rye into a wheat background via a 1RS/1BL translocation. This study was conducted to genetically map Dn7 and to characterize the type of resistance the gene confers. The resistant line '94M370' was crossed with a susceptible wheat cultivar that also contains a pair of 1RS/1BL translocation chromosomes. The F₂ progeny from this cross segregated for resistance in a ratio of 3 resistant: 1 susceptible, indicating a single dominant gene. One-hundred and eleven RFLP markers previously mapped on wheat chromosomes 1A, 1B and 1D, barley chromosome 1H and rye chromosome 1R, were used to screen the parents for polymorphism. A genetic map containing six markers linked to Dn7, encompassing 28.2 cM, was constructed. The markers flanking Dn7 were Xbcd1434 and XksuD14, which mapped 1.4 cM and 7.4 cM from Dn7, respectively. Dn7 confers antixenosis, and provides a higher level of resistance than that provided by Dn4. The applications of Dn7 and the linked markers in wheat breeding are discussed.Communicated by J. Dvorak     

Identification and Physical Mapping of New PCR-Based Markers Specific for the Long Arm of Rye(Secale cereale L.) Chromosome 6

To effectively use elite genes on the long arm of rye chromosome 6(the 6RL arm) in wheat breeding programs,precise and fast identification of 6RL chromatin in wheat backgrounds is necessary.PCR-based 6RL-specific markers can facilitate the detection of elite genes on 6RL in wheat breeding.However,only a limited number of 6RL-specific markers have been developed.In the present study.300 new PCR-based 6RL-specific markers were identified using specific length amplified fragment sequencing(SLAF-seq) technology,and were further physically mapped to four regions on the 6RL arm using 6R and 6RL deletion lines.Interestingly,127 of the 300 markers were physically localized to a region from the site between 2.3 and 2.5 to the telomere,the same region where the powdery mildew resistance gene was mapped.In addition,95 of the 300 markers exhibit polymorphisms,which can be used to investigate the diversity of rye 6RL arms.The markers developed in this study can be used to identify given segments of 6RL in wheat backgrounds and accelerate the utilization of elite genes on 6RL in wheat breeding.     

Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew

The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology‐based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single‐cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3‐ and Pm8‐like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co‐evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.