Rapid verification of wheat-Hordeum introgressions by direct staining of SCAR, STS, and SSR amplicons.

A range of single tagged site (STS), simple sequence repeat (SSR), and sequence-characterized amplified region (SCAR) markers were screened for their utility in detecting Hordeum vulgare and H. chilense chromosomes in a wheat background. PCR conditions were optimized for specific amplification of the targeted sequences and to avoid cross-species amplification. Two H. vulgare derived STSs, six H. vulgare derived SSRs, and nine H. chilense derived SCARs were usable for the detection of five H. vulgare and three H. chilense chromosomes by direct ethidium bromide staining of the PCR products in test tubes, avoiding the more costly and time-consuming DNA electrophoresis step. The practical application of the method is illustrated by the identification of a monotelosomic substitution of H. vulgare chromosome 6HS in tritordeum and a monosomic addition of H. chilense chromosome 6Hch in durum wheat.     

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.     

Molecular cytogenetic analysis of durum wheat x tritordeum hybrids.

Southern and in situ hybridization were used to examine the chromosome constitution, genomic relationships, repetitive DNA sequences, and nuclear architecture in durum wheat x tritordeum hybrids (2n = 5x = 35), where tritordeum is the fertile amphiploid (2n = 6x = 42) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense, enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed that the chromosomes of H. chilense origin were in individual domains with only limited intermixing with chromosomes of wheat origin. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps owing to methylation. Both techniques gave complementary information, extending that available from phenotypic, chromosome morphology, or isozyme analysis, and perhaps are useful for following chromosomes or chromosome segments during further crossing of the lines in plant breeding programs.     

Identification of intergenomic translocations involving wheat,Hordeum vulgare and Hordeum chilense chromosomes by FISH

Intergenomic translocations between wheat, Hordeum chilense and Hordeum vulgare have been obtained in tritordeum background. Advanced lines from the crosses between three disomic chromosome addition lines for chromosome 2Hv, 3Hv, and 4Hv of barley (Hordeum vulgare) in Triticum aestivum cv. Chinese Spring (CS) and hexaploid tritordeum (2n = 6x = 42, AABBHchHch) were analyzed. Multicolor FISH using both genomic DNA from H. chilense and H. vulgare were used to establish the presence and numbers of H. vulgare introgressions into tritordeum. Interspecific H. vulgare/H. chilense and intergeneric wheat/H. vulgare and wheat/H. chilense translocations were identified. Frequencies of plants containing different kinds of intergenomic translocations between chromosome arms are presented. These lines can be useful for introgressing into tritordeum characters of interest from H. vulgare.     

Comparative FISH mapping of two highly repetitive DNA sequences in Hordeum chilense (Roem. et Schult.)

Hordeum chilense Roem. et Schult. (2n = 14) is an autogamous wild barley from Chile and Argentina included in the section Anisolepis Nevski. This species shows interesting agronomic traits that can be incorporated into crop plant species. Hordeum chilense has been successfully crossed with species of the genus Aegilops. Among the amphiploids obtained, the hexaploid tritordeum (2n = 6x = 42, AABBHchHch) is outstanding and shows good agronomic characteristics, suggesting its potential either as a new crop or as a bridge species to introgress interesting traits into cultivated cereals. The aim of the present work was to study the hybridization patterns of the two repetitive DNA probes pAs1 and pSc119.2 to evaluate their utility for the identification of H. chilense chromosomes. Fourteen lines of H. chilense were analyzed with fluorescent in situ hybridization using probes pSc119.2 and pAs1. The probe pAs1 was more widely dispersed than pSc119.2 over the H. chilense (Hch) genome. We found 89 different signals for pAs1, distributed evenly over the whole genome, and 10 for pSc119.2, located mainly over the telomeric regions. Five distinct hybridization signals were found for pAs1 and four distinct signals for pSc119.2. These signals allow the identification of different H. chilense lines. For example, centromeric signals for pAs1 on the short arms of chromosomes 1 and 7 identify line H46, and a telomeric signal for pSc119.2 on the short arm of chromosome 2 identifies line H1. A high degree of polymorphism in the hybridization patterns was found, confirming the extensive variability present in H. chilense. This work provides tools for the identification of H. chilense chromosomes in different genetic backgrounds.     

Prospects for exploitation of disease resistance from Hordeum chilense in cultivated cereals

Hordeum chilense is a South American wild barley with high potential for cereal breeding given its high crossability with other members of the Triticeae. In the present paper we consider the resistance of H. chilense to several fungal diseases and the prospects for its transference to cultivated cereals. All H. chilense accessions studied are resistant to the barley, wheat and rye brown rusts, the powdery mildews of wheat, barley, rye and oat, to Septoria leaf blotch, common bunt and to loose smuts, which suggests that H. chilense is a non-host of these diseases. There are also lines resistant to wheat and barley yellow rust, stem rust and to Agropyron leaf rust, as well as lines giving moderate levels of resistance to Septoria glume blotch, tan spot and Fusarium head blight. Some H. chilense lines display pre-appressorial avoidance to brown rust. Lines differ in the degree of haustorium formation by rust and mildew fungi they permit, and in the degree to which a hypersensitive response occurs after haustoria are formed. Unfortunately, resistance of H. chilense to rust fungi is not expressed in tritordeum hybrids, nor in chromosome addition lines in wheat. In tritordeum, H. chilense contributes quantitative resistance to wheat powdery mildew, tan spot and loose smut. The resistance to mildew, expressed as a reduced disease severity, is not associated with macroscopically visible necrosis. Hexaploid tritordeums are immune to Septoria leaf blotch and to common bunt although resistance to both is slightly diluted in octoploid tritordeums. Studies with addition lines in wheat indicate that the resistance of H. chilense to powdery mildew, Septoria leaf blotch and common bunt is of broad genetic basis, conferred by genes present on various chromosomes.     

Genomic differentiation of Hordeum chilense from H. vulgare as revealed by repetitive and EST sequences

Hordeum vulgare, cultivated barley, and its wild relative, H. chilense, have several important traits that might be useful for wheat improvement. Here, in situ hybridization and barley expressed sequence tag (EST) markers were used to characterize and compare the chromosomes of H. chilense with those of H. vulgare. FISH with four repetitive DNA sequences, AG, AAG, 5S rDNA and 45S rDNA, was applied to the mitotic chromosomes of H. vulgare, H. chilense and available wheat-H. chilense addition and substitution lines. FISH with the AAG repeat differentiated the individual chromosomes of H. chilense and H. vulgare. The patterns of FISH signals in the two species differed greatly. The 45S rDNA signals were observed on two pairs of chromosomes in both species, while the 5S rDNA signals were observed on four pairs of chromosomes in H. vulgare and on one pair in H. chilense. The AG repeat showed FISH signals at the centromeric regions of all chromosomes of H. vulgare but none of the chromosomes of H. chilense. These results indicate that the chromosomes of the two species are highly differentiated. To study the homoeology between the two species, 209 EST markers of H. vulgare were allocated to individual chromosomes of H. chilense. One hundred and forty of the EST markers were allocated to respective chromosomes of H. chilense using the wheat-H. chilense addition and substitution lines. Twenty-six EST markers on average were allocated to each chromosome except to the chromosome 2H(ch)S, to which only 10 markers were allocated. Ninety percent of the allocated EST markers in H. chilense were placed on H. vulgare chromosomes of the same homo-eologous group, indicating that the expressed sequences of the two species were highly conserved. These EST markers would be useful for detecting chromatin introgressed from these species into the wheat genome.     

Introgression of 1Dx5+1Dy10 into Tritordeum

The uses of hexaploid tritordeum as a crop for human consumption require improvement of its bread-making quality. For this purpose chromosome 1D of bread wheat with the Glu-D1 allele encoding for high-molecular-weight glutenin subunits Dx5+Dy10 was introgressed into tritordeum. Different primary tritordeums were crossed with wheats carrying subunits Dx5+Dy10. The hybrids were backcrossed to tritordeum and seeds for the next backcross (or selfing) were selected for the presence of chromosome 1D using SDS-PAGE. Forty two chromosome plants carrying subunits Dx5+Dy10 were obtained after two backcrosses and selfing. Chromosome characterization of these plants using fluorescence in situ hybridisation (FISH) proved that either chromosome substitution 1H(ch)/1D or 1A/1D had been obtained. A homozygous plant with a translocation of the entire 1DL arm to 1H(ch)S was also obtained. The complete chromosome substitution lines have better agronomic characteristics than the lines with translocations.     

Development of wild barley (Hordeum chilense)-derived DArT markers and their use into genetic and physical mapping

Diversity arrays technology (DArT) genomic libraries were developed from H. chilense accessions to support robust genotyping of this species and a novel crop comprising H. chilense genome (e.g., tritordeums). Over 11,000 DArT clones were obtained using two complexity reduction methods. A subset of 2,209 DArT markers was identified on the arrays containing these clones as polymorphic between parents and segregating in a population of 92 recombinant inbred lines (RIL) developed from the cross between H. chilense accessions H1 and H7. Using the segregation data a high-density map of 1,503 cM was constructed with average inter-bin density of 2.33 cM. A subset of DArT markers was also mapped physically using a set of wheat-H. chilense chromosome addition lines. It allowed the unambiguous assignment of linkage groups to chromosomes. Four segregation distortion regions (SDRs) were found on the chromosomes 2H(ch), 3H(ch) and 5H(ch) in agreement with previous findings in barley. The new map improves the genome coverage of previous H. chilense maps. H. chilense-derived DArT markers will enable further genetic studies in ongoing projects on hybrid wheat, seed carotenoid content improvement or tritordeum breeding program. Besides, the genetic map reported here will be very useful as the basis to develop comparative genomics studies with barley and model species.     

Development of SCARs by direct sequencing of RAPD products: a practical tool for the introgression and marker-assisted selection of wheat

RAPD markers generated by mixtures of two different primers were developed for octoploid × Tritordeum (amphiploid Hordeum chilense × Triticum aestivum) and its parents. Addition lines were used to identify 21 specific RAPD markers for the H. chilense chromosomes detectable in a wheat background. Ten RAPD bands were selected and eight of them were converted into dominant SCAR markers by direct sequencing of the RAPD products, avoiding the costly and time-consuming cloning step. The methodology overcomes some of the pitfalls associated with the election of the right clones when developing SCARs from RAPD markers. The SCARs generated have maintained both the chromosome specificity and the possibility of detection in a wheat background. This strategy provides a rapid method for the characterization of RAPD markers and for the development of PCR-based markers for both the characterization of the introgression of H. chilense in bread and durum wheat, as well as the efficient and reliable screening of tritordeum lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

普通小麦—簇毛麦异附加系和异代换系的C—分带鉴定

用改良的C-分带技术鉴定南京农业大学细胞遗传研究室获得的普通小麦的簇毛麦V_2、V_3、V_4、V_6、V_7染色体异附加系和V_2、V_5异代换系,得到与N-分带和染色体配对分析一致的结果,并且由于C-分带可同时鉴别小麦全部21对染色体,鉴定出V_2异代换系中被代换掉的小麦染色体为1A。     

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.     

Effect of the 5R(5A) alien chromosome substitution on the growth habit and winter hardiness of wheat

The growth habit, ear emergence time, and frost tolerance of wheat/rye substitution lines have been studied in cultivars Rang and Mironovskaya Krupnozernaya whose chromosome 5A is substituted with chromosome 5R of Onkhoyskaya rye. Hybrid analysis has demonstrated that the spring habit of the recipient cultivars Rang and Mironovskaya Krupnozernaya is controlled by dominant gene Vrn-A1 located in chromosome 5A. Onokhoyskaya rye has a dominant gene for the spring habit (Sp1) located in chromosome 5R. It has been found that the resultant 5R(5A) alien-substitution lines have a winter type of development and ears do not emerge during summer in plants sown in spring. The change in growth habit has been shown to be related to the absence of the rye Spl gene expression in the substitution lines. The winter hardiness of winter 5R(5A) alien-substitution lines has been studied under the environmental conditions of Novosibirsk. Testing the lines in the first winter demonstrated that their winter survival is 20-27%. The possible presence of the frost resistance gene homeoallelic to the known genes Fr1 and Fr2 of the common wheat located on chromosomes 5A and 5D, respectively, is discussed.     

The origin of spelt and free-threshing hexaploid wheat

It is widely believed that hexaploid wheat originated via hybridization of hulled tetraploid emmer with Aegilops tauschii (genomes DD) and that the nascent hexaploid was spelt, from which free-threshing wheat evolved by mutations. To reassess the role of spelt in the evolution of Triticum aestivum, 4 disomic substitution lines of Ae. tauschii chromosome 2D in Chinese Spring wheat were developed and one of them was used to map the Tg locus, which controls glume tenacity in Ae. tauschii, relative to simple sequence repeat (SSR) and expressed sequence tag loci on wheat chromosome 2D. The segregation of SSR markers was used to assess the presence of Tg alleles in 11 accessions of spelt, both from Europe and from Asia. Ten of them had an inactive tg allele in the D genome and most had an active Tg allele in the B genome. This is consistent with spelt being derived from free-threshing hexaploid wheat by hybridization of free-threshing wheat with hulled emmer. It is proposed that the tetraploid parent of hexaploid wheat was not hulled emmer but a free-threshing form of tetraploid wheat.     

Production of alloplasmic and euplasmic wheat-barley ditelosomic substitution lines 7H<Superscript>1</Superscript>L<Superscript>mar</Superscript>(7D) and analysis of the 18S/5S mitochondrial repeat in these lines

Alloplasmic lines of common wheat with disomic substitution of chromosome 7D for telocentric chromosome 7H¹L^mar of barley H. marinum subsp. gussoneanum Hudson were isolated from the plants of generation BC₃, produced as a result of backcrossing of barley-wheat hybrids H. marinum subsp. gussoneanum (2n = 28) × T. aestivum (2n = 42), Pyrotrix, cultivar, with 28 common wheat cultivars Pyrotrix 28 and Novosibirskaya 67. Chromosome substitution pattern was determined using SSR analysis and C-banding. In preliminary genomic in situ hybridization experiments, telocentric chromosomes were assigned to wild barley was established. In the BC₃F₈ generations of three alloplasmic lines with the 7H¹L^mar(7D) substitution type the differences in fertility manifestation were observed: most of the L-32(1) plants were sterile, in line L-32(2) only sporadic plants were sterile, and line L-32(3) was fertile. Simultaneously with these experiments, using selfpollinated progeny of the hybrids obtained in crosses of common wheat cultivar Saratovskaya 29 (2n = 41), monosomic for chromosome 7D, with common wheat cultivar Pyrotrix 28 with addition of pair of telocentric chromosomes 7H¹L^mar (7D) of barley H. marinum subsp. gussoneanum, euplasmic wheat-barley ditelosomic substitution 7H¹L^mar (7D) lines were isolated. The lines obtained had normal fertility. PCR analysis of the 18S/5S mitochondrial repeat (hereafter, mtDNA sequence) in alloplasmic and euplasmic ditelosomic substitution lines 7H¹L^mar(7D) was performed. In the plants from alloplasmic sterile line L-32(1), the sequences only of the barley (maternal) type were revealed, while the plants from alloplasmic fertile lines L-32(2) and L-32(3) demonstrated heteroplasmy (the presence of barley- and wheat-like sequences within one individual). In euplasmic ditelosomic substitution lines the presence of only wheat-like 18S/5S mitochondrial repeat sequences was observed. The results indicate that the presence of barley-like mtDNA sequences in alloplasmic substitution lines was not associated with the presence of barley chromosomes in their nuclear genomes.     

IRAP,REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species

Retrotransposon (RTN)-based markers, such as the inter-retrotransposon amplified polymorphism (IRAP) and the retrotransposon-microsatellite amplified polymorphism (REMAP), are highly informative, multilocus, and reveal insertion polymorphisms among individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, and detection of genetic rearrangements induced by allopolyploidization. The hexaploid tritordeum (H^chH^chAABB; 2n?=?6x?=?42) is an allopolyploid produced from crosses between wild barley (Hordeum chilense Roem. et Schultz.) (H^chH^ch; 2n?=?2x?=?14) and durum wheat (Triticum turgidum L. conv. durum) (AABB; 2n?=?4x?=?28). With this study, we carried out the DNA fingerprinting of two newly formed hexaploid tritordeum lines (HT22 and HT27) and their respective parents, line H1 of H. chilense and line T81 of durum wheat, based on IRAPs, REMAPs and inter-simple sequence repeats (ISSRs), in order to detect potential rearrangements in tritordeum derived from polyploidization. The amphiploid nature of the HT22 and HT27 individuals was successfully confirmed after fluorescence in situ hybridization (FISH), which was performed on their mitotic chromosome spreads with genomic DNA from H. chilense and 45S ribosomal DNA (rDNA), simultaneously, as probes. Six combinations of LTR (long terminal repeat) primers and seven combinations of one LTR and one SSR (simple sequence repeat) primers successfully produced IRAPs and REMAPs, respectively, in both tritordeum lines, and their respective parents. ISSRs were produced with three SSR primers (8081, 8082, and 8564). The analysis of the presence/absence of bands among the tritordeum lines and the respective parents allowed the detection of polymorphic bands: (1) shared by tritordeum and one of the parents; (2) exclusively amplified in tritordeum; and (3) exclusively present in one of the parents. Once no polymorphism was detected among the individuals of each parental species, the polymorphic bands that fit into the second and third cases probably constituted rearrangements in the newly formed tritordeums that arose in response to allopolyploidization, which resulted from the loss of parental bands or, conversely, from the appearance of novel bands not seen in the parental species. Most of the polymorphic IRAPs in tritordeum were shared with the female parent (H. chilense), while most of the polymorphic REMAPs and ISSRs were common to the male parent (durum wheat), but globally, most of the bands inherited by tritordeum had a wheat origin. In conclusion, these dominant markers were successful for DNA fingerprinting and detection of rearrangements in newly formed tritordeum derived from responses to allopolyploidization.     

Map-based analysis of the tenacious glume gene Tg-B1 of wild emmer and its role in wheat domestication

The domestication of wheat was instrumental in spawning the civilization of humankind, and it occurred through genetic mutations that gave rise to types with non-fragile rachises, soft glumes, and free-threshing seed. Wild emmer (Triticum turgidum ssp. dicoccoides), the tetraploid AB-genome progenitor of domesticated wheat has genes that confer tenacious glumes (Tg) that underwent genetic mutations to give rise to free-threshing wheat. Here, we evaluated disomic substitution lines involving chromosomes 2A and 2B of wild emmer accessions substituted for homologous chromosomes in tetraploid and hexaploid backgrounds. The results suggested that both chromosomes 2A and 2B of wild emmer possess genes that inhibit threshability. A population of recombinant inbred lines derived from the tetraploid durum wheat variety Langdon crossed with a Langdon — T. turgidum ssp. dicoccoides accession PI 481521 chromosome 2B disomic substitution line was used to develop a genetic linkage map of 2B, evaluate the genetics of threshability, and map the gene derived from PI 481521 that inhibited threshability. A 2BS linkage map comprised of 58 markers was developed, and markers delineated the gene to a 2.3 cM interval. Comparative analysis with maps containing the tenacious glume gene Tg-D1 on chromosome arm 2DS from Aegilops tauschii, the D genome progenitor of hexaploid wheat, revealed that the gene inhibiting threshability in wild emmer was homoeologous to Tg-D1 and therefore designated Tg-B1. Comparative analysis with rice and Brachypodium distachyon indicated a high level of divergence and poorly conserved colinearity, particularly near the Tg-B1 locus. These results provide a foundation for further studies involving Tg-B1, which, together with Tg-D1, had profound influences on wheat domestication.     

小麦新种质4844中外源P染色质的GISH与SSR分析

采用基因组原位杂交(GISH)检测和染色体组成分析方法,对大穗多花小麦新种质4844后代的15个株系进行遗传分析。结果发现,4844-12是1个稳定的异附加系,4844-2和4844-8是稳定的异代换系;对异代换系进行SSR分析表明,代换系中小麦的6D染色体被1对P染色体代换,说明这对冰草染色体与小麦6D染色体有部分同源关系,由此确定4844中的冰草染色体为6P;同时筛选出冰草6P染色体的4个SSR标记。     

Cytogenetic analysis of forms produced by crossing hexaploid triticale with common wheat

Summary Hexaploid triticales were crossed with common wheats, and the resultant froms were selected for either triticale (AD 213/5-80) or common wheat (lines 381/80, 391/80, 393/80). The cytogenetic analysis showed that all forms differ in their chromosome composition. Triticale AD 213/5-80 and wheat line 381/80 were stable forms with 2n = 6x = 42. Lines 391/80 and 393/80 were cytologically unstable. In triticale AD 213/5-80, a 2R (2D) chromosome substitution was found. Each of the three wheat lines had a chromosome formed by the translocation of the short arm of IR into the long arm of the IB chromosome. In line 381/80, this chromosome seems to be inherited from the Kavkaz wheat variety. In lines 391/80 and 393/80, this chromosome apparently formed de novo since the parent forms did not have it. The karyotype of line 381/80 was found to contain rye chromosomes 4R/7R, 5R and 7R/4R. About 15% of the cells in line 391/80 contained an isochromosome for the 5R short arm and also a chromosome which arose from the translocation of the long arms of the 5D and 5R chromosomes. About one-third of the cells in the common wheat line 393/80 contained the 5R chromosome. This chromosome was normal or rearranged. Practical applications of the C-banding technique in the breeding of triticale is discussed.