High-resolution cytological mapping of the long arm of chromosome 5A in common wheat using a series of deletion lines induced by gametocidal (Gc) genes of Aegilops speltoides

Gametocidal (Gc) genes of Aegilops in the background of the wheat genome lead to breakage of wheat chromosomes. The Q gene of wheat was used as a marker to select 19 deletion lines for the long arm of chromosome 5A of common wheat, Triticum aestivum cv. Chinese Spring (CS). The extents of deleted segments were cytologically estimated by the C-banding technique. The DNAs of deletion lines were hybridized with 22 DNA probes recognizing sites on the long arm of the chromosome (5AL) to determine their physical order. Based on the breeding behavior of the deletion lines, the location of a novel gene (Pv, pollen viability) affecting the viability of the male gamete was deduced. The segment translocated from 4AL to 5AL in CS was cytologically estimated to represent 13% of the total length of 5AL. Although DNA markers were almost randomly distributed along the chromosome arm, DNA markers located around the centromere and C-banded regions were obtained only rarely. Some deletion lines were highly rearranged in chromosome structure due to the effect(s) of the Gc gene. Applications of Gc genes for manipulating wheat chromosomes are discussed.     

A new PCR-based marker on chromosome 4AL for resistance to pre-harvest sprouting in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Pre-harvest sprouting (PHS) is a complex trait controlled by multiple genes with strong interaction between environment and genotype that makes it difficult to select breeding materials by phenotypic assessment. One of the most important genes for pre-harvest sprouting resistance is consistently identified on the long arm of chromosome 4A. The 4AL PHS tolerance gene has therefore been targeted by Australian white-grained wheat breeders. A new robust PCR marker for the PHS QTL on wheat chromosome 4AL based on candidate genes search was developed in this study. The new marker was mapped on 4AL deletion bin 13-0.59-0.66 using 4AL deletion lines derived from Chinese Spring. This marker is located on 4AL between molecular markers Xbarc170 and Xwg622 in the doubled-haploid wheat population Cranbrook × Halberd. It was mapped between molecular markers Xbarc170 and Xgwm269 that have been previously shown to be closely linked to grain dormancy in the doubled haploid wheat population SW95-50213 × Cunningham and was co-located with Xgwm269 in population Janz × AUS1408. This marker offers an additional efficient tool for marker-assisted selection of dormancy for white-grained wheat breeding. Comparative analysis indicated that the wheat chromosome 4AL QTL for seed dormancy and PHS resistance is homologous with the barley QTL on chromosome 5HL controlling seed dormancy and PHS resistance. This marker will facilitate identification of the gene associated with the 4A QTL that controls a major component of grain dormancy and PHS resistance.     

Molecular cytogenetic characterization of alien introgressions with gene Fhb3 for resistance to Fusarium head blight disease of wheat

Fusarium head blight (FHB) resistance was identified in the alien species Leymus racemosus, and wheat-Leymus introgression lines with FHB resistance were reported previously. Detailed molecular cytogenetic analysis of alien introgressions T01, T09, and T14 and the mapping of Fhb3, a new gene for FHB resistance, are reported here. The introgression line T09 had an unknown wheat-Leymus translocation chromosome. A total of 36 RFLP markers selected from the seven homoeologous groups of wheat were used to characterize T09 and determine the homoeologous relationship of the introgressed Leymus chromosome with wheat. Only short arm markers for group 7 detected Leymus-specific fragments in T09, whereas 7AS-specific RFLP fragments were missing. C-banding and genomic in situ hybridization results indicated that T09 has a compensating Robertsonian translocation T7AL·7Lr#1S involving the long arm of wheat chromosome 7A and the short arm of Leymus chromosome 7Lr#1 substituting for chromosome arm 7AS of wheat. Introgression lines T01 (2n = 44) and T14 (2n = 44) each had two pairs of independent translocation chromosomes. T01 had T4BS·4BL-7Lr#1S + T4BL-7Lr#1S·5Lr#1S. T14 had T6BS·6BL-7Lr#1S + T6BL·5Lr#1S. These translocations were recovered in the progeny of the irradiated line Lr#1 (T5Lr#1S·7Lr#1S). The three translocation lines, T01, T09, and T14, and the disomic addition 7Lr#1 were consistently resistant to FHB in greenhouse point-inoculation experiments, whereas the disomic addition 5Lr#1 was susceptible. The data indicated that at least one novel FHB resistance gene from Leymus, designated Fhb3, resides in the distal region of the short arm of chromosome 7Lr#1, because the resistant translocation lines share a common distal segment of 7Lr#1S. Three PCR-based markers, BE586744-STS, BE404728-STS, and BE586111-STS, specific for 7Lr#1S were developed to expedite marker-assisted selection in breeding programs.     

RFLP-based maps of the homoeologous group-6 chromosomes of wheat and their application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat

Genetic maps of the homoeologous group-6 chromosomes of bread wheat, Triticum aestivum, have been constructed spanning 103 cM on 6A, 90 cM on 6B and 124 cM on 6D. These maps were transferred to a Chinese Spring (CS) x line #31 cross to locate a dominant powdery mildew resistance gene, Pm12, introgressed into line #31 from Aegilops speltoides. Pm12 was shown to lie on the short arm of translocation chromosome 6BS-6SS.6SL in line #31, but could not be mapped more precisely due to the lack of recombination between the 6S Ae. speltoides segment and chromosome 6B. Possible strategies to reduce the size of the alien segment, which probably encompasses the complete long arm and more than 82% of the short arm of chromosome 6B, are discussed.     

Engineering of interstitial foreign chromosome segments containing the K+/Na+ selectivity gene Kna1 by sequential homoeologous recombination in durum wheat

Targeted homoeologous recombination mediated by the absence of the Ph1 locus is currently the most efficient technique by which foreign genes can be introgressed into polyploid wheat species. Because intra-arm homoeologous double cross-overs are rare, introgressed foreign genes are usually on terminal foreign chromosome segments. Since the minimum length of such a segment is determined by the position of a gene in the chromosome, large chromosome segments with undesirable genetic effects are often introgressed. Introgression of foreign genes on short interstitial segments based on two cycles of homoeologous recombination is described here. The utility of the technique is demonstrated by the introgression of the Kna1 locus, which controls K⁺/Na⁺ selectivity in T. aesivum L., on short interstitial segments of chromosome 4D into chromosome 4B of Triticum turgidum L. The level of recombination in a homoeologous segment is not significantly affected by a juxtaposed proximal homologous segment in the absence of the Ph1 locus.     

Association between presence of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> introgression and D-genome retention in hexaploid/tetraploid wheat crosses

The 2G Triticum timopheevii introgression harbours genes for multiple disease resistance and quality traits in bread wheat. In order to transfer this segment from bread wheat into durum, the bread wheat line Sunguard, which carries this introgressed 2G segment was crossed with three tetraploid durum parents. A significant difference was observed in the segregation ratio of the 2G segment in the different crosses at the F₂ generation with two of the three populations indicating segregation distortion against the hexaploid 2G segment. In these populations, the presence of the 2G segment was strongly correlated with the presence of D-genome chromosomes. These results were confirmed in the F₄ generation of these populations. Six plants were identified in the F₄ generation, which had retained the introgressed 2G segment in a homozygous condition and did not have a complete D-genome set. Two of these lines only had two non-homologous D-genome chromosomes in the F₅ generation. Thus, the 2G segment and possibly other translocations can be transferred into durum wheat through hexaploid/tetraploid hybridisation.     

The introgressed segment carrying rust resistance genes Yr17, Lr37 and Sr38 in wheat can be assayed by a cloned disease resistance gene-like sequence

A cloned gene sequence (Vrga1D), with features of the nucleotide-binding-site leucine-rich repeat class of disease resistance (R) gene sequence super family, was previously shown to belong to a family of five gene members derived from a Triticum ventricosum Ces. (syn. Aegilops ventricosa Tausch) segment in wheat (Triticum aestivum L.). This gene family was introgressed, together with the linked rust resistance genes Yr17, Lr37 and Sr38 from T. ventricosum, to wheat chromosome 2AS. An independently derived T. ventricosum segment carrying a leaf rust resistance gene in a French wheat cultivar, was shown to exhibit a rust resistance response equivalent to Lr37 as well as Yr17 and Sr38. DNA probes from different regions of the Vrga1D clone consistently detected the presence of RFLPs associated with the introgressed segment carrying the resistance genes Yr17, Lr37 and Sr38 present in diverse wheat genotypes from Australia, Canada, France and the UK. Our results showed that the transfer of the T. ventricosum- derived Vrga1 gene members and the rust resistance genes were always accompanied by the loss of a corresponding set of Vrga1-related gene members in recipient wheat cultivars presumed to be of homoeoallelic origin. A PCR assay, based on sequences from the 3"-untranslated region of a Vrga1 gene member isolated from the T. ventricosum donor line of the introgressed segment, was developed. The PCR assay detected the presence of the introgressed rust resistance genes across the diverse wheat backgrounds and should be useful in marker- assisted selection in wheat breeding. Received: 24 December 1999 / Accepted: 13 June 2000     

RFLP analysis of the size of chromosomal segments retained around the Tm-2 locus of tomato during backcross breeding

Summary Genes introduced into cultivated plants by backcross breeding programs are flanked by introgressed segments of DNA derived from the donor parent. This phenomenon is known as linkage drag and is frequently thought to affect traits other than the one originally targeted. The Tm-2 gene of Lycopersicon peruvianum, which confers resistance to tobacco mosaic virus, was introduced into several different tomato cultivars (L. esculentum) by repeated backcrossing. We have measured the sizes of the introgressed segments flanking the Tm-2 locus in several of these cultivars using a high density map of restriction fragment length polymorphic (RFLP) markers. The smallest introgressed segment is estimated to be 4 cM in length, while the longest is over 51 cM in length and contains the entire short arm of chromosome 9. Additionally, RFLP analysis was performed on remnant seed from different intermediate generations corresponding to two different backcross breeding programs for TMV resistance. The results reveal that plants containing desirable recombination near the resistance gene were rarely selected during backcrossing and, as a result, the backcross breeding method was largely ineffective in reducing the size of linked DNA around the resistance gene. We propose that, by monitoring recombination around genes of interest with linked RFLP markers, one can quickly and efficiently reduce the amount of linkage drag associated with introgression. Using such a procedure, it is estimated that an introgressed segment can be obtained in two generations that is as small as that which would otherwise require 100 backcross generations without RFLP selection.     

An introgression on wheat chromosome 4DL in RL6077 (Thatcher*6/PI 250413) confers adult plant resistance to stripe rust and leaf rust (Lr67)

Adult plant resistance (APR) to leaf rust and stripe rust derived from the wheat (Triticum aestivum L.) line PI250413 was previously identified in RL6077 (=Thatcher*6/PI250413). The leaf rust resistance gene in RL6077 is phenotypically similar to Lr34 which is located on chromosome 7D. It was previously hypothesized that the gene in RL6077 could be Lr34 translocated to another chromosome. Hybrids between RL6077 and Thatcher and between RL6077 and 7DS and 7DL ditelocentric stocks were examined for first meiotic metaphase pairing. RL6077 formed chain quadrivalents and trivalents relative to Thatcher and Chinese Spring; however both 7D telocentrics paired only as heteromorphic bivalents and never with the multivalents. Thus, chromosome 7D is not involved in any translocation carried by RL6077. A genome-wide scan of SSR markers detected an introgression from chromosome 4D of PI250413 transferred to RL6077 through five cycles of backcrossing to Thatcher. Haplotype analysis of lines from crosses of Thatcher × RL6077 and RL6058 (Thatcher*6/PI58548) × RL6077 showed highly significant associations between introgressed markers (including SSR marker cfd71) and leaf rust resistance. In a separate RL6077-derived population, APR to stripe rust was also tightly linked with cfd71 on chromosome 4DL. An allele survey of linked SSR markers cfd71 and cfd23 on a set of 247 wheat lines from diverse origins indicated that these markers can be used to select for the donor segment in most wheat backgrounds. Comparison of RL6077 with Thatcher in field trials showed no effect of the APR gene on important agronomic or quality traits. Since no other known Lr genes exist on chromosome 4DL, the APR gene in RL6077 has been assigned the name Lr67.     

Fine mapping, phenotypic characterization and validation of non-race-specific resistance to powdery mildew in a wheat–Triticum militinae introgression line

Introgression of several genomic loci from tetraploid Triticum militinae into bread wheat cv. T?hti has increased resistance of introgression line 8.1 to powdery mildew in seedlings and adult plants. In our previous work, only a major quantitative trait locus (QTL) on chromosome 4AL of the line 8.1 contributed significantly to resistance, whereas QTL on chromosomes 1A, 1B, 2A, 5A and 5B were detected merely on a suggestive level. To verify and characterize all QTLs in the line 8.1, a mapping population of double haploid lines was established. Testing for seedling resistance to 16 different races/mixtures of Blumeria graminis f. sp. tritici revealed four highly significant non-race-specific resistance QTL including the main QTL on chromosome 4AL, and a race-specific QTL on chromosome 5B. The major QTL on chromosome 4AL (QPm.tut-4A) as well as QTL on chromosome 5AL and a newly detected QTL on 7AL were highly effective at the adult stage. The QPm.tut-4A QTL accounts on average for 33-49 % of the variation in resistance in the double haploid population. Interactions between the main QTL QPm.tut-4A and the minor QTL were evaluated and discussed. A population of 98 F(2) plants from a cross of susceptible cv. Chinese Spring and the line 8.1 was created that allowed mapping the QPm.tut-4A locus to the proximal 2.5-cM region of the introgressed segment on chromosome 4AL. The results obtained in this work make it feasible to use QPm.tut-4A in resistance breeding and provide a solid basis for positional cloning of the major QTL.     

High-resolution cytological mapping of the long arm of chromosome 5A in common wheat using a series of deletion lines induced by gametocidal (Gc) genes of Aegilops speltoides

Gametocidal (Gc) genes of Aegilops in the background of the wheat genome lead to breakage of wheat chromosomes. The Q gene of wheat was used as a marker to select 19 deletion lines for the long arm of chromosome 5A of common wheat, Triticum aestivum cv. Chinese Spring (CS). The extents of deleted segments were cytologically estimated by the C-banding technique. The DNAs of deletion lines were hybridized with 22 DNA probes recognizing sites on the long arm of the chromosome (5AL) to determine their physical order. Based on the breeding behavior of the deletion lines, the location of a novel gene (Pv, pollen viability) affecting the viability of the male gamete was deduced. The segment translocated from 4AL to 5AL in CS was cytologically estimated to represent 13% of the total length of 5AL. Although DNA markers were almost randomly distributed along the chromosome arm, DNA markers located around the centromere and C-banded regions were obtained only rarely. Some deletion lines were highly rearranged in chromosome structure due to the effect(s) of the Gc gene. Applications of Gc genes for manipulating wheat chromosomes are discussed.     

Molecular mapping of resistance to <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> race 5 and sensitivity to Ptr ToxB in wheat

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an economically important foliar disease in the major wheat growing areas of the world. Multiple races of the pathogen have been characterized based on their ability to cause necrosis and/or chlorosis in differential wheat lines. Isolates of race 5 cause chlorosis only, and they produce a host-selective toxin designated Ptr ToxB that induces chlorosis when infiltrated into sensitive genotypes. The international Triticeae mapping initiative (ITMI) mapping population was used to identify genomic regions harboring QTLs for resistance to fungal inoculations of Ptr race 5 and to determine the chromosomal location of the gene conditioning sensitivity to Ptr ToxB. The toxin-insensitivity gene, which we are designating tsc2, mapped to the distal tip of the short arm of chromosome 2B. This gene was responsible for the effects of a major QTL associated with resistance to the race 5 fungus and accounted for 69% of the phenotypic variation. Additional minor QTLs were identified on the short arm of 2A, the long arm of 4A, and on the long arm of chromosome 2B. Together, the major QTL on 2BS identified by tsc2 and the QTL on 4AL explained 73% of the total phenotypic variation for resistance to Ptr race 5. The results of this research indicate that Ptr ToxB is a major virulence factor, and the markers closely linked to tsc2 and the 4A QTL should be useful for introgression of resistance into adapted germplasm.     

Tagging of an Aegilops speltoides Derived Leaf Rust Resistance Gene Lr 28 with a Microsatellite Marker in Wheat

Leaf rust resistance gene Lr28 has been transferred form Aegilops speltoides into bread wheat on chromosome 4AL. To identify the molecular markers linked to Lr28 the available microsatellite markers for wheat chromosome arm 4AL were surveyed on near isogenic lines (NILs) of Triticum aestivum cultivars having Lr28 gene, other Lrgenes and susceptible cultivars. A null allele of Xgwm 160 marker was found to be associated with Lr28. Linkage between the marker and the Lr28 resistance gene was confirmed using F₂ mapping population of cross PBW343 and HD2329 + Lr28.     

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication‐related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.     

Cereal cyst nematode resistance gene <Emphasis Type="Italic">CreV</Emphasis> effective against <Emphasis Type="Italic">Heterodera filipjevi</Emphasis> transferred from chromosome 6VL of <Emphasis Type="Italic">Dasypyrum villosum</Emphasis> to bread wheat

Cereal cyst nematodes (CCN) are a global economic problem for cereal production. Heterodera filipjevi is one of the most commonly identified and widespread CCN species found in many wheat production regions of the world. Transferring novel genes for resistance to H. filipjevi from wild relatives of wheat is a promising strategy for protection of wheat crops. A set of wheat–Dasypyrum villosum chromosome addition lines, T6V#4S·6AL translocation lines and their donor parental lines were tested for their response to the nematode. D. villosum and wheat–D. villosum disomic addition line DA6V#4 were resistant. As T6V#4S·6AL translocation lines were susceptible, resistance was presumed to be located on chromosome 6V#4L. The objective of this study was to produce and characterize wheat–6V#4L translocations and confirm the chromosome location of the resistance. Introgression lines T6V#4L·6AS, T6V#4L-4BL·4BS and DT6V#4L were developed and subjected to molecular cytogenetic analysis. These and four additional wheat–6V#4 introgression lines were tested for response to H. filipjevi in the greenhouse. The results indicated that introgression lines DA6V#4, T6V#4L·6AS, T6V#4L-4BL·4BS, T6V#4L·6V#4S-7BS and DT6VL#4 had higher levels of H. filipjevi resistance than their recurrent parent. However, Del6V#4L-1 and translocation line T6V#4S·6AL were equally susceptible to wheat cv. Chinese Spring. The CCN resistance gene, temporarily named CreV, was therefore physically mapped to chromosome arm 6V#4L FL 0.80–1.00. Translocation chromosomes T6V#4L·6AS transferred to a modern wheat cv. Aikang 58 with its co-dominant molecular markers could be utilized as a novel germplasm for CCN resistance breeding in wheat.     

Identification of novel QTLs for seedling and adult plant leaf rust resistance in a wheat doubled haploid population

Pyramiding of genes that confer partial resistance is a method for developing wheat (Triticum aestivum L.) cultivars with durable resistance to leaf rust caused by Puccinia triticina. In this research, a doubled haploid population derived from the cross between the synthetic hexaploid wheat (SHW) (×Aegilotriticum spp.) line TA4152-60 and the North Dakota breeding line ND495 was used for identifying genes conferring partial resistance to leaf rust in both the adult plant and seedling stages. Five QTLs located on chromosome arms 3AL, 3BL, 4DL, 5BL and 6BL were associated with adult plant resistance with the latter four representing novel leaf rust resistance QTLs. Resistance effects of the 4DL QTL were contributed by ND495 and the effects of the other QTLs were contributed by the SHW line. The QTL on chromosome arm 3AL had large effects and also conferred seedling resistance to leaf rust races MJBJ, TDBG and MFPS. The other major QTL, which was on chromosome arm 3BL, conferred seedling resistance to race MFPS and was involved in a significant interaction with a locus on chromosome arm 5DS. The QTLs and the associated molecular markers identified in this research can be used to develop wheat cultivars with potentially durable leaf rust resistance.     

Genetic analysis of the traits introgressed from <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch. to bread wheat and determined by chromosome 5A genes

A winter bread wheat accession from the Arsenal collection was genetically examined to study the results of introgression, which substantially changed the physiological and morphological traits of the original spring cultivar Rodina. Apart from its winter habit, the accession was characterized by awned speltoid spikes, suggesting introgression into chromosome 5A, which carries marker genes in the order Vrn-A1-Q-B1. Genetic analysis showed that the chromosome fragment introgressed from Aegilops speltoides recombined well with the homeologous region of bread wheat chromosome 5A in the region between the Vrn-A1 and Q genes. Recombination between the Vrn-A1 and B1 genes was not detected, and it was assumed that the order of the marker genes of chromosome 5A was inverted to produce Q-Vrn-A1-B1. When the winter introgression line was crossed with Triticum spelta L., an interaction of two dominant genes determining the spike character was for the first time detected in F₁, increasing the spike length and the number of spikelets, and followed with transgression in F₂. It was assumed that Ae. speltoides had a homeoallelic speltoid gene, which was designated as Q ^S .     

Identification and mapping of PmG16, a powdery mildew resistance gene derived from wild emmer wheat

The gene-pool of wild emmer wheat, Triticum turgidum ssp. dicoccoides, harbors a rich allelic repertoire for disease resistance. In the current study, we made use of tetraploid wheat mapping populations derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16) to identify and map a new powdery mildew resistance gene derived from wild emmer wheat. Initially, the two parental lines were screened with a collection of 42 isolates of Blumeria graminis f. sp. tritici (Bgt) from Israel and 5 isolates from Switzerland. While G18-16 was resistant to 34 isolates, Langdon was resistant only to 5 isolates and susceptible to 42 isolates. Isolate Bgt#15 was selected to differentiate between the disease reactions of the two genotypes. Segregation ratio of F_2-3 and recombinant inbreed line (F₇) populations to inoculation with isolate Bgt#15 indicated the role of a single dominant gene in conferring resistance to Bgt#15. This gene, temporarily designated PmG16, was located on the distal region of chromosome arm 7AL. Genetic map of PmG16 region was assembled with 32 simple sequence repeat (SSR), sequence tag site (STS), Diversity array technology (DArT) and cleaved amplified polymorphic sequence (CAPS) markers and assigned to the 7AL physical bin map (7AL-16). Using four DNA markers we established colinearity between the genomic region spanning the PmG16 locus within the distal region of chromosome arm 7AL and the genomic regions on rice chromosome 6 and Brachypodium Bd1. A comparative analysis was carried out between PmG16 and other known Pm genes located on chromosome arm 7AL. The identified PmG16 may facilitate the use of wild alleles for improvement of powdery mildew resistance in elite wheat cultivars via marker-assisted selection.