Analysis of leaf and stripe rust severities reveals pathotype changes and multiple minor QTLs associated with resistance in an Avocet?×?Pastor wheat population

Leaf rust and stripe rust are important diseases of wheat world-wide and deployment of cultivars with genetic resistance is an effective and environmentally sound control method. The use of minor, additive genes conferring adult plant resistance (APR) has been shown to provide resistance that is durable. The wheat cultivar ‘Pastor’ originated from the CIMMYT breeding program that focuses on minor gene-based APR to both diseases by selecting and advancing generations alternately under leaf rust and stripe rust pressures. As a consequence, Pastor has good resistance to both rusts and was used as the resistant parent to develop a mapping population by crossing with the susceptible ‘Avocet’. All 148 F₅ recombinant inbred lines were evaluated under artificially inoculated epidemic environments for leaf rust (3 environments) and stripe rust (4 environments, 2 of which represent two evaluation dates in final year due to the late build-up of a new race virulent to Yr31) in Mexico. Map construction and QTL analysis were completed with 223 polymorphic markers on 84 randomly selected lines in the population. Pastor contributed Yr31, a moderately effective race-specific gene for stripe rust resistance, which was overcome during this study, and this was clearly shown in the statistical analysis. Linked or pleiotropic chromosomal regions contributing to resistance against both pathogens included Lr46/Yr29 on 1BL, the Yr31 region on 2BS, and additional minor genes on 5A, 6B and 7BL. Other minor genes for leaf rust resistance were located on 1B, 2A and 2D and for stripe rust on 1AL, 1B, 3A, 3B, 4D, 6A, 7AS and 7AL. The 1AL, 1BS and 7AL QTLs are in regions that were not identified previously as having QTLs for stripe rust resistance. The development of uniform and severe epidemics facilitated excellent phenotyping, and when combined with multi-environment analysis, resulted in the relatively large number of QTLs identified in this study.     

Identification,mapping, and marker development of stem rust resistance genes in durum wheat ‘Lebsock’

Wheat production in many wheat-growing regions is vulnerable to stem rust, caused by Puccinia graminis f. sp. tritici (Pgt). Several previous studies showed that most of the durum cultivars adapted to the upper Great Plains in the USA have good resistance to the major Pgt pathotypes, including the Ug99 race group. To identify the stem rust resistance (Sr) genes in the durum cultivar ‘Lebsock’, a tetraploid doubled haploid (DH) population derived from a cross between Lebsock and Triticum turgidum ssp. carthlicum PI 94749 was screened with the Pgt races TTKSK, TRTTF, and TTTTF. The stem rust data and the genotypic data previously developed were used to identify quantitative trait loci (QTL) associated with resistance. We identified one QTL each on chromosome arms 4AL, 6AS, 6AL, and 2BL. Based on marker and race-specification analysis, we postulated that the QTL on 4AL, 6AS, 6AL, and 2BL correspond to Sr7a, Sr8155B1, Sr13, and likely Sr9e, respectively. The results indicated that most of the US durum germplasm adapted to the upper Great Plains likely harbors the four major Sr genes characterized in this study. Among these genes, Sr8155B1 was recently identified and shown to be unique in that it conferred susceptibility to TTKSK but resistance to variant race TTKST. Two, three, and one thermal asymmetric reverse PCR (STARP) markers were developed for Sr7a, Sr8155-B1, and Sr13, respectively. Knowledge of the Sr genes in durum germplasm and the new STARP markers will be useful to pyramid and deploy multiple Sr genes in future durum and wheat cultivars.     

Mapping of novel salt tolerance QTL in an Excalibur × Kukri doubled haploid wheat population

Key message

Novel QTL for salinity tolerance traits have been detected using non-destructive and destructive phenotyping in bread wheat and were shown to be linked to improvements in yield in saline fields.

Abstract

Soil salinity is a major limitation to cereal production. Breeding new salt-tolerant cultivars has the potential to improve cereal crop yields. In this study, a doubled haploid bread wheat mapping population, derived from the bi-parental cross of Excalibur?×?Kukri, was grown in a glasshouse under control and salinity treatments and evaluated using high-throughput non-destructive imaging technology. Quantitative trait locus (QTL) analysis of this population detected multiple QTL under salt and control treatments. Of these, six QTL were detected in the salt treatment including one for maintenance of shoot growth under salinity (QG_(1–5).asl-7A), one for leaf Na⁺ exclusion (QNa.asl-7A) and four for leaf K⁺ accumulation (QK.asl-2B.1, QK.asl-2B.2, QK.asl-5A and QK:Na.asl-6A). The beneficial allele for QG_(1–5).asl-7A (the maintenance of shoot growth under salinity) was present in six out of 44 mainly Australian bread and durum wheat cultivars. The effect of each QTL allele on grain yield was tested in a range of salinity concentrations at three field sites across 2 years. In six out of nine field trials with different levels of salinity stress, lines with alleles for Na⁺ exclusion and/or K⁺ maintenance at three QTL (QNa.asl-7A, QK.asl-2B.2 and QK:Na.asl-6A) excluded more Na⁺ or accumulated more K⁺ compared to lines without these alleles. Importantly, the QK.asl-2B.2 allele for higher K⁺ accumulation was found to be associated with higher grain yield at all field sites. Several alleles at other QTL were associated with higher grain yields at selected field sites.

     

Mapping QTLs with epistatic effects and QTL×environment interactions for plant height using a doubled haploid population in cultivated wheat

Quantitative trait loci (QTLs) for plant height in wheat (Triticum aestivum L.) were studied using a set of 168 doubled haploid (DH) lines, which were derived from the cross Huapei 3/Yumai 57. A genetic linkage map was constructed using 283 SSR and 22 EST-SSR markers. The DH population and the parents were evaluated for wheat plant height in 2005 and 2006 in Tai'an and 2006 in Suzhou. QTL analyses were performed using the software of QTLNetwork version 2.0 based on the mixed linear model. Four additive QTLs and five pairs of epistatic effects were detected, which were distributed on chromosomes 3A, 4B, 4D, 5A, 6A, 7B, and 7D. Among them, three additive QTLs and three pairs of epistatic QTLs showed QTLxenvironment interactions (QEs). Two major QTLs, QphAB and Qph4D, which accounted for 14.51 % and 20.22% of the phenotypic variation, were located similar to the reported locations of the dwarfing genes Rhtl and Rht2, respectively. The Qph3A-2 with additive effect was not reported in previous linkage mapping studies. The total QTL ef fects detected for the plant height explained 85.04% of the phenotypic variation, with additive effects 46.07%, epistatic effects 19.89%, and QEs 19.09%. The results showed that both additive effects and epistatic effects were important genetic bases of wheat plant height, which were subjected to environmental modifications, and caused dramatic changes in phenotypic effects. The information obtained in this study will be useful for manipulating the QTLs for wheat plant height by molecular marker-assisted selection (MAS).     

Mapping QTLs with epistatic effects and QTL×environment interactions for plant height using a doubled haploid population in cultivated wheat

Quantitative trait loci (QTLs) for plant height in wheat (Triticum aestivum L.) were studied using a set of 168 doubled haploid (DH) lines, which were derived from the cross Huapei 3/Yumai 57. A genetic linkage map was constructed using 283 SSR and 22 EST-SSR markers. The DH population and the parents were evaluated for wheat plant height in 2005 and 2006 in Tai’an and 2006 in Suzhou. QTL analyses were performed using the software of QTLNetwork version 2.0 based on the mixed linear model. Four additive QTLs and five pairs of epistatic effects were detected, which were distributed on chromosomes 3A, 4B, 4D, 5A, 6A, 7B, and 7D. Among them, three additive QTLs and three pairs of epistatic QTLs showed QTL×environment interactions (QEs). Two major QTLs, Qph4B and Qph4D, which accounted for 14.51% and 20.22% of the phenotypic variation, were located similar to the reported locations of the dwarfing genes Rht1 and Rht2, respectively. The Qph3A-2 with additive effect was not reported in previous linkage mapping studies. The total QTL effects detected for the plant height explained 85.04% of the phenotypic variation, with additive effects 46.07%, epistatic effects 19.89%, and QEs 19.09%. The results showed that both additive effects and epistatic effects were important genetic bases of wheat plant height, which were subjected to environmental modifications, and caused dramatic changes in phenotypic effects. The information obtained in this study will be useful for manipulating the QTLs for wheat plant height by molecular marker-assisted selection (MAS).     

Genetics of resistance to yellow rust in PBW343 × Kenya Kudu recombinant inbred line population and mapping of a new resistance gene YrKK

Yellow or stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important disease of common wheat (Triticum aestivum L.) worldwide. A recombinant inbred line (RIL) population, derived from the cross PBW343 × Kenya Kudu, was phenotyped for yellow rust reaction in the field at the CIMMYT research station near Toluca, Mexico, during 2010 and 2011. Segregation results indicated the presence of a race-specific resistance gene, temporarily designated as YrKK, in Kenya Kudu that conferred immunity to adult plants in field trials, despite conferring only slight reductions in seedling reactions in greenhouse tests with three Mexican pathotypes. A minimum of four minor genes having additive effects also segregated in the population and were likely derived from both parents. A total of 635 simple sequence repeat (SSR) primers were screened for polymorphism surveys on the parents, and resistant (YrKK-possessing RILs) and susceptible (YrKK-lacking RILs) bulks identified four polymorphic markers. These markers were located on the short arm of chromosome 2B. Genotyping of the entire RIL population identified Xgwm148 and Xwmc474 as the most closely linked proximal and distal flanking SSR markers, with respective genetic distances of 3.6 and 1.8 cM from YrKK. Four yellow rust resistance genes (Yr27, Yr31, Yr41, and YrP81) are located on chromosome 2BS; however, their specificity to pathogen pathotypes and host reactions in seedling and adult plants indicate that YrKK is a new resistance gene.     

QTL analysis of the spring wheat “Chapio” identifies stable stripe rust resistance despite inter-continental genotype × environment interactions

Chapio is a spring wheat developed by CIMMYT in Mexico by a breeding program that focused on multigenic resistances to leaf rust and stripe rust. A population consisting of 277 recombinant inbred lines (RILs) was developed by crossing Chapio with Avocet. The RILs were genotyped with DArT markers (137 randomly selected RILs) and bulked segregant analysis conducted to supplement the map with informative SSR markers. The final map consisted of 264 markers. Phenotyping against stripe rust was conducted for three seasons in Toluca, Mexico and at three sites over two seasons (total of four environments) in Sichuan Province, China. Significant loci across the two inter-continental regions included Lr34/Yr18 on 7DS, Sr2/Yr30 on 3BS, and a QTL on 3D. There were significant genotype × environment interactions with resistance gene Yr31 on 2BS being effective in most of the Toluca environments; however, a late incursion of a virulent pathotype in 2009 rendered this gene ineffective. This locus also had no effect in China. Conversely, a 5BL locus was only effective in the Chinese environments. There were also complex additive interactions. In the Mexican environments, Yr31 suppressed the additive effect of Yr30 and the 3D locus, but not of Lr34/Yr18, while in China, the 3D and 5BL loci were generally not additive with each other, but were additive when combined with other loci. These results indicate the importance of maintaining diverse, multi-genic resistances as Chapio had stable inter-continental resistance despite the fact that there were QTLs that were not effective in either one or the other region.     

Advanced backcross QTL analysis of a hard winter wheat × synthetic wheat population

Advanced backcross quantitative trait locus (AB-QTL) analysis was used to identify QTLs for yield and yield components in a backcross population developed from a cross between hard red winter wheat (Triticum aestivum L.) variety Karl 92 and the synthetic wheat line TA 4152-4. Phenotypic data were collected for agronomic traits including heading date, plant height, kernels per spike, kernel weight, tiller number, biomass, harvest index, test weight, grain yield, protein content, and kernel hardness on 190 BC₂F_2:4 lines grown in three replications in two Kansas environments. Severity of wheat soilborne mosaic virus (WSBMV) reaction was evaluated at one location. The population was genotyped using 151 microsatellite markers. Of the ten putative QTLs identified, seven were located on homoeologous group 2 and group 3 chromosomes. The favorable allele was contributed by cultivated parent Karl 92 at seven QTLs including a major one for WSBMV resistance, and by the synthetic parent at three QTLs: for grain hardness, kernels per spike, and tiller number. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Powdery mildew resistance and Lr34/Yr18 genes for durable resistance to leaf and stripe rust cosegregate at a locus on the short arm of chromosome 7D of wheat

The incorporation of effective and durable disease resistance is an important breeding objective for wheat improvement. The leaf rust resistance gene Lr34 and stripe rust resistance gene Yr18 are effective at the adult plant stage and have provided moderate levels of durable resistance to leaf rust caused by Puccinia triticina Eriks. and to stripe rust caused by Puccinia striiformis Westend. f. sp. tritici. These genes have not been separated by recombination and map to chromosome 7DS in wheat. In a population of 110 F₇ lines derived from a Thatcher × Thatcher isogenic line with Lr34/Yr18, field resistance to leaf rust conferred by Lr34 and to stripe rust resistance conferred by Yr18 cosegregated with adult plant resistance to powdery mildew caused by Blumeria graminis (DC) EO Speer f. sp. tritici. Lr34 and Yr18 were previously shown to be associated with enhanced stem rust resistance and tolerance to barley yellow dwarf virus infection. This chromosomal region in wheat has now been linked with resistance to five different pathogens. The Lr34/Yr18 phenotypes and associated powdery mildew resistance were mapped to a single locus flanked by microsatellite loci Xgwm1220 and Xgwm295 on chromosome 7DS.     

QTL mapping of resistance to Sclerotinia midstalk rot in RIL of sunflower population NDBLOSsel × CM625

Midstalk rot caused by Sclerotinia sclerotiorum is an important disease of sunflower in its main areas of cultivation. The objectives of this study were to (1) verify quantitative trait loci (QTL) for midstalk-rot resistance found in F₃ families of the NDBLOS_sel × CM625 population in recombinant inbred lines (RIL) derived from the same cross; (2) re-estimate their position and genetic effects; (3) draw inferences about the predictive quality of QTL for midstalk-rot resistance identified in the F₃ families as compared to those in the RIL. Phenotypic data for three resistance (leaf lesion, stem lesion, and speed of fungal growth) and two morphological traits (leaf length and leaf length with petiole) were obtained from 317 RIL following artificial infection in field experiments across two environments. For genotyping the 248 RIL, we selected 41 simple sequence repeat (SSR) markers based on their association with QTL for Sclerotinia midstalk-rot resistance in an earlier study. The resistance traits showed intermediate to high heritabilities and were significantly correlated with each other Genotypic correlations between F₃ families and the RIL were highly significant and ranged between 0.50 for leaf length and 0.64 for stem lesion. For stem lesion, two genomic regions on linkage group (LG) 8 and LG16 explaining 26.5% of the genotypic variance for Sclerotinia midstalk-rot resistance were consistent across generations. For this trait, the genotypic correlation between the observed performance and its prediction based on QTL positions and effects in F₃ families was surprisingly high The genetic effects and predictive quality of these two QTL are promising for application in marker-assisted selection to Sclerotinia midstalk-rot resistance.     

Linkage mapping of genes for resistance to leaf,stem and stripe rusts and ω-secalins on the short arm of rye chromosome 1R

Summary The genes controlling resistance to three wheat rusts, viz., leaf rust (Lr26), stem rust (Sr31) and stripe or yellow rust (Yr9), and -secalins (Sec1), located on the short arm of rye chromosome 1R, were mapped with respect to each other and the centromere. Analysis of 214 seeds (or families derived from them) from testcrosses between a 1BL.1RS/1R heterozygote and Chinese Spring ditelocentric 1BL showed no recombination between the genes for resistance to the three rusts, suggesting very tight linkage or perhaps a single complex locus conferring resistance to the three rusts. The rust resistance genes were located 5.4 ± 1.7 cM from the Sec1 locus, which in turn was located 26.1 ± 4.3 cM from the centromere; the gene order being centromere — Sec1 — Lr26/Sr31/Yr9 — telomere. In a second test-cross, using a different 1BL.1RS translocation which had only stem rust resistance (SrR), the above gene order was confirmed despite a very large proportion of aneuploids (45.8%) among the progeny. Furthermore, a map distance of 16.0 ± 4.8 cM was estimated for SrR and the telomeric heterochromatin (C-band) on 1RS. These results suggest that a very small segment of 1RS chromatin is required to maintain resistance to all three wheat rusts. It should be possible but difficult to separate the rust resistance genes from the secalin gene(s), which are thought to contribute to dough stickiness of wheat-rye translocation lines carrying 1RS.     

Genotyping-by-sequencing to remap QTL for type II Fusarium head blight and leaf rust resistance in a wheat–tall wheatgrass introgression recombinant inbred population

Fusarium graminearum Schwabe (Fusarium head blight, FHB) and Puccinia triticina Eriks (leaf rust) are two major fungal pathogens posing a continuous threat to the wheat crop; consequently, identifying resistance genes from various sources is always of importance to wheat breeders. We identified tightly linked single nucleotide polymorphism (SNP) markers for the FHB resistance quantitative trait locus (QTL) Qfhs.pur-7EL and the leaf rust resistance locus Lr19 using genotyping-by-sequencing (GBS) in a wheat–tall wheatgrass introgression-derived recombinant inbred line (RIL) population. One thousand and seven hundred high-confidence SNPs were used to conduct the linkage and QTL analysis. Qfhs.pur-7EL was mapped to a 2.9 cM region containing four markers within a 43.6 cM segment of wheatgrass chromosome 7el₂ that was translocated onto wheat chromosome 7DL. Lr19 from 7el₁ was mapped to a 1.21 cM region containing two markers in the same area, in repulsion. Five lines were identified with the resistance-associated SNP alleles for Qfhs.pur-7EL and Lr19 in coupling. Two SNP markers in the Qfhs.pur-7EL region were converted into PCR-based KASP markers. Investigation of the genetic characteristics of the parental lines of this RIL population indicated that they are translocation lines in two different wheat cultivar genetic backgrounds instead of 7E–7D substitution lines in Thatcher wheat background, as previously reported in the literature.     

Chromosomal location of genes for stem rust resistance derived from ‘Waldron’ wheat

The chromosomal locations of genes for resistance to stem rust (Puccinia graminis Pers.: Pers. f. sp. tritici Eriks. & E. Henn.) in the wheat (Triticum aestivum L.) cultivar ‘Waldron’ (WDR) were determined by monosomic analyses. Wheat lines WDR-B1, -C2, -E4, and -F1,which have single genes for resistance to stem rust derived previously from WDR sel. ‘Little Club’, were crossed onto a complete set of 21 ‘Chinese Spring’ monosomics. The F₂ and backcross-F₁ (BC₁F₁) seedlings from each of the 84 crosses were tested for reaction to culture 111-SS2 (CRL-LCBB) of stem rust, and a few selected segregants were analyzed cytologically for chromosome number. The F₂ from 2 crosses of WDR-C2, -E4 and -F1 and the BC₁F₁ from 2 crosses of WDR-F1 were tested also with culture Or11c (CRL-QBCN). Significant deviations from disomic ratios towards monosomic ratios in the F₂ and BC₁F₁ were used to determine which chromosomes carried the genes for resistance. Cytological analyses of certain BC₁F₁ and susceptible F₂ plants were used to help identify the location of the genes for rust resistance. WDR-B1 has a gene, herein designated Sr41, for resistance on chromosome 4D. WDR-C2 has a gene on chromosome 7 A that may be the same as one previously designated SrWld2. WDR-E4 has a gene on chromosome 2A, possibly SrWld1, which is effective against most or all North American stem rust cultures. WDR-F1 has a gene on chromosome 6B that is the same as or similar to Sr11.     

QTL mapping for grain filling rate and yield-related traits in RILs of the Chinese winter wheat population Heshangmai × Yu8679

A set of 142 winter wheat recombinant inbred lines (RILs) deriving from the cross Heshangmai x Yu8679 were tried in four ecological environments during the seasons 2006 and 2007. Nine agronomic traits comprising mean grain filling rate (GFR(mean)), maximum grain filling rate (GFR(max)), grain filling duration (GFD), grain number per ear (GNE), grain weight per ear (GWE), flowering time (FT), maturation time (MT), plant height (PHT) and thousand grain weight (TGW) were evaluated in Beijing (2006 and 2007), Chengdu (2007) and Hefei (2007). A genetic map comprising 173 SSR markers and two EST markers was generated. Based on the genetic map and phenotypic data, quantitative trait loci (QTL) were mapped for these agronomic traits. A total of 99 putative QTLs were identified for the nine traits over four environments except GFD, PHT and MT, measured in two environments (BJ07 and CD07), respectively. Of the QTL detected, 17 for GFR(mean), 16 for GFR(max), 21 for TGW and 10 for GWE involving the chromosomes 1A, 1B, 2A, 2D, 3A, 3B, 3D, 4A, 4D, 5A, 5B, 6D and 7D were identified. Moreover, 13 genomic regions showing pleiotropic effects were detected in chromosomes 1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 4B, 4D, 5B, 6D and 7D; these QTL revealing pleiotropic effects may be informative for a better understanding of the genetic basis of grain filling rate and other yield-related traits, and represent potential targets for multi-trait marker aided selection in wheat.     

Genetic mapping of QTL for resistance to Fusarium head blight spread (type 2 resistance) in a Triticum dicoccoides × Triticum durum backcross-derived population

Improvement of resistance to Fusarium head blight (FHB) is a continuous challenge for durum wheat breeders, particularly due to the limited genetic variation within this crop species. We accordingly generated a backcross-derived mapping population using the type 2 FHB resistant Triticum dicoccoides line Mt. Gerizim #36 as donor and the modern Austrian T. durum cultivar Helidur as recipient; 103 BC₁F_6:7 lines were phenotyped for type 2 FHB resistance using single-spikelet inoculations and genotyped with 421 DNA markers (SSR and AFLP). QTL mapping revealed two highly significant QTL, mapping to chromosomes 3A and 6B, respectively. For both QTL the T. dicoccoides allele improved type 2 FHB resistance. Recombinant lines with both favorable alleles fixed conferred high resistance to FHB similar to that observed in the T. dicoccoides parent. The results appear directly applicable for durum wheat resistance breeding.     

Genetics and mapping of seedling resistance to Ug99 stem rust in Canadian wheat cultivars ‘Peace’ and ‘AC Cadillac’

Stem rust (caused by Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn.) has re-emerged as a threat to wheat production with the evolution of new pathogen races, namely TTKSK (Ug99) and its variants, in Africa. Deployment of resistant wheat cultivars has provided long-term control of stem rust. Identification of new resistance genes will contribute to future cultivars with broad resistance to stem rust. The related Canadian cultivars Peace and AC Cadillac show resistance to Ug99 at the seedling stage and in the field. The purpose of this study was to elucidate the inheritance and genetically map resistance to Ug99 in these two cultivars. Two populations were produced, an F_2:3 population from LMPG/AC Cadillac and a doubled haploid (DH) population from RL6071/Peace. Both populations showed segregation at the seedling stage for a single stem rust resistance (Sr) gene, temporarily named SrCad. SrCad was mapped to chromosome 6DS in both populations with microsatellite markers and a marker (FSD_RSA) that is tightly linked to the common bunt resistance gene Bt10. FSD_RSA was the closest marker to SrCad (≈1.6 cM). Evaluation of the RL6071/Peace DH population and a second DH population, AC Karma/87E03-S2B1, in Kenya showed that the combination of SrCad and leaf rust resistance gene Lr34 provided a high level of resistance to Ug99-type races in the field, whereas in the absence of Lr34 SrCad conferred moderate resistance. A survey confirmed that SrCad is the basis for all of the seedling resistance to Ug99 in Canadian wheat cultivars. While further study is needed to determine the relationship between SrCad and other Sr genes on chromosome 6DS, SrCad represents a valuable genetic resource for producing stem rust resistant wheat cultivars.     

The cytological and genetic characterisation of doubled haploid lines derived from triticale×wheat hybrids

Summary Anther culture, when applied to hexaploid triticale×wheat hybrids, offers the opportunity to re-assort wheat D genome and rye R genome chromosomes into homozygous doubled haploid lines in a single generation. The characterisation of such lines is the first step in their utilisation in wheat improvement. Two lines, M24 and M25 from the cross of Beagle×Kedong 58, and one line, M27, from the cross Beagle×Jinghua No. 1 have been characterised using different methods including conventional cytology and chromosome banding, and by using marker systems for storage protein composition (glutenins and gliadins), isozymes (-amylase, aminopeptidase, glutamate oxaloacetic transaminase (GOT)) and RFLP markers. The results from all approaches were consistent in proving that M24 is a whole chromosome 6R/6D substitution line, while M25 and M27 are whole chromosome 1R/1D substitution lines. The relative advantages and disadvantages of each method of identification are also discussed.