Mapping of quantitative trait loci for root hair length in wheat identifies loci that co-locate with loci for yield components.

Root hairs are fast growing, ephemeral tubular extensions of the root epidermis. They arise in the unsuberized maturation zone of the root, effectively increasing the root surface area in the region over which nutrient and water uptake occur. Variation in root hair length (RHL) between varieties has been shown to be genetically determined, and could, therefore, have consequences for nutrient capture and yield potential in crops. We describe the development of a medium-to-high throughput screening method for assessing RHL in wheat at the seedling stage. This method was used to screen a number of wheat mapping population parental lines for variation in RHL. Parents of two populations derived from inter-varietal crosses differed for RHL: Spark vs Rialto and Charger vs Badger. We identified quantitative trait loci (QTLs) for RHL in the populations derived from these crosses. In Spark × Rialto, QTLs on chromosomes 1A, 2A and 6A were associated with variation in RHL, whilst in Charger × Badger, a QTL for RHL was identified on 2BL. The QTLs on 2A and 6A co-localized with previously described QTLs for yield components. Longer root hairs may confer an advantage by exploiting limiting mineral and water resources. This first QTL analysis of root hair length in wheat identifies loci that could usefully be further investigated for their role in tolerance to limiting conditions.

Wheat antixenosis, antibiosis, and tolerance to infestation by Delphacodes kuscheli (Hemiptera: Delphacidae), a vector of "Mal de Río Cuarto" in Argentina.

"Mal de Rio Cuarto", is the most important virus disease of corn, Zea mays L., in Argentina. It is caused by the Mal de Rio Cuarto virus (family Reoviridae, genus Fijivirus. MRCV), which is a persistent virus transmitted by Delphacodes kuscheli (Fennah 1955) (Hemiptera: Delphacidae). Because corn is not a natural host of D. kuscheli, it has little protection from this pest. In contrast, wheat, Triticum aestivum L., is one of the main hosts of this vector and a reservoir of MRCV. The aim of this work was to identify genes involved in antixenosis, antibiosis, and tolerance of infestation by D. kuscheli in wheat, which might be used to reduce the population level of this vector on corn. A set of recombinant dihaploid (RDH) lines for chromosome 6A derived from the F1 cross between 'Chinese Spring' (CS) X 'Chinese Spring (Synthetic 6A)' (S6A) substitution line, was used for mapping. The S6A parental line is resistant to the MRCV vector. Antixenosis, antibiosis, and tolerance were evaluated using conventional tests in controlled environmental conditions. Most of the RDH and S6A showed higher levels of antixenosis against D. kuscheli than the parental line CS. The RDH lines showed highly significant antibiosis in terms of the duration of first, third, and fifth nymphal instars, developmental time (days), survival and fecundity. There were highly significant differences in the tolerance to D. kuscheli based on the chlorophyll content of the first and second leaves, foliar area, and aboveground fresh and dry weights. The duration of the fifth nymphal instar and the developmental period were significantly associated with Xgwm1017 marker loci, located at 48 cM on 6AL. Another quantitative trait locus accounting for the variation in chlorophyll content of the first leaf was associated with the interval between loci Xgwm459 and Xgwm334a, located in the telomeric region of the 6AS chromosome arm. The alleles with positive effects came from S6A. Antibiotic resistance of RDH could be useful for controlling the population increase of the MRCV vector on wheat, because prolonging the duration of development increases the period between two subsequent generations, so reducing the abundance of infective populations colonizing corn.

Development and genetic mapping of sequence-tagged microsatellites (STMs) in bread wheat (Triticum aestivum L.).

The density of SSRs on the published genetic map of bread wheat (Triticum aestivum L.) has steadily increased over the last few years. This has improved the efficiency of marker-assisted breeding and certain types of genetic research by providing more choice in the quality of SSRs and a greater chance of finding polymorphic markers in any cross for a chromosomal region of interest. Increased SSR density on the published wheat genetic map will further enhance breeding and research efforts. Here, sequence-tagged microsatellite profiling (STMP) is demonstrated as a rapid technique for the economical development of anonymous genomic SSRs to increase marker density on the wheat genetic map. A total of 684 polymorphic sequence-tagged microsatellites (STMs) were developed, and 380 were genetically mapped in three mapping populations, with 296 being mapped in the International Triticeae Mapping Initiative W7984 x Opata85 recombinant inbred cross. Across the three populations, a total of 479 STM loci were mapped. Several technological advantages of STMs over conventional SSRs were also observed. These include reduced marker deployment costs for fluorescent-based SSR analysis, and increased genotyping throughput by more efficient electrophoretic separation of STMs and a high amenability to multiplex PCR.

Major genetic changes in wheat with potential to affect disease tolerance.

ABSTRACT Selection through plant breeding has resulted in most elite winter wheat germplasm in the United Kingdom containing the Rht-D1b semi-dwarfing allele, the 1BL.1RS chromosome arm translocation with rye, and an allele conferring suppression of awns. Near-isogenic lines (NILs) were used to test whether these major genetic changes have had any effect on disease tolerance. The ability of the NILs to tolerate epidemics of Septoria leaf blotch or stripe rust was measured in four field experiments over two seasons. Tolerance was quantified as yield loss per unit of green canopy area lost to disease. There was a trend for the presence of the 1BL.1RS translocation to decrease tolerance; however, this was not consistent across experiments and there was no effect of semi-dwarfing. The awned NIL exhibited decreased tolerance compared with the unawned NIL. There were significant differences in tolerance between the cultivar backgrounds in which the NILs were developed. Tolerance was lower in the modern genetic background of Weston, released in 1996, than in the genetic background of Maris Hunstman, released in 1972. The data suggest that certain physiological traits were associated with the tolerance differences among the backgrounds in these experiments. Potential yield, accumulation of stem soluble carbohydrate reserves, and grain sink capacity were negatively correlated with tolerance, whereas flag leaf area was positively correlated.

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques.

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations.

Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70-78% of the plant lines causing 14-21% of the loci to contain only the mid to right border part of a T-DNA. In 53-66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60-80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53-66% of loci) were generally a greater limitation to the production of marker-free T(1) plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).

A new approach to extending the wheat marker pool by anchored PCR amplification of compound SSRs.

A study was undertaken to determine the utility in bread wheat of anchored PCR for the development of single locus SSR markers targeted at compound repeat motifs. In anchored PCR, microsatellite amplification is achieved using a single primer complementary to the flanking sequence, and one which anchors to the repeat junction of the compound SSR. The recovery rate of useable markers was found to be similar (43%) to that reported for conventionally generated SSRs. Thus, anchored PCR can be used to reduce the costs of marker development, since it requires that only half the number of primers be synthesised. Where fluorescence-based platforms are used, marker deployment costs are lower, since only the anchoring primers need to be labelled. In addition, anchored PCR improves the recovery of useful markers, as it allows assays to be generated from microsatellite clones with repeat sequences located close to their ends, a situation where conventional PCR amplification fails as two flanking primers cannot be designed. Strategies to permit the large-scale development of compound SSR markers amplified by anchored PCR are discussed.

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding.

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.

Mapping genes affecting flowering time and frost resistance on chromosome 5B of wheat.

Two populations of single chromosome recombinant lines were used to map genes controlling flowering time on chromosome 5B of wheat, and one of the populations was also used to map a new frost resistance gene. Genetic maps were developed, mainly using microsatellite markers, and QTL analysis was applied to phenotypic data on the performance of each population collected from growth-room tests of flowering time and frost tolerance. Using a recombinant substitution-line mapping population derived from a cross between the substitution-line 'Chinese Spring' ('Cheyenne' 5B) and 'Chinese Spring' (CS), the gene Vrn-B1, affecting vernalization response, an earliness per se locus, Eps-5BL1, and a gene, Fr-B1, affecting frost resistance, were mapped. Using a 'Hobbit Sib' ('Chinese Spring' 5BL) x 'Hobbit Sib' recombinant substitution line mapping population, an earliness per se locus, Eps-5BL2 was mapped. The Vrn-B1 locus was mapped on the distal portion of the long arm of chromosome 5B, to a region syntenous with the segments of chromosomes 5A and 5D containing Vrn-A1 and Vrn-D1 loci, respectively. The two Eps-5BL loci were mapped close to the centromere with a 16-cM distance from each other, one in agreement with the position of a homoeologous locus previously mapped on chromosome 5H of barley, and suggested by the response of 'Chinese Spring' deletion lines. The Fr-B1 gene was mapped on the long arm of chromosome 5B, 40 cM from the centromeric marker. Previous comparative mapping data with rice chromosome 9 would suggest that this gene could be orthologous to the other Fr genes mapped previously by us on chromosomes 5A or 5D of wheat, although in a more proximal position. This study completes the mapping of these homoeoallelic series of vernalization requirement genes and frost resistance genes on the chromosomes of the homoeologous group 5 in wheat.

Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup.

Transgene integration, expression level and stability have been studied, across two generations, in a population of rice plants transformed using a new dual binary vector system: pGreen/pSoup. pGreen is a small Ti binary vector unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. We engineered both pGreen and pSoup to contain each a different T-DNA. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units (no transgene in pSoup) or with a pSoup vector containing an aphIV and gfp expression units (no transgene in pGreen). High plant transformation frequencies (up to 40%) were obtained using herbicide resistance ( bar) or antibiotic resistance ( aphIV) genes. Around 80% of the independently transformed plants expressed unselected reporter genes ( gusA or gfp) present in the vectors. Backbone sequences transfer was frequent (45% of lines) and occurred often in multicopy lines. Around 15-20% of the rice plant lines contained a single T-DNA integration without backbone. Integration of additional transgene copies did not improve expression levels in either T(0) plants or T(1) progenies. Nearly all multicopy lines contained transgenes integrated at several loci in the plant genome, showing that T-DNAs from either pGreen or pSoup frequently integrated at unlinked loci. Precise determination of loci number required the analysis of transgene presence in progeny. Segregation of transgene phenotype was generally misleading and tended to underestimate the real number of transgenic loci. The contribution of this new dual-binary vector system to the development of high-throughput rice transformation systems and to the production of marker-free transgenic rice plants is discussed.

An update of the Courtot x Chinese Spring intervarietal molecular marker linkage map for the QTL detection of agronomic traits in wheat.

We made an update of the intervarietal molecular marker linkage map of the wheat genome developed using a doubled-haploid (DH) population derived from the cross between the cultivars "Courtot" and "Chinese Spring". This map was constructed using 187 DH lines and 659 markers. The genome was well covered (more than 95%) except for chromosomes from homoeologous group 4 and chromosomes 5D and 7D, which had gaps slightly larger than 50 cM. A core-map based on a set of 200 anchor loci (one marker each 18.4 cM) was developed. The total length of this map was 3,685 cM which is similar to the size of the international reference map of the ITMI population (3,551 cM). Map coverage was identical for the three genomes (A, B and D) and for the number of anchor loci, as well as for the size of the map. Using this map, QTLs for several agronomic traits were detected on phenotypic data from the population grown in Clermont-Ferrand (France) under natural field conditions over 6 years, and in Norwich (UK) in controlled conditions and under natural field conditions in 1 year. Almost all of the 21 chromosomes were involved in at least one trait. However, several regions seemed to contain gene clusters either for grain traits (and thus bread-making quality) or plant development traits.

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.

The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants.

Segregating T(1), T(2) and T(3) transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected beta-glucuronidase ( gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing the likelihood of an additive effect on transgene expression level is discussed.

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum.

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/ H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines.

An efficient method for the physical mapping of transgenes in barley using in situ hybridization.

The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes. The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which yielded optimal hybridization. minimal fluorescent background, and accurate physical location of the transgene.

Detection of QTLs for heading time and photoperiod response in wheat using a doubled-haploid population.

The genetic basis of heading time in wheat (Triticum aestivum L.) was investigated through the study of flowering under normal autumn sown field conditions as well as photoperiod responses under a controlled environment. Quantitative trait loci (QTLs) for these traits were mapped in a doubled-haploid (DH) population derived from a cross between the wheat cultivars 'Courtot' and 'Chinese Spring'. A molecular marker linkage map of this cross that was previously constructed based on 187 DH lines and 380 markers was used for QTL mapping. The genome was well covered (85%) except for chromosomes 1D and 4D, and a set of anchor loci regularly spaced over the genome (one marker each 15.5 cM) was chosen for marker regression analysis. The presence of a QTL was declared at a significance threshold of alpha = 0.005. The population was grown under field conditions in Clermont-Ferrand, France during two years (1994-1995), in Norwich, U.K. over one year (1998), and also under controlled environments in Norwich. For each trait, between 2 and 4 QTLs were identified with individual effects ranging between 6.3% and 44.4% of the total phenotypic variation. Two QTLs were detected that simultaneously affected heading time and photoperiod response. For heading time, these two QTLs were detected in more than one year. One QTL located on chromosome arm 2BS near the locus Xfbb121-2B, co-segregated with the gene Ppd-B1 known to be involved in photoperiod response. This chromosome region explained a large part of the variation (23.4-44.4% depending on the years or the traits). Another region located on chromosome arm 7BS between the loci Xfbb324-7B and Xfbb53-7B also had a strong effect (7.3-15.3%). This region may correspond to a QTL for earliness per se.

Physical characterization of the homoeologous group 5 chromosomes of wheat in terms of rice linkage blocks, and physical mapping of some important genes.

The wheat homoeologous Group 5 chromosomes were characterized physically in terms of rice linkage blocks using a deletion mapping approach. All three chromosomes, 5A, 5B, and 5D, were shown to have a similar structure, apart from the 4A-5A translocation on the distal end of chromosome arm 5AL. The physical mapping of rice markers on the deletion lines revealed that the whole of rice chromosome 9 is syntenous to a large block, proximal to the centromere, on the long arm. Likewise, a small segment of the distal end of the long arm showed conserved synteny with the distal one-third end of the long arm of rice chromosome 3. In between those conserved regions, there is a region on the long arm of the Group 5 chromosomes which shows broken synteny. The proximal part of the short arms of the Group 5 chromosomes showed conserved synteny with a segment of the short arm of rice chromosome 11 and the distal ends showed conserved synteny with a segment of rice chromosome 12. The physical locations of flowering time genes (Vrn and earliness per se) and the gene for grain hardness (Ha) on the Group 5 chromosomes were determined. These results indicate that comparative mapping using the deletion mapping approach is useful in the study of genome relationships, the physical location of genes, and can determine the appropriate gene cloning strategy.

Induction and characterization of Ph1 wheat mutants.

The cloning of genes for complex traits in polyploid plants that possess large genomes, such as hexaploid wheat, requires an efficient strategy. We present here one such strategy focusing on the homologous pairing suppressor (Ph1) locus of wheat. This locus has been shown to affect both premeiotic and meiotic processes, possibly suggesting a complex control. The strategy combined the identification of lines carrying specific deletions using multiplex PCR screening of fast-neutron irradiated wheat populations with the approach of physically mapping the region in the rice genome equivalent to the deletion to reveal its gene content. As a result, we have located the Ph1 factor controlling the euploid-like level of homologous chromosome pairing to the region between two loci (Xrgc846 and Xpsr150A). These loci are located within 400 kb of each other in the rice genome. By sequencing this region of the rice genome, it should now be possible to define the nature of this factor.

'Green revolution' genes encode mutant gibberellin response modulators.

World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

A simple PCR-based method for scoring the ph1b deletion in wheat.

The amplified fragment length polymorphism (AFLP) technique was used to isolate DNA sequences present in the euploid wheat Chinese Spring but not in the Chinese Spring ph1b mutant (which has a deletion of the Ph1 gene, a suppressor of homoeologous chromosome pairing). The polymorphic DNA fragments identified by AFLP were then cloned, sequenced, and used to design two primer pairs. These primers were used in a PCR-based assay to specifically amplify products from the Chinese Spring euploid but not from the ph1b mutant. This PCR assay can be carried out from extracted genomic DNA or directly from alkaline-treated wheat leaves, and the reaction products can be scored on a plus-minus basis, making the screening amenable to automation. The reliability of the assay was tested using a F1-derived doubled-haploid population of 55 lines which segregate for the ph1b deletion. This PCR-screening technique is less time and labour consuming, and more accurate and reliable, than cytologically based conventional methods.