Wild relatives of common wheat, Triticum aestivum, and related species are an important source of disease and pest resistance and several useful traits have been transferred from these species to wheat. C-banding and in situ hybridization analyses are powerful cytological techniques allowing the detection of alien chromatin in wheat. C-banding permits identification of the wheat and alien chromosomes involved in wheat-alien translocations, whereas genomic in situ hybridization analysis allows determination of their size and breakpoint positions. The present review summarizes the available data on wheat-alien transfers conferring resistance to diseases and pests. Ten of the 57 spontaneous and induced wheat-alien translocations were identified as whole arm translocations with the breakpoints within the centromeric regions. The majority of transfers (45) were identified as terminal translocations with distal alien segments translocated to wheat chromosome arms. Only two intercalary wheat-alien transloctions were identified, one induced by radiation treatment with a small segment of rye chromosome 6RL (H25) inserted into the long arm of wheat chromosome 4A, and the other probably induced by homoeologous recombination with a segment derived from the long arm of a group 7 Agropyron elongatum chromosome with Lr19 inserted into the long arm of 7D. The presented information should be useful for further directed chromosome engineering aimed at producing superior germplasm.

Characterization of wheat-alien translocations conferring resistance to diseases and pests: current status

The development and use of molecular markers for the detection and exploitation of DNA polymorphism is one of the most significant developments in the field of molecular genetics. The presence of various types of molecular markers, and differences in their principles, methodologies, and applications require careful consideration in choosing one or more of such methods. No molecular markers are available yet that fulfill all requirements needed by researchers. According to the kind of study to be undertaken, one can choose among the variety of molecular techniques, each of which combines at least some desirable properties. This article provides detail review for 11 different molecular marker methods: restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), sequence characterized regions (SCARs), sequence tag sites (STSs), cleaved amplified polymorphic sequences (CAPS), microsatellites or simple sequence repeats (SSRs), expressed sequence tags (ESTs), single nucleotide polymorphisms (SNPs), and diversity arrays technology (DArT).

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An overview of molecular marker methods for plants

Wheat provides 20% of calories and protein consumed by humans. Recent genetic gains are <1% per annum (p.a.), insufficient to meet future demand. The Wheat Yield Consortium brings expertise in photosynthesis, crop adaptation and genetics to a common breeding platform. Theory suggest radiation use efficiency (RUE) of wheat could be increased ∼50%; strategies include modifying specificity, catalytic rate and regulation of Rubisco, up-regulating Calvin cycle enzymes, introducing chloroplast CO2 concentrating mechanisms, optimizing light and N distribution of canopies while minimizing photoinhibition, and increasing spike photosynthesis. Maximum yield expression will also require dynamic optimization of source: sink so that dry matter partitioning to reproductive structures is not at the cost of the roots, stems and leaves needed to maintain physiological and structural integrity. Crop development should favour spike fertility to maximize harvest index so phenology must be tailored to different photoperiods, and sensitivity to unpredictable weather must be modulated to reduce conservative responses that reduce harvest index. Strategic crossing of complementary physiological traits will be augmented with wide crossing, while genome-wide selection and high throughput phenotyping and genotyping will increase efficiency of progeny screening. To ensure investment in breeding achieves agronomic impact, sustainable crop management must also be promoted through crop improvement networks.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-3040.2012.02588.x

Achieving yield gains in wheat.

Race Ug99, or TTKSK, of fungus Puccinia graminis tritici, causing stem or black rust disease on wheat (Triticum aestivum), first identified in Uganda in 1998 has been recognized as a major threat to wheat production. Its spread in 2006 to Yemen and Sudan and further spread towards North Africa, Middle East and West-South Asia is predicted -aided by predominant wind currents and large areas of wheat varieties that are susceptible and grown under environments favorable for survival and multiplication of the pathogen. This has raised serious concerns of major epidemics that could destroy the wheat crop in these primary risk areas. Detection in Kenya of a new variant TTKST in 2006 with virulence to gene Sr24, which caused severe epidemics in 2007 in some regions of Kenya and rendered about half of the previously known Ug99-resistant global wheat materials susceptible, has further increased the vulnerability globally. Rigorous screening since 2005 in Kenya and Ethiopia of wheat materials from 22 countries and International Centers has identified low frequency of resistant materials that have potential to replace susceptible cultivars. Diverse sources of resistance, both race-specific and adult-plant type, are now available in high-yielding wheat backgrounds and are being used in breeding. The proposed strategy is to deploy spring wheat varieties possessing durable, adult plant resistance in East Africa and other primary risk areas to reduce inoculum and selection of new virulences capable of overcoming undefeated race-specific resistance genes. Race-specific resistance genes can then be deployed in secondary risk areas preferably in combinations. We believe that Ug99 threat in most countries can be reduced to low levels by urgently identifying, releasing and providing seed of new high yielding, resistant varieties.

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Will Stem Rust Destroy the World’s Wheat Crop

Stem or black rust, caused by Puccinia graminis tritici, has historically caused severe losses to wheat (Triticum aestivum) production worldwide. Successful control of the disease for over three decades through the use of genetic resistance has resulted in a sharp decline in research activity in recent years. Detection and spread in East Africa of race TTKS, commonly known as Ug99, is of high significance as most wheat cultivars currently grown in its likely migration path, i.e. to North Africa through Arabian Peninsula and then to Middle East and Asia, are highly susceptible to this race and the environment is conducive to disease epidemics. Identifying/developing adapted resistant cultivars in a relatively short time and replacing the susceptible cultivars before rust migrates out of East Africa is the strategy to mitigate potential losses. Although several alien genes will provide resistance to this race, the long-term strategy should focus on rebuilding the ‘Sr2-complex’ (combination of slow rusting gene Sr2 with other unknown additive genes of similar nature) to achieve long-term durability. A Global Rust Initiative has been launched to monitor the further migration of this race, facilitate field testing in Kenya or Ethiopia of wheat cultivars and germplasm developed by wheat breeding programmes worldwide, understand the genetic basis of resistanceespecially the durable type, carry out targeted breeding to incorporate diverse resistance genes into key cultivars and germplasm, and enhance the capacity of national programmes. A few wheat genotypes that combine stem rust resistance with high yield potential and other necessary traits have been identified but need rigorous field testing to determine their adaptation in target areas.

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Current status, likely migration and strategies to mitigate the threat to wheat production from race Ug99 (TTKS) of stem rust pathogen

Amplified fragment length polymorphism (AFLP) markers were used to enrich the map of the wheat chromosomal region containing the Thinopyrum-derived Lr19 leaf rust resistance gene. The region closest to Lr19 was targeted through the use of deletion and recombinant lines of the translocated segment. One of the AFLP bands thus identified was converted into a sequence-tagged-site (STS) marker. This assay generated a 130-bp PCR fragment in all Lr19-carrying lines tested, except for one deletion mutant, while non-carrier template failed to amplify any product. This sequence represents the first marker to map on the distal side of Lr19 on chromosome 7el1. The conversion process of AFLP fragments to STS markers was technically difficult, mainly because of the presence of contaminating fragments. Various approaches were taken to reduce the frequency of false positives and to identify the correct clone. We were able to formulate a general verification strategy prior to clone sequencing. Various other factors causing problems with converting AFLP bands to an STS assays are also discussed.

AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.

Conversion of a RAPD-generated PCR product, containing a novel dispersed repetitive element, into a fast and robust assay for the presence of rye chromatin in wheat.

The amount of alien chromatin introgressed in eight wheat/Ae. longissima Pm13 recombinant lines, involving breakpoints on the short arms of wheat chromosomes 3B and 3D, was evaluated by cytogenetic and molecular approaches. For each line the residual homologous synaptic ability of the recombinant chromosome in its proximal wheat and distal alien portion was estimated through meiotic analyses. Subsequently, telocentric and RFLP mapping were used to assess the genetic distance from the wheat centromere to the wheat/Ae. longissima breakpoints. One 3B recombinant line was distinguished from the other four by the chromosome pairing and telocentric mapping analyses. RFLP analysis succeeded in differentiating the remaining four lines into two groups. Chromosome pairing and telocentric mapping of the three 3D recombinant lines suggested that all had distinct breakpoints. However, the RFLP data could not discriminate between the two more proximal translocations. Physical locations for some RFLP loci were determined by a comparison of genotypes and C-banding karyotypes. This showed a considerable expansion of the genetic map compared to its physical length.

Cytogenetic and molecular mapping of the wheat-Aegilops longissima chromatin breakpoints in powdery mildew-resistant introgression lines.

Two wheat consensus primer sets, directed to ”early-methionine-labelled” (Em) gene sequences, were tested for their ability to amplify beyond their original source. A range of widely diverse templates, including other Triticeae species and sample monocot and dicot species, was assayed. Primer set EMC5/EMC3, amplifying the entire coding region with its intron and part of the 3’ untranslated region, targets Triticeae and sorghum Em sequences. The other set, EMC5/EMCO31, directed to the coding region and its intron, amplifies templates from all the grass species. Both primer sets fail to amplify Em sequences from more distant monocots and the dicots. Using set EMC5/EMC3, we isolated and sequenced ten members of the rye Em gene family from five different rye sources. Significant DNA sequence variation between wheat and rye sequences in the non-coding regions was found, and this was used to develop seven sequence-specific primers. Twelve primer combinations were analysed, 7 of which were Em-R1-specific, amplifying a product in at least one of the tested rye or rye-carrying genotypes but not in wheat. Four sets exhibited clear amplification length polymorphisms which allowed discrimination between and within the rye sources. The primers also discriminated between wheat-rye recombinants with proximal 1RL rye chromatin and those carrying distal 1RL rye chromatin. These results show that wheat consensus primer sets can be used to isolate orthologous sequences, especially from species that are used for alien gene transfer in wheat. Subsequently, species-specific assays can be designed that are useful tools for this application.

R. M. D. Koebner

Papers

AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat

Conversion of a RAPD-generated PCR product, containing a novel dispersed repetitive element, into a fast and robust assay for the presence of rye chromatin in wheat.

Cytogenetic and molecular mapping of the wheat-Aegilops longissima chromatin breakpoints in powdery mildew-resistant introgression lines.

The applicability of consensus PCR primers across species and genera: the use of wheat Em sequences to develop markers for orthologues in rye