J. W. Snape

‘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.

Raising yield potential in wheat

Recent advances in crop research have the potential to accelerate genetic gains in wheat, especially if co-ordinated with a breeding perspective. For example, improving photosynthesis by exploiting natural variation in Rubiscos catalytic rate or adopting C(4) metabolism could raise the baseline for yield potential by 50% or more. However, spike fertility must also be improved to permit full utilization of photosynthetic capacity throughout the crop life cycle and this has several components. While larger radiation use efficiency will increase the total assimilates available for spike growth, thereby increasing the potential for grain number, an optimized phenological pattern will permit the maximum partitioning of the available assimilates to the spikes. Evidence for underutilized photosynthetic capacity during grain filling in elite material suggests unnecessary floret abortion. Therefore, a better understanding of its physiological and genetic basis, including possible signalling in response to photoperiod or growth-limiting resources, may permit floret abortion to be minimized for a more optimal source:sink balance. However, trade-offs in terms of the partitioning of assimilates to competing sinks during spike growth, to improve root anchorage and stem strength, may be necessary to prevent yield losses as a result of lodging. Breeding technologies that can be used to complement conventional approaches include wide crossing with members of the Triticeae tribe to broaden the wheat genepool, and physiological and molecular breeding strategically to combine complementary traits and to identify elite progeny more efficiently.

RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat

A population of single chromosome recombinant lines was developed from the cross between a frost-sensitive, vernalization-insensitive substitution line, ‘Chinese Spring’ (Triticum spelta 5A) and a frost-tolerant, vernalization-sensitive line, ‘Chinese Spring’ (‘Cheyenne’ 5A), and used to map the genes Vrn1 and Fr1 controlling vernalization requirement and frost tolerance, respectively, relative to RFLP markers located on this chromosome. The Vrn1 and Fr1 loci were located closely linked on the distal portion of the long arm of 5AL, but contrary to previous observations, recombination between them was found. Three RFLP markers, Xpsr426, Xcdo504 and Xwg644 were tightly linked to both. The location of Vrn1 suggests that it is homoeologous to other spring habit genes in related species, particularly the Sh2 locus on chromosome 7 (5H) of barley and the Sp1 locus on chromosome 5R of rye.

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 Genetic Framework for Grain Size and Shape Variation in Wheat

Using large-scale quantitative analysis, this work reveals that grain shape and size are independent traits in both modern and primitive wheat and are under the control of distinct genetic components. Moreover, the phenotypic diversity in grain morphology found in modern commercial wheat is the result of a recent and severe bottleneck. Grain morphology in wheat (Triticum aestivum) has been selected and manipulated even in very early agrarian societies and remains a major breeding target. We undertook a large-scale quantitative analysis to determine the genetic basis of the phenotypic diversity in wheat grain morphology. A high-throughput method was used to capture grain size and shape variation in multiple mapping populations, elite varieties, and a broad collection of ancestral wheat species. This analysis reveals that grain size and shape are largely independent traits in both primitive wheat and in modern varieties. This phenotypic structure was retained across the mapping populations studied, suggesting that these traits are under the control of a limited number of discrete genetic components. We identified the underlying genes as quantitative trait loci that are distinct for grain size and shape and are largely shared between the different mapping populations. Moreover, our results show a significant reduction of phenotypic variation in grain shape in the modern germplasm pool compared with the ancestral wheat species, probably as a result of a relatively recent bottleneck. Therefore, this study provides the genetic underpinnings of an emerging phenotypic model where wheat domestication has transformed a long thin primitive grain to a wider and shorter modern grain.

WAITING FOR FINE TIMES: GENETICS OF FLOWERING TIME IN WHEAT

To maximise yield potential in any environment, wheat cultivars must have an appropriate flowering time and life cycle duration which ‘fine-tunes’ the life cycle to the target environment. This in turn, requires a detailed knowledge of the genetical control of the key components of the life cycle. This paper discusses our current knowledge of the genetical control of the three key groups of genes controlling life-cycle duration in wheat, namely those controlling vernalization response, photoperiod response and developmental rate (“earliness per se”, Eps genes). It also discuses how our ability to carry out comparative mapping of these genes across Triticeae species, and particularly with barley, is indicating new target genes for discovery in wheat. Major genes controlling vernalization response, the Vrn-1 series, have now been located both genetically and physically on the long arms of the homoeologous group five chromosomes. These genes are homoeologous to each other and to the vernalization genes on chromosomes 5H of barley and 5R of rye. Comparative analysis with barley also indicates that other series of vernalization response genes may exit on chromosomes of homoeologous groups 4 (4B, 4D, 5A) and 1. The major genes controlling photoperiod response in wheat, the Ppd-1 genes, are located on the homoeologous group 2 chromosomes, and are homoeologous to a gene on barley chromosome 2H. Mapping in barley also indicates a photoperiod response locus on barley 1H and 6H, indicating that a homoeologous series should exist on wheat group 1 and 6 chromosomes. In wheat, only a few “earliness per se” loci have been located, such as on chromosomes of homoeologous group 2. However, in barley, all chromosomes appear to carry such loci, indicating that several series of loci that affect developmental rate independent of environment remain to be discovered. Overall, comparative studies indicate that there are probably twenty-five loci, controlling the duration of the life-cycle, Vrn,Ppd and Eps genes, that remain to be mapped in wheat. There are major gaps in our knowledge of the detailed physiological effects of genes discovered to date on the timing of the life cycle from different sowing dates. This is being addressed by studying the phenology of isogenic and deletion lines in both field and controlled environmental conditions. This has indicated that the vernalization genes have major effects on the rate of primodia production, whilst the photoperiod genes affect the timing of terminal spikelet production and stem elongation, and these effects interact with sowing date.

Mapping quantitative trait loci for flag leaf senescence as a yield determinant in winter wheat under optimal and drought-stressed environments

The timing of flag leaf senescence (FLS) is an important determinant of yield under stress and optimal environments. A doubled haploid population derived from crossing the photo period-sensitive variety Beaver,with the photo period-insensitive variety Soissons, varied significantly for this trait, measured as the percent green flag leaf area remaining at 14 days and 35 days after anthesis. This trait also showed a significantly positive correlation with yield under variable environmental regimes. QTL analysis based on a genetic map derived from 48 doubled haploid lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, revealed the genetic control of this trait. The coincidence of QTL for senescence on chromosomes 2B and 2D under drought-stressed and optimal environments, respectively, indicate a complex genetic mechanism of this trait involving the re-mobilisation of resources from the source to the sink during senescence.

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

Abstract  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.

Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-IΔD86) for nematode resistance in transgenic rice plants

Abstract We have used a genotype-independent transformation system involving particle gun bombardment of immature embryos to genetically engineer rice as part of a programme to develop resistance to nematodes. Efficient tissue culture, regeneration, DNA delivery and selection methodologies have been established for elite African varieties (‘ITA212’, ‘IDSA6’, ‘LAC23’, ‘WAB56-104’). Twenty-five transformed clones containing genes coding for an engineered cysteine proteinase inhibitor (oryzacystatin-IΔD86, OC-IΔD86), hygromycin resistance (aphIV) and β-glucuronidase (gusA) were recovered from the four varieties. Transformed plants were regenerated from all clones and analysed by PCR, Southern and western blot. Detectable levels of OC-IΔD86 (up to 0.2% total soluble protein) in plant roots were measured in 12 out of 25 transformed rice lines. This level of expression resulted in a significant 55% reduction in egg production by Meloidogyne incognita.

Genetic analysis of a photoperiod response gene on the short arm of chromosome 2(2H) of Hordeum vulgare (barley)

RFLP mapping of 94 doubled haploid lines from a winter x spring barley cross (Igri x Triumph) identified a previously undescribed major photoperiod response locus (Ppd-H1) on the short arm of barley chromosome 2(2H). Lines with the Igri allele (winter parent) flowered 10 days earlier under long days in a glasshouse experiment. The Ppd-H1 locus also had a major effect in field experiments, giving differences of 12 days (spring sowing) and 7 days (autumn sowing). The Ppd-H1 locus accounted for 60 per cent of the genetic variation in flowering time in the spring sowing and 46 per cent in the autumn sowing. The most likely location of Ppd-H1 was in the 6 cM interval between the RFLP loci XMWG858 and XpsrB9, 1 cM proximal to XMWG858. The map position of Ppd-H1 suggests that it may be a homoeoallele of the wheat photoperiod response gene Ppdl. Field data also showed that the barley Ppd-H1 locus was associated with highly significant effects on plant height, biomass and yield components which were probably the direct results of the variation in flowering time. Flowering time and other agronomic characters were also significantly affected by an additional developmental rate gene on the same chromosome.