Doubled haploid ramets via embryogenesis of haploid tissue cultures

Tissue culture in the oil palm business is generally concerned with the multiplication (clonal production) of dura, pisifera and tenera palms. These are all normal diploids (2n=2x=36). Sumatra Bioscience has pioneered haploid tissue culture of oil palm (n=x=18). Haploid oil palm is the first step in producing doubled haploid palms which in turn provide parental lines for F1 hybrid production. Chromosome doubling is known to occur during embryogenesis in other haploid cultures, e.g. barley anther culture. Haploid tissue cultures in oil palm were therefore set up to investigate and exploit spontaneous chromosome doubling during embryogenesis. Flow cytometry of embryogenic tissue showed the presence of both haploid (n) and doubled haploid (2n) cells indicating spontaneous doubling. Completely doubled haploid ramets were regenerated suggesting that doubling occurred during the first mitoses of embryogenesis. This is the first report of doubled haploid production in oil palm via haploid tissue culture. The method provides a means of producing a range of doubled haploids in oil palm from the 1,000 plus haploids available at Sumatra Bioscience, in addition the method also produced doubled haploid (and haploid) clones. 1.

Methods of producing haploid and doubled haploid oil palms

The present invention relates to haploid oil palm plants and homozygous doubled haploid oil palm plants. The invention also relates to methods for producing and selecting haploid and doubled haploid plants. More particularly, but not exclusively, the method may be used for selecting haploid and doubled haploid oil palm plants. Haploid and doubled haploid plants are selected by a large-scale screening based on a combination of the phenotype with the use of molecular methods combined with flow cytometry techniques to identify haploid and doubled haploid plants. More particularly, a method for selecting haploid and doubled haploid plants is described comprising: (a) germinating seeds; (b) selecting seedlings with atypical phenotype; (c) assessing heterozygosity using markers; (d) isolating cells from the seedlings and determining the DNA content of the cells; and (e) isolating and purifying the DNA and using defined molecular markers to characterise the genotype of the plant. The haploid oil palm plants may be used for producing homozygous doubled haploid oil palms: doubled haploids may be intercrossed to produce uniform F.sub.1 hybrids of superior properties.

A novel transcriptomic approach to identify candidate genes for grain quality traits in wheat

A novel methodology is described in which transcriptomics is combined with the measurement of bread-making quality and other agronomic traits for wheat genotypes grown in different environments (wet and cool or hot and dry conditions) to identify transcripts associated with these traits. Seven doubled haploid lines from the Spark x Rialto mapping population were selected to be matched for development and known alleles affecting quality. These were grown in polytunnels with different environments applied 14 days post-anthesis, and the whole experiment was repeated over 2 years. Transcriptomics using the wheat Affymetrix chip was carried out on whole caryopsis samples at two stages during grain filling. Transcript abundance was correlated with the traits for approximately 400 transcripts. About 30 of these were selected as being of most interest, and markers were derived from them and mapped using the population. Expression was identified as being under cis control for 11 of these and under trans control for 18. These transcripts are candidates for involvement in the biological processes which underlie genotypic variation in these traits.

Mapping quantitative trait loci for flag leave 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.

Effects of genotype and environment on free amino acid levels in wheat grain: implications for acrylamide formation during processing

Acrylamide forms from free asparagine and reducing sugars during cooking, with asparagine concentration being the key parameter determining the formation in foods produced from wheat flour. In this study free amino acid concentrations were measured in the grain of varieties Spark and Rialto and four doubled haploid lines from a Spark x Rialto mapping population. The parental and doubled haploid lines had differing levels of total free amino acids and free asparagine in the grain, with one line consistently being lower than either parent for both of these factors. Sulfur deprivation led to huge increases in the concentrations of free asparagine and glutamine, and canonical variate analysis showed clear separation of the grain samples as a result of treatment (environment, E) and genotype (G) and provided evidence of G x E interactions. Low grain sulfur and high free asparagine concentration were closely associated with increased risk of acrylamide formation. G, E, and G x E effects were also evident in grain from six varieties of wheat grown at field locations around the United Kingdom in 2006 and 2007. The data indicate that progress in reducing the risk of acrylamide formation in processed wheat products could be made immediately through the selection and cultivation of low grain asparagme varieties and that further genetically driven improvements should be achievable. However, genotypes that are selected should also be tested under a range of environmental conditions.

Production of haploids and doubled haploids in oil palm

Background: Oil palm is the world’s most productive oil-food crop despite yielding well below its theoretical maximum. This maximum could be approached with the introduction of elite F1 varieties. The development of such elite lines has thus far been prevented by difficulties in generating homozygous parental types for F1 generation. Results: Here we present the first high-throughput screen to identify spontaneously-formed haploid (H) and doubled haploid (DH) palms. We secured over 1,000 Hs and one DH from genetically diverse material and derived further DH/mixoploid palms from Hs using colchicine. We demonstrated viability of pollen from H plants and expect to generate 100% homogeneous F1 seed from intercrosses between DH/mixoploids once they develop female inflorescences. Conclusions: This study has generated genetically diverse H/DH palms from which parental clones can be selected in sufficient numbers to enable the commercial-scale breeding of F1 varieties. The anticipated step increase in productivity may help to relieve pressure to extend palm cultivation, and limit further expansion into biodiverse rainforest.

Reduced height (Rht) and photoperiod insensitivity (Ppd) allele associations with establishment and early growth of wheat in contrasting production systems

Near isogenic lines (NILs) varying for genes for reduced height (Rht) and photoperiod insensitivity (Ppd-D1a) in a cv. Mercia background (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c + Ppd-D1a, Rht-D1c, Rht12) were compared at one field site but within contrasting ('organic' vs. 'conventional') rotational and agronomic contexts, in each of 3 years. In the final year, further NILs (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht-B1b + Rht-D1b, Rht-D1b + Rht-B1c) in both Maris Huntsman and Maris Widgeon backgrounds were added together with 64 lines of a doubled haploid (DH) population [Savannah (Rht-D1b) x Renesansa (Rht-8c + Ppd-D1a)]. Assessments included laboratory tests of germination and coleoptile length, and various field measurements of crop growth between emergence and pre jointing [plant population, tillering, leaf length, ground cover (GC), interception of photosynthetically active radiation (PAR), crop dry matter (DM) and nitrogen accumulation (N), far red: red reflectance ratio (FR:R), crop height, and weed dry matter]. All of the dwarfing alleles except Rht12 in the Mercia background and Rht8c in the DHs were associated with reduced coleoptile length. Most of the dwarfing alleles (depending on background) reduced seed viability. Severe dwarfing alleles (Rht-B1c, Rht-D1c and Rht12) were routinely associated with fewer plant numbers and reduced early crop growth (GC, PAR, DM, N, FR:R), and in 1 year, increased weed DM. In the Mercia background and the DHs the semi-dwarfing allele Rht-D1b was also sometimes associated with reductions in early crop growth; no such negative effects were associated with the marker for Rht8c. When significant interactions between cropping system and genotype did occur it was because differences between lines were more exaggerated in the organic system than in the conventional system. Ppd-D1a was associated positively with plant numbers surviving the winter and early crop growth (GC, FR:R, DM, N, PAR, height), and was the most significant locus in a QTL analysis. We conclude that, within these environmental and system contexts, genes moderating development are likely to be more important in influencing early resource capture than using Rht8c as an alternative semi-dwarfing gene to Rht-D1b.

Effects of reduced height (Rht) and photoperiod insensitivity (Ppd) alleles on yield of wheat in contrasting production systems

Near isogenic lines (NILs) varying for reduced height (Rht) and photoperiod insensitivity (Ppd-D1) alleles in a cv. Mercia background (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c+Ppd-D1a, Rht-D1c, Rht12) were compared for interception of photosynthetically active radiation (PAR), radiation use efficiency (RUE), above-ground biomass (AGB), harvest index (HI), height, weed prevalence, lodging and grain yield, at one field site but within contrasting (‘organic’ v ‘conventional’) rotational and agronomic contexts, in each of three years. In the final year, further NILs (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht-B1b+Rht-D1b, Rht-D1b+Rht-B1c) in Maris Huntsman and Maris Widgeon backgrounds were added together with 64 lines of a doubled haploid (DH) population [Savannah (Rht-D1b) × Renesansa (Rht-8c+Ppd-D1a)]. There were highly significant genotype × system interactions for grain yield, mostly because differences were greater in the conventional system than in the organic system. Quadratic fits of NIL grain yield against height were appropriate for both systems when all NILs and years were included. Extreme dwarfing was associated with reduced PAR, RUE, AGB, HI, and increased weed prevalence. Intermediate dwarfing was often associated with improved HI in the conventional system, but not in the organic system. Heights in excess of the optimum for yield were associated particularly with reduced HI and, in the conventional system, lodging. There was no statistical evidence that optimum height for grain yield varied with system although fits peaked at 85cm and 96cm in the conventional and organic systems, respectively. Amongst the DH lines, the marker for Ppd-D1a was associated with earlier flowering, and just in the conventional system also with reduced PAR, AGB and grain yield. The marker for Rht-D1b was associated with reduced height, and again just in the conventional system, with increased HI and grain yield. The marker for Rht8c reduced height, and in the conventional system only, increased HI. When using the System × DH line means as observations grain yield was associated with height and early vegetative growth in the organic system, but not in the conventional system. In the conventional system, PAR interception after anthesis correlated with yield. Savannah was the highest yielding line in the conventional system, producing significantly more grain than several lines that out yielded it in the organic system.

Reduced height alleles (Rht) and Hagberg falling number of wheat

Near isogenic lines varying for alleles for reduced height (Rht) and photoperiod insensitivity (Ppd-D1) in cv. Mercia (2005/6 to 2010/11; rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c+Ppd-D1a, Rht-D1c, Rht12) and cvs Maris Huntsman and Maris Widgeon (2007/8 to 2010/11; rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht-B1b+Rht-D1b, Rht-D1b+Rht-B1c) were compared at one field site, but within different systems (‘organic’, O, 2005/6 to 2007/8 v ‘intensive’, I, 2005/6 to 2010/11). Further experiments at the site (2006/7 to 2008/9) compared 64 lines of a doubled haploid (DH) population [Savannah (Rht-D1b) × Renesansa (Rht-8c+Ppd-D1a)]. Gibberellin (GA) insensitive dwarfing alleles (Rht-B1b; Rht-B1c; Rht-D1b; Rht-D1c) could reduce α-amylase activity and/or increase Hagberg falling number (HFN) but effects depended greatly on system, background and season. Only Rht-B1c increased grain dormancy despite producing plants taller than Rht-D1c. The GA-sensitive Rht8c+Ppd-D1a in Mercia was associated with reduced HFN but analysis of the DH population suggested this was more closely linked with Ppd-D1a, rather than Rht8c. The severe GA-sensitive dwarfing allele Rht12 was associated with reduced HFN. Instability in HFN over season tended to increase with degree of dwarfing. There was a negative association between mean grain weight and HFN that was in addition to effects of Rht and Ppd-D1 allele.

Shoot zinc (Zn) concentration varies widely within Brassica oleracea L. and is affected by soil Zn and phosphorus (P) levels

The low availability of zinc (Zn) in soils and crops affects dietary Zn intake worldwide. This study sought to determine if the natural genetic variation in shoot Zn concentrations (Zn(shoot)) is sufficient to pursue a crop improvement breeding strategy in a leafy vegetable crop. The gene-pool of Brassica oleracea L. was sampled using a large (n = 376) diversity foundation set (DFS), representing almost all species-wide common allelic variation, and 74 commercial varieties (mostly F(1)). The DFS genotypes were grown at low and high soil phosphorus (P) levels under glasshouse and field conditions, and also in a Zn-deficient soil, with or without Zn-fertilisation, in a glasshouse. Despite the large variation in Zn(shoot) among genotypes, environment had a profound effect on Zn(shoot) The heritability of Zn(shoot) was significant, but relatively low, among 90 doubled-haploid (DH) lines from a mapping population. While several quantitative trait loci (QTL) associated with Zn(shoot) occurred on chromosomes C2, C3, C5, C7, and C9, these were generally weak and conditional upon growth conditions. Breeding for Zn(shoot) in B. oleracea is therefore likely to be challenging. Shoot P concentrations increased substantially in all genotypes under low soil Zn conditions. Conversely, only some genotypes had increased Zn(shoot) at low soil P levels. Sufficient natural genetic variation may therefore exist to study some of the interactions between Zn and P nutrition.

Shoot yield drives phosphorus use efficiency in Brassica oleracea and correlates with root architecture traits

The environmental and financial costs of using inorganic phosphate fertilizers to maintain crop yield and quality are high. Breeding crops that acquire and use phosphorus (P) more efficiently could reduce these costs. The variation in shoot P concentration (shoot-P) and various measures of P use efficiency (PUE) were quantified among 355 Brassica oleracea L. accessions, 74 current commercial cultivars, and 90 doubled haploid (DH) mapping lines from a reference genetic mapping population. Accessions were grown at two or more external P concentrations in glasshouse experiments; commercial and DH accessions were also grown in replicated field experiments. Within the substantial species-wide diversity observed for shoot-P and various measures of PUE in B. oleracea, current commercial cultivars have greater PUE than would be expected by chance. This may be a consequence of breeding for increased yield, which is a significant component of most measures of PUE, or early establishment. Root development and architecture correlate with PUE; in particular, lateral root number, length, and growth rate. Significant quantitative trait loci associated with shoot-P and PUE occur on chromosomes C3 and C7. These data provide information to initiate breeding programmes to improve PUE in B. oleracea.

The identification of QTL controlling ergot sclerotia size in hexaploid wheat implicates a role for the Rht dwarfing alleles

The fungal pathogen Claviceps purpurea infects ovaries of a broad range of temperate grasses and cereals, including hexaploid wheat, causing a disease commonly known as ergot. Sclerotia produced in place of seed carry a cocktail of harmful alkaloid compounds that result in a range of symptoms in humans and animals, causing ergotism. Following a field assessment of C. purpurea infection in winter wheat, two varieties ‘Robigus’ and ‘Solstice’ were selected which consistently produced the largest differential effect on ergot sclerotia weights. They were crossed to produce a doubled haploid mapping population, and a marker map, consisting of 714 genetic loci and a total length of 2895 cM was produced. Four ergot reducing QTL were identified using both sclerotia weight and size as phenotypic parameters; QCp.niab.2A and QCp.niab.4B being detected in the wheat variety ‘Robigus’, and QCp.niab.6A and QCp.niab.4D in the variety ‘Solstice’. The ergot resistance QTL QCp.niab.4B and QCp.niab.4D peaks mapped to the same markers as the known reduced height (Rht) loci on chromosomes 4B and 4D, Rht-B1 and Rht-D1, respectively. In both cases, the reduction in sclerotia weight and size was associated with the semi-dwarfing alleles, Rht-B1b from ‘Robigus’ and Rht-D1b from ‘Solstice’. Two-dimensional, two-QTL scans identified significant additive interactions between QTL QCp.niab.4B and QCp.niab.4D, and between QCp.niab.2A and QCp.niab.4B when looking at sclerotia size, but not between QCp.niab.2A and QCp.niab.4D. The two plant height QTL, QPh.niab.4B and QPh.niab.4D, which mapped to the same locations as QCp.niab.4B and QCp.niab.4D, also displayed significant genetic interactions.

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

The effect of doubled CO2 and model basic state biases on the monsoon-ENSO system. II: Changing ENSO regimes

Integrations of a fully-coupled climate model with and without flux adjustments in the equatorial oceans are performed under 2×CO2 conditions to explore in more detail the impact of increased greenhouse gas forcing on the monsoon-ENSO system. When flux adjustments are used to correct some systematic model biases, ENSO behaviour in the modelled future climate features distinct irregular and periodic (biennial) regimes. Comparison with the observed record yields some consistency with ENSO modes primarily based on air-sea interaction and those dependent on basinwide ocean wave dynamics. Simple theory is also used to draw analogies between the regimes and irregular (stochastically forced) and self-excited oscillations respectively. Periodic behaviour is also found in the Asian-Australian monsoon system, part of an overall biennial tendency of the model under these conditions related to strong monsoon forcing and increased coupling between the Indian and Pacific Oceans. The tropospheric biennial oscillation (TBO) thus serves as a useful descriptor for the coupled monsoon-ENSO system in this case. The presence of obvious regime changes in the monsoon-ENSO system on interdecadal timescales, when using flux adjustments, suggests there may be greater uncertainty in projections of future climate, although further modelling studies are required to confirm the realism and cause of such changes.