The transcriptional landscape of polyploid wheat.

The coordinated expression of highly related homoeologous genes in polyploid species underlies the phenotypes of many of the world's major crops. Here we combine extensive gene expression datasets to produce a comprehensive, genome-wide analysis of homoeolog expression patterns in hexaploid bread wheat. Bias in homoeolog expression varies between tissues, with ~30% of wheat homoeologs showing nonbalanced expression. We found expression asymmetries along wheat chromosomes, with homoeologs showing the largest inter-tissue, inter-cultivar, and coding sequence variation, most often located in high-recombination distal ends of chromosomes. These transcriptionally dynamic genes potentially represent the first steps toward neo- or subfunctionalization of wheat homoeologs. Coexpression networks reveal extensive coordination of homoeologs throughout development and, alongside a detailed expression atlas, provide a framework to target candidate genes underpinning agronomic traits in wheat.

Fine mapping of LrSV2, a race-specific adult plant leaf rust resistance gene on wheat chromosome 3BS.

KEY MESSAGE: Fine mapping permits the precise positioning of genes within chromosomes, prerequisite for positional cloning that will allow its rational use and the study of the underlying molecular action mechanism. Three leaf rust resistance genes were identified in the durable leaf rust resistant Argentinean wheat variety Sinvalocho MA: the seedling resistance gene Lr3 on distal 6BL and two adult plant resistance genes, LrSV1 and LrSV2, on chromosomes 2DS and 3BS, respectively. To develop a high-resolution genetic map for LrSV2, 10 markers were genotyped on 343 F2 individuals from a cross between Sinvalocho MA and Gama6. The closest co-dominant markers on both sides of the gene (3 microsatellites and 2 STMs) were analyzed on 965 additional F2s from the same cross. Microsatellite marker cfb5010 cosegregated with LrSV2 whereas flanking markers were found at 1 cM distal and at 0.3 cM proximal to the gene. SSR markers designed from the sequences of cv Chinese Spring BAC clones spanning the LrSV2 genetic interval were tested on the recombinants, allowing the identification of microsatellite swm13 at 0.15 cM distal to LrSV2. This delimited an interval of 0.45 cM around the gene flanked by the SSR markers swm13 and gwm533 at the subtelomeric end of chromosome 3BS.

Radiation hybrid QTL mapping of Tdes2 involved in the first meiotic division of wheat.

Since the dawn of wheat cytogenetics, chromosome 3B has been known to harbor a gene(s) that, when removed, causes chromosome desynapsis and gametic sterility. The lack of natural genetic diversity for this gene(s) has prevented any attempt to fine map and further characterize it. Here, gamma radiation treatment was used to create artificial diversity for this locus. A total of 696 radiation hybrid lines were genotyped with a custom mini array of 140 DArT markers, selected to evenly span the whole 3B chromosome. The resulting map spanned 2,852 centi Ray with a calculated resolution of 0.384 Mb. Phenotyping for the occurrence of meiotic desynapsis was conducted by measuring the level of gametic sterility as seeds produced per spikelet and pollen viability at booting. Composite interval mapping revealed a single QTL with LOD of 16.2 and r (2) of 25.6 % between markers wmc326 and wPt-8983 on the long arm of chromosome 3B. By independent analysis, the location of the QTL was confirmed to be within the deletion bin 3BL7-0.63-1.00 and to correspond to a single gene located ~1.4 Mb away from wPt-8983. The meiotic behavior of lines lacking this gene was characterized cytogenetically to reveal striking similarities with mutants for the dy locus, located on the syntenic chromosome 3 of maize. This represents the first example to date of employing radiation hybrids for QTL analysis. The success achieved by this approach provides an ideal starting point for the final cloning of this interesting gene involved in meiosis of cereals.

A multiple resistance locus on chromosome arm 3BS in wheat confers resistance to stem rust (Sr2), leaf rust (Lr27) and powdery mildew.

Sr2 is the only known durable, race non-specific adult plant stem rust resistance gene in wheat. The Sr2 gene was shown to be tightly linked to the leaf rust resistance gene Lr27 and to powdery mildew resistance. An analysis of recombinants and mutants suggests that a single gene on chromosome arm 3BS may be responsible for resistance to these three fungal pathogens. The resistance functions of the Sr2 locus are compared and contrasted with those of the adult plant resistance gene Lr34.

Genetic diversity and linkage disequilibrium studies on a 3.1-Mb genomic region of chromosome 3B in European and Asian bread wheat (Triticum aestivum L.) populations.

Genetic diversity and linkage disequilibrium (LD) were investigated in 376 Asian and European accessions of bread wheat (Triticum aestivum L.). After a first and rapid screening about diversity and genetic structure at the whole genome scale using 70 simple sequence repeats (SSRs), we focused on a sequenced contig (ctg954) of 3.1 Mb located on the short arm of chromosome 3B of cv. Chinese Spring, using 32 SSRs and 10 single nucleotide polymorphisms. This contig is part of a multiple fungal resistance region. Mean polymorphism information content value on the 32 SSRs was slightly higher in the Asian genepool (0.396) than that for the European (0.329) pool. Compared with results at the whole genome scale, data from this 3.1-Mb region indicated similar trends in genetic diversity indices between both genepools. Population structure and molecular variance analyses demonstrated significant genetic differentiation and geographical subdivision in both groups of accessions. Concerning LD at the contig level, the European population had a significantly higher mean r(2) value (0.23) than the Asian population (0.18), indicating a stronger LD in the European material. With a mean of 1 marker every 74 kb, the resolution reached here allowed to perform a detailed comparative analysis of the LD and genetic diversity along the complete 3.1-Mb region in both genepools. A sliding-window approach revealed some interesting regions of the contig where LD is increasing when genetic diversity is decreasing. This study provides an in-depth understanding of molecular population genetics in European and Asian wheat gene pools, and prospects for association mapping of important sources of fungal disease resistance.

Evaluation of the genetic variability of homoeologous group 3 SSRs in bread wheat.

Thorough characterization of the genetic variability in bread wheat (Triticum aestivum L.) is important for a better improvement of this key crop and to increase cereal yield in the context of sustainable agriculture to face human needs in the next decades. To study the genetic variability of SSRs on wheat homoeologous group 3 chromosomes, we characterized 38 hexaploid and two tetraploid wheat lines using a set of 165 microsatellites that we cytogenetically assigned to the 17 deletion bins for chromosomes group 3. A comparative analysis of the genetic variability through the PIC value study, allele numbers and SSR lengths indicated that there were no statistically significant differences (p > 0.05) between the three chromosomes of this homoeologous group despite the fact that SSRs from chromosome 3B exhibited slightly more alleles per locus compared to chromosomes 3A and 3D as well as slightly higher PIC values compared to chromosome 3D. However, there was a stronger correlation between SSR length and allele number on the short arms compared to the long arms and the correlation increased from the centromeres toward the telomeres. We did not find statistically significant differences in allele numbers and PIC values for SSRs located in more distal bins on 3A and 3B chromosomes. On the contrary, for chromosome 3D, we observed significant differences (p < 0.05) between the PIC values determined for SSRs assigned to deletion bin 3DL3-0.81-1.00 bin that is located distal compare to the more proximal region (C-3DL3-0.81). These results suggest that recombination which is higher in the telomeric regions does not contribute to increase a lot the variability of the SSRs.

Mating system and recombination affect molecular evolution in four Triticeae species.

Mating systems and recombination are thought to have a deep impact on the organization and evolution of genomes. Because of the decline in effective population size and the interference between linked loci, the efficacy of selection is expected to be reduced in regions with low recombination rates and in the whole genome of self-fertilizing species. At the molecular level, relaxed selection is expected to result in changes in the rate of protein evolution and the pattern of codon bias. It is increasingly recognized that recombination also affects non-selective processes such as the biased gene conversion towards GC alleles (bGC). Like selection, this kind of meiotic drive in favour of GC over AT alleles is expected to be reduced in weakly recombining regions and genomes. Here, we investigated the effect of mating system and recombination on molecular evolution in four Triticeae species: two outcrossers (Secale cereale and Aegilops speltoides) and two selfers (Triticum urartu and Triticum monococcum). We found that GC content, possibly driven by bGC, is affected by mating system and recombination as theoretically predicted. Selection efficacy, however, is only weakly affected by mating system and recombination. We investigated the possible reasons for this discrepancy. A surprising one is that, in outcrossing lineages, selection efficacy could be reduced because of high substitution rates in favour of GC alleles. Outcrossers, but not selfers, would thus suffer from a 'GC-induced' genetic load. This result sheds new light on the evolution of mating systems.

[Neonatal screening of congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Lille experience 1980-1996]. / Le dépistage néonatal de l'hyperplasie congénitale des surrénales par déficit en 21-hydroxylase: expérience lilloise 1980-1996.

AIM: The results of the neonatal screening of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by 17-hydroxyprogesterone measurement from blood spot on blotting-paper in 408,138 newborns in the French Nord-Pas-de-Calais region from 1980 to 1996 are reported. METHODS: This measurement successively used a tracer tritium labelled (RIA H3), 125 iodine (RIA I125), then immunofluorometric method (Delfia). From 1992, sampling was systematically performed at the third day of life. RESULTS: Thirty-three cases were detected and confirmed (20 boys and 13 girls). Diagnosis was made before recalling on a clinical basis in three boys and eight girls. In 22 cases (17 boys and five girls) when diagnosis was not made before recalling, it could have been suspected in three girls because of a sex ambiguity once associated with dehydration and in eight boys because of failure to thrive (six times) or a marked dehydration (twice). Lack of sex ambiguity in two girls characterized non classical form of the illness. These two patients benefited from the early detection of the illness on growth data. Out of 49 subjects who died before recall, three could be suspected of bearing 21-hydroxylase deficiency. One single false negative case was found, which led to decrease cut-off value. On the other hand, false positive cases were frequent (0.37%), mainly in premature newborns (88% of cases). CONCLUSION: Although decrease of median age for recall at 7 days did not prevent the occurrence of two cases of dehydration, neonatal screening of 21-hydroxylase deficiency appears to be efficient, as far as diagnostic strategy is considered.

Preparation of the first European working standard for phenylalanine determination in dried blood spots.

OBJECTIVE: To develop a standard preparation for measurement of phenylalanine in blood spots in phenylketonuria against which manufacturers of kits for phenylalanine determination could calibrate their own blood spot calibrators. METHODS: As most of the variation of phenylalanine concentration in dried blood spots is due to the way the blood spot calibrators are prepared, a minimum set of prerequisites for the preparation of blood spot calibrators was established by a group of scientific experts and manufacturers. A "European working standard for phenylalanine in blood spots" (EWS-Phe-01) was prepared and distributed to manufacturers taking part in this project. RESULTS: The control of the homogeneity, linearity, and stability of the EWS-Phe-01 batch gave results within expected ranges. The preliminary results of the recalibration of calibrators for phenylalanine against EWS-Phe-01 by the participating manufacturers were promising; the differences between measurements were much decreased. CONCLUSION: Manufacturers have agreed to use EWS-Phe-01, and since November 1996 EWS-Phe calibrators have been routinely available from these companies. This pilot study may act as a model for minimising discrepancies found in other screening tests.

Need for a standardized procedure in the preparation of phenylalanine calibrators.

INTRODUCTION: Sensitive screening for phenylketonuria (PKU) and other disorders requires accurate measurement of analytes in blood eluted from filter paper. The development of new quantitative methods for phenylalanine measurement in dried blood spots for PKU screening has introduced an increased number of calibrators provided with the kits. We compared the phenylalanine values of seven different sets of calibrators (six from commercial sources and one from a screening laboratory), all measured with the same technique. METHODS: Two types of filter papers, S&S #903 and S&S #2992 were used. Phenylalanine was measured by high performance liquid chromatography (HPLC). RESULTS: There was a wide variation in the phenylalanine values assigned to the seven different sets of calibrators used, with the ratios of HPLC to assigned value ranging from 0.68-1.62. This could not be only explained by the use of different types of filter papers. However, factors in calibrator preparation did influence the measured concentration. These included higher values when 100 microliters rather than 35 microliters of blood was spotted, when the red blood cells were intact rather than lysed, and as the hematocrit increased. DISCUSSION: These observations emphasize the need to establish recommendations for preparation of dried blood calibrators in order to appropriately compare new techniques and exchange screening experiences among centers.

[Evaluation of the detection of phenylketonuria and hypothyroidism at the Lille Regional Center]. / Bilan du dépistage de la phénylcétonurie et de l'hypothyroïdie par le Centre régional de Lille.

During the past 20 years, neonatal screening for metabolic diseases has been developed and implemented in the Nord-Pas de Calais region. The regional centre (established in 1972) has screened more than 1,000,000 babies for phenylketonuria (PKU) and more than 700,000 for congenital hypothyroidism (CH). Ninety-two PKU and 182 CH patients were discovered and treated. The recent introduction of a computerised system for the clerical works in the centre has allowed a systematic survey of some parameters linked to the screening practices: collection, handling and follow-up of samples. The analysis of these parameters showed that 50% of samples were collected on the 5th day of life but 35% were collected after that date, and that 4.7% of specimen reached the laboratory 10 days or more after the day of collection. These delays can introduce a significant delay in the final diagnosis of affected children. The average age at which the diagnosis was suspected was 12.2 days in PKU and 20.6 days in CH. The difference was not only explained by the technical delay for TSH measurement but also by a later date of sampling. This observation suggests that, if blood collection was performed before nursery discharge, there were some pathologic factors justifying a later discharge. Of the control specimen (second test) which have been requested, 16% were not received; this represents a high risk of missing a case.