Integrated consensus genetic and physical maps of flax (Linum usitatissimum L.).

Three linkage maps of flax (Linum usitatissimum L.) were constructed from populations CDC Bethune/Macbeth, E1747/Viking and SP2047/UGG5-5 containing between 385 and 469 mapped markers each. The first consensus map of flax was constructed incorporating 770 markers based on 371 shared markers including 114 that were shared by all three populations and 257 shared between any two populations. The 15 linkage group map corresponds to the haploid number of chromosomes of this species. The marker order of the consensus map was largely collinear in all three individual maps but a few local inversions and marker rearrangements spanning short intervals were observed. Segregation distortion was present in all linkage groups which contained 1-52 markers displaying non-Mendelian segregation. The total length of the consensus genetic map is 1,551 cM with a mean marker density of 2.0 cM. A total of 670 markers were anchored to 204 of the 416 fingerprinted contigs of the physical map corresponding to ~274 Mb or 74 % of the estimated flax genome size of 370 Mb. This high resolution consensus map will be a resource for comparative genomics, genome organization, evolution studies and anchoring of the whole genome shotgun sequence.

Simple sequence repeat marker development from bacterial artificial chromosome end sequences and expressed sequence tags of flax (Linum usitatissimum L.).

Flax is an important oilseed crop in North America and is mostly grown as a fibre crop in Europe. As a self-pollinated diploid with a small estimated genome size of ~370 Mb, flax is well suited for fast progress in genomics. In the last few years, important genetic resources have been developed for this crop. Here, we describe the assessment and comparative analyses of 1,506 putative simple sequence repeats (SSRs) of which, 1,164 were derived from BAC-end sequences (BESs) and 342 from expressed sequence tags (ESTs). The SSRs were assessed on a panel of 16 flax accessions with 673 (58 %) and 145 (42 %) primer pairs being polymorphic in the BESs and ESTs, respectively. With 818 novel polymorphic SSR primer pairs reported in this study, the repertoire of available SSRs in flax has more than doubled from the combined total of 508 of all previous reports. Among nucleotide motifs, trinucleotides were the most abundant irrespective of the class, but dinucleotides were the most polymorphic. SSR length was also positively correlated with polymorphism. Two dinucleotide (AT/TA and AG/GA) and two trinucleotide (AAT/ATA/TAA and GAA/AGA/AAG) motifs and their iterations, different from those reported in many other crops, accounted for more than half of all the SSRs and were also more polymorphic (63.4 %) than the rest of the markers (42.7 %). This improved resource promises to be useful in genetic, quantitative trait loci (QTL) and association mapping as well as for anchoring the physical/genetic map with the whole genome shotgun reference sequence of flax.

Fine-mapping of the leaf rust Lr34 locus in Triticum aestivum (L.) and characterization of large germplasm collections support the ABC transporter as essential for gene function.

Leaf rust resistance gene Lr34 is likely the most important leaf rust gene characterized to date. It has been characterized as an adult plant resistance gene and is known to enhance the resistance of other leaf rust resistance genes and to condition resistance to a number of other diseases. Located on chromosome 7D, this gene was identified to be one of six co-located genes of which, an ABC transporter was shown to be the only valid candidate. Ten new molecular markers were developed spanning the Lr34 locus, including six novel microsatellite markers (cam), one insertion site-based polymorphism marker (caISBP), two single nucleotide polymorphisms (caSNP), and one gene-specific marker (caIND). Using these new markers and others that were previously published, a comparative fine map of the locus was constructed from five segregating populations representing 1,742 lines. Identification of a susceptible line with a recombination in the 4.9 kb interval between caSNP4 located in the ABC transporter gene and cam8 located just upstream of this gene provided further evidence to support the identity of the ABC transporter as Lr34 by ruling out four of the adjacent genes. Originally, three mutations forming two haplotypes had been described for the ABC transporter gene. A third combination of the three mutations and an additional rare mutation in exon 22 were subsequently described. We identified an additional novel mutation in exon 10 that would cause a frameshift and is likely non-functional. This mutation was only found in Lr34- lines and constituted a novel molecular haplotype. Characterization of two germplasm collections of 700 Triticum aestivum lines permitted us to gain an understanding of the frequency of the ABC haplotypes characterized to date and their distribution in germplasm from and around the world. In addition to the four haplotypes previously described, a fifth haplotype was found in two of the 700 lines from the germplasm collections. These lines displayed the deletion in indel 11 characteristic of Lr34+ lines, but are likely susceptible to leaf rust. Mapping and haplotyping data suggest that of all the markers described herein, marker caIND11 is the best diagnostic marker for marker-assisted selection of Lr34 because it is co-dominant, robust and with the exception of 2/700 lines, it is highly diagnostic. Other markers are also described to provide alternatives for laboratories with different technologies.

Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers.

A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L. 'Glenlea'. Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA. The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1. A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones. Ninety-six percent of the clones had inserts. The insert size ranged from 5 to 189 kb with an average of 79 kb. The entire library was gridded onto 24 high-density filters using a 5 x 5 array. A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2%. The genome coverage was estimated to be 3.1 x haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence. BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS. These markers were either locus-specific amplicons or microsatellites. A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1x genome coverage. An example using the gene encoding the HMW glutenin Bx7 is illustrated.