ABSTRACT
Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype-dependency. Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) chimeric gene. Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low- and high-copy-number integration events were recovered in the T0 population with various levels of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome. A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary screening of transgene quality and GE events in the T0 population of plants regenerated through the co-delivery of GRF-GIF can allow for the propagation of the best candidates for further phenotypic analysis.
ABSTRACT
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.
Subject(s)
Flax/genetics , Physical Chromosome Mapping/methods , Contig Mapping , Genetic Linkage , Genetic MarkersABSTRACT
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.
