Diversity in somatic DNA repair efficiencies between commercial inbred maize lines and native Central American purple landraces

Maize is a staple food all over the world. Models for climate change suggest that, in the future, cloud formation might be reduced in the tropics increasing the exposure to Ultraviolet-B (UV-B) radiation, a DNA-damaging agent. UV-B (290 to 320 nm) has been shown to affect yield in maize. In this project we have determined the differences in DNA repair efficiencies between U.S. inbred lines B73 and Mo17, and Central American purple landraces from Guatemala and Costa Rica. Our results from single cell electrophoresis experiments (Comet Assay) suggest that the landrace Pujagua Santa Cruz (P1, Costa Rican) was resistant to damage caused by the radiomimetic agent zeocin (24h/100 micrograms per mL), while landrace Pujagua La Cruz (P2, Costa Rican) was able to repair DNA damage after one hour. On the other hand, the homozygous line Mo17 (Missouri, USA) was unable to repair the damage, while the homozygous line B73 (Iowa, USA) and the landraces Jocopilas (Guatemalan), Orotina Congo (Costa Rican) and Talamanca (Costa Rican) were partially able to repair DNA damage. High Resolution Melting (HRM) curve analysis of putative homologous DNA repair gene ZeaATM1 showed that both P1 and P2 had differences in the melting temperatures for this gene compared to the inbred lines B73 and Mo17, while P1 showed additional differences in ZeaSOG1, ZeaRAD51 and ZeaBRCA1, suggesting that in this landrace the presence of polymorphisms may be common among key genes for this pathway. Taken together our results suggest that key adaptive differences in DNA repair efficiencies exist between inbred lines and landraces of maize and that some Central America landraces could be used as a valuable pool of alleles for plant breeding aiming to increase tolerance to radiation.

A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals structural genome variation in rainbow trout

Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2N=64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.Article SummaryA de-novo genome assembly was generated for the Arlee homozygous line of rainbow trout to enable identification and characterization of genome variants towards developing a rainbow trout pan-genome reference. The new assembly was generated using the PacBio sequencing technology and scaffolding with Hi-C contact maps and Bionano optical mapping. A contiguous genome assembly was obtained, with the contig and scaffold N50 over 15.6 Mb and 39 Mb, respectively, and 95% of the assembly in chromosome sequences. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes.

Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2

The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2ts) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2ts SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.

DH-plant production in culture of unpollinated ovules of cucumber (Cucumis sativus L.)

Relevance. The development of F1 hybrids distinguishing it from cultivars by high productivity, plant uniformity in ripening date, fruit sizes and quality is the promising trend in breeding program in cucumber (Cucumis sativus L.).The aim of the study was to optimize the gynogenesis induction condition in culture of unpollinated ovules in vitro in order to broad the generation of new breeding forms and to accelerate homozygous line production.Materials and methods. Eight promising cucumber accessions from Laboratory of Cucurbit Breeding and Seed Production (FSBSI FSVC) were taken for the study. The protocol developed in Laboratory of Biotechnology (FSBSI FSVC) for production of doubled haploid in Cucurbitaceae family was used in the experiment. The medium IMC with 30 g/L sucrose and 7g/L agar supplemented with 200 mg/L ampicillin and 0.2 mg/L thidiazuron (TDZ) was applied to induce gynogenic development.Results. The half-open bud or flower was shown to be the most suitable to be taken as an explant for cultivation. Highest number of embryo-like structures in all accessions developed from ovaries 2.1-2.6 cm long. Exposure to sterilization solution of sodium hypochlorite for 15 min made ovary wall softer and ovules can be then easily extracted without traumatizing. The traumatized ovule resulted in inhibited gynogenic development. Embryoids and calli had developed in all studied cucumber accessions, but well-formed plants were only obtained in six accessions. In total 26 plants were produced. The maximum gynogenesis induction equal to 63.1% was achieved in accession 1810. Maximum number of plant produced was twelve in accession 1763, but the greatest plant outcome 7.7% of the ovules with induced gynogenesis was observed in accession 1807.

Rapid development of homozygous lines through culture of isolated microspores in leafy crops of Brassicaceae Burnett

Relevance Biotechnological methods are generally used to speed up breeding programs and to enhance genetic diversity, so the culture of isolated microspore in vitro can be regarded as one of very suitable methods. Nontraditional and uncommon vegetable crops belonging to Brassicaceae Burnett. are becoming more popular. Methods Accessions of sarepta mustard (Brassica juncea L. Czern.) and rocket salad (Eruca sativa Mill.) were taken for the study with the aim to optimize the basic protocol for these species. Results As a result of the study the optimum cultivation conditions have been determined for the species. Sizes of buds 2.5-3.5 mm long for sarepta mustard and 7.0-7.5 long for rocket salad which were used for cultivation had been experimentally defined. It was also shown that the cold pretreatment had improved the embryo yield. The nutritional NLN-13 medium with pH 6.1 and pretreatment at 32°C during a cultivation day had been shown to be more favourable for all accessions. All conditions that had been used were suitable for embryo formation. First divisions had been seen after 4 days of cultivation, while the embryos at primary cotyledonary stage only appeared after 2 weeks of cultivation. The embryo yield per 5 buds reached 25-30 and 5-7 in the sarepta mustard and the rocket salad, respectively. It is worth noticing that the root formation and plant adaptation had passed better and faster in sarepta mustard than in rocket salad. Thus, whole process of homozygous line developing can be completed for 4-5 months, making the breeding program 3 times shorter.

Over-expression of a high-affinity phosphate transporter in transgenic barley plants does not enhance phosphate uptake rates

Transgenic barley plants that over-express the gene encoding a phosphate transporter were generated and used to test the hypothesis that manipulation of transporters may lead to improved phosphate uptake by plant roots. Replicate T2 seedlings from a homozygous line with a single locus insertion were grown in dilute flow culture. The phosphate contents and uptake rates of these plants were compared with control transgenic and wild-type plants. When external phosphate concentration was maintained at 10 μM, all plants including the transgenic over-expressing line displayed low rates of phosphate uptake and contained high levels of phosphate in the shoot tissue. When external phosphate concentration was maintained at 2 μM, the uptake rates increased to a similar level in all plant lines. Three transgenic over-expressing lines were then grown in soil at a range of phosphate concentrations and the dry weights and total phosphorus contents of the shoots were measured and compared to a transgenic control line. The results showed that over-expression of the gene encoding a phosphate transporter did not improve the uptake of phosphate under any of the conditions tested. Transporter activity is likely to be influenced by post-transcriptional mechanisms and will require further investigation before this strategy can be applied to improving plant nutrition.