Detection of segregation distortion loci in triticale (x Triticosecale Wittmack) based on a high-density DArT marker consensus genetic linkage map

<p>Abstract</p> <p>Background</p> <p>Triticale is adapted to a wide range of abiotic stress conditions, is an important high-quality feed stock and produces similar grain yield but more biomass compared to other crops. Modern genomic approaches aimed at enhancing breeding progress in cereals require high-quality genetic linkage maps. Consensus maps are genetic maps that are created by a joint analysis of the data from several segregating populations and different approaches are available for their construction. The phenomenon that alleles at a locus deviate from the Mendelian expectation has been defined as segregation distortion. The study of segregation distortion is of particular interest in doubled haploid (DH) populations due to the selection pressure exerted on the plants during the process of their establishment.</p> <p>Results</p> <p>The final consensus map, constructed out of six segregating populations derived from nine parental lines, incorporated 2555 DArT markers mapped to 2602 loci (1929 unique). The map spanned 2309.9 cM with an average number of 123.9 loci per chromosome and an average marker density of one unique locus every 1.2 cM. The R genome showed the highest marker coverage followed by the B genome and the A genome. In general, locus order was well maintained between the consensus linkage map and the component maps. However, we observed several groups of loci for which the colinearity was slightly uneven. Among the 2602 loci mapped on the consensus map, 886 showed distorted segregation in at least one of the individual mapping populations. In several DH populations derived by androgenesis, we found chromosomes (2B, 3B, 1R, 2R, 4R and 7R) containing regions where markers exhibited a distorted segregation pattern. In addition, we observed evidence for segregation distortion between pairs of loci caused either by a predominance of parental or recombinant genotypes.</p> <p>Conclusions</p> <p>We have constructed a reliable, high-density DArT marker consensus genetic linkage map as a basis for genomic approaches in triticale research and breeding, for example for multiple-line cross QTL mapping experiments. The results of our study exemplify the tremendous impact of different DH production techniques on allele frequencies and segregation distortion covering whole chromosomes.</p

Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L.

Until recently, only a few microsatellites have been available for Cucurbita, thus their development is highly desirable. The Austrian oil-pumpkin variety Gleisdorfer Ölkürbis (C. pepo subsp. pepo) and the C. moschata cultivar Soler (Puerto Rico) were used for SSR development. SSR-enriched partial genomic libraries were established and 2,400 clones were sequenced. Of these 1,058 (44%) contained an SSR at least four repeats long. Primers were designed for 532 SSRs; 500 primer pairs produced fragments of expected size. Of these, 405 (81%) amplified polymorphic fragments in a set of 12 genotypes: three C. moschata, one C. ecuadorensis, and eight C. pepo representing all eight cultivar groups. On an average, C. pepo and C. moschata produced 3.3 alleles per primer pair, showing high inter-species transferability. There were 187 SSR markers detecting polymorphism between the USA oil-pumpkin variety “Lady Godiva” (O5) and the Italian crookneck variety “Bianco Friulano” (CN), which are the parents of our previous F2 mapping population. It has been used to construct the first published C. pepo map, containing mainly RAPD and AFLP markers. Now the updated map comprises 178 SSRs, 244 AFLPs, 230 RAPDs, five SCARs, and two morphological traits (h and B). It contains 20 linkage groups with a map density of 2.9 cM. The observed genome coverage (Co) is 86.8%

Transferability of SSR markers in the grass family

Simple sequence repeat (SSR) markers are a valuable tool suitable for many genetic studies but their initial development is costly, time consuming and labor intensive. Based on the conservation of gene content and order among closely related species, transferability, application of existing SSR markers from one species in another species, is considered as an alternative way to obtain SSR markers. Our objectives were to examine (1) the transferability of SSR markers among wheat (Triticum aestivum L.), rye ( Secale cereale L.) and triticale (x Triticosecale Wittmack); (2) the genetic diversity of hexaploid triticale accessions using wheat and rye SSR markers; and (3) transferability in 11 agronomically important grass genera using SSR markers from rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and wheat. Transferability of 148 wheat SSR markers to rye was 17%, whereas 25% of 28 rye markers were amplifiable in wheat. In triticale, 58% and 39% transferability was achieved for wheat and rye markers, respectively. In the genetic diversity study of 80 hexaploid triticale accessions, 43 wheat and 14 rye SSR markers detected an average gene diversity of 0.54. Cluster analysis grouped the accessions into 5 clusters that were generally consistent with the available pedigree information, country of origin, growth habit, and release year. Every larger cluster, however, included lines with unrelated pedigrees, different countries of origin, growth habit, and release year. Transferability of 20 SSR markers each of rice, sorghum and wheat in 11 grass genera yielded none to multiple amplification products in non-donor genera. The alignment of sequences amplified by Xcup61, a sorghum marker, showed the homologous fragment amplified in sugar cane (Saccharum officinarum L.), a closely related genus, but not in distant genera, wheat, triticale and barley (Hordeum vulgare L.). Overall results indicated that the SSR markers were transferable in the closely related species and could be used in the genetic studies. However, the amplification products of non-donor species should be sequenced to ensure their homology

Development of Real-Time Fluorescence CRISPR/Cas12a-Based Detection as a Portable Diagnostic System Using Integrated Circuits

A solution for achieving high-performance measurements in a space-constrained experimental setup was developed as a portable incubating instrument for real-time fluorescence detection of AvrPi9 gene in rice blast fungus by using a calibrated spectrometer in CRISPR-Cas12a detection. The system demonstrates accurate temperature control with low energy consumption and low deviation of ±0.16 °C from the setpoint temperatures, with high sensitivity and accurate detection within 10 min. The CRISPR-Cas12a detection reaction was demonstrated using AvrPi9 PCR product, crRNAs, LbCas12a and fluorescence-quencher reporter incubating at 37 °C for 10 min. Calibrated C12666MA spectrometer with 480 nm and 520 nm LEDs vs HR4000 reference exhibits low RMS of 0.54 and 1.30 and drift of 6.4 nm and 4.84 nm, respectively indicating high accuracy and reliability in fluorescence detection. Fluorescence signals were observed under an LED transilluminator, while real-time analysis was conducted through spectrometric measurements upon excitation by a 480 nm high-intensity blue LED source. Accuracy of detection between positive, non-template and non-target control was reported with no incidence of false positives observed. The instrument exhibits reliable quantitative detection capabilities with a limit of detection of 3.8 ng of DNA targets that are comparable to when running the same reaction on a commercial real-time PCR, with a detection limit of 1 ng. This study demonstrates that the CRISPR-Cas12a detection method represents a significant breakthrough in molecular diagnostics due to its advantages of rapidity, high sensitivity, and convenience allowing for the development of a compact, and energy-efficient platform that can facilitate real-time on-site diagnostics with accurate temperature control