Tyramide-FISH mapping of single genes for development of an integrated recombination and cytogenetic map of chromosome 5 ofAllium cepa

Abstract

Chromosome 5 of onion carries major quantitative trait loci (QTL) that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors, all of which are of interest to breeders. SNPs, SSRs, and RFLPs in expressed regions of the onion genome have been genetically mapped, and we used these clones and sequences from the NCBI database to develop DNA probes for in situ hybridization to integrate the genetic and physical maps of onion chromosome 5. We produced genomic amplicons from expressed regions of the onion genome that carried both exons and introns in order to increase the hybridization specificity of the probes and to enlarge the target DNA sizes. Tyramide-FISH technique was used to increase the detection sensitivity of relatively short target DNA regions, which range from 950 to 2100 bp. Through the integration of genetic and chromosomal maps, we were able to estimate the distribution of recombination events along onion chromosome 5. We demonstrated the efficiency of chromosomal in situ mapping of exon-intron genomic clones for the extremely large genome of onion.

Comparative Tyramide-FISH mapping of the genes controlling flavor and bulb color in Allium species revealed an altered gene order

“…We performed PCR amplification and cloning of intron-exon parts of the genes with the length varying from 1100 to 1200 bp. The use of genomic amplicons from expressed regions that carried both exons and introns allows for increased hybridization specificity of the probes and enlarging of the target DNA sizes 29,30 . Individual clones, verified by sequencing, were labeled by Nick-translation and used for tyr-FISH experiments (see Material and Methods for details).…”

“…But still there were some problems such as detection frequency of the small size of the target chromosomal DNA and precarious hybridization site for different probes 28 . Recently we developed a new approach for the probe preparation 29 as well as a method for preparing high quality plant chromosomes. In the current study with tyr-FISH, we mapped members of the alliinase multigene family on the physical chromosomes of two widely cultivated onion crops, A .…”

Exploring the use of wild relatives in potato breeding through integrated cytogenetic and genomic approaches

“…Both explanations are congruent with the minor breaks in homeologous pairing observed in the triploid hybrids with potato. Fine-scale analysis of these regions requires a denser set of BAC probes Achenbach et al 2010;, the use of smaller probes (Han et al 2015;Aliyeva-Schnorr et al 2016), advanced FISH with enhanced resolution and sensitivity (Kirov et al 2015;Romanov et al 2015;Khrustaleva et al 2016) or comparative genomics including Mummer analyses (e.g., .…”