cTBP: A Successful Intron Length Polymorphism (ILP)-Based Genotyping Method Targeted to Well Defined Experimental Needs

Abstract

There seem to be a certain degree of reluctance in accepting ILP-based methods as part of the range of molecular markers that are classically used for plant genotyping. Indeed, since DNA polymorphism results from difference in length of fragments amplified from specific gene loci, not anonymous sequences, the number of markers that can be generated is sometime inadequate for classical phylogeny studies. Yet, ILP-based markers have many other useful advantages that should not go neglected. We support this statement by presenting a large variety of data we have been collecting for a long while regarding the use of cTBP, an ILP marker based on difference in length of the introns present within the members of the plant beta-tubulin gene family

Intron length polymorphism of β‐tubulin genes of Aegilops biuncialis Vis

“…We have not isolated and cloned the fragments amplified from Ae. biuncialis , since the previous sequence study of amplicons obtained by h‐TBP method from different plants revealed that each amplified band corresponded to a specific β‐tubulin isotype (Bardini et al, ; Breviario et al, ; Braglia et al, , Galasso et al, ).…”

“…The length of Ae. biuncialis amplified fragments were always longer than 300 bp, what corresponds to the location of primer pairs within the coding sequence (Breviario et al, ; Braglia et al, ). The fragments longer then 1000–1200 bp for TBP analysis and 2000 bp for h‐TBP analysis are reproducible and are involved in differentiation of hetero‐ and/or homoduplex DNA populations, what may be a possible explanation for these high‐molecular fragments formation during the amplification.…”

“…Variable introns are surrounded by conservative exon sites. The assay based on a simple Exon-Primed Intron-Crossing (EPIC-PCR) reaction that uses one single primer combination for any plant DNA is fast, convenient, reliable, reproducible, providing a ready-to use, and clearly intelligible results (Bardini et al, 2004;Wang et al, 2005;Braglia et al, 2010;Pirko, 2011;Gavazzi et al, 2016).…”

“…Some species of Brassica L., Lotus L., Coffea L., Eleusine Gaertn., Rosa L., etc. genera were analyzed using this system (Bardini et al, 2004;Breviario et al, 2007;Braglia et al, 2010;Galasso et al, 2010;Pirko, 2011;Gavazzi et al, 2016). At the same time, data on TBP use for the evaluation of the genetic diversity of monocotyledonous plants, particularly cereals, is incomplete.…”

Intron length polymorphism of beta-tubulin genes in Ulmus pumila L. plants in the Steppe Prydniprov'ia

“…В той же час, як джерело ДНК-поліморфізму все більшої популярності набувають інтрони. Встановлено, що інтрони беруть участь у численних важливих молекулярних подіях -контролі експресії генів через механізм альтернативного сплайсингу та інших процесах [15][16][17][18][19]. Еволюція екзонних і інтронних послідовностей йде з різною швидкістю [20,21], при цьому інтрони є гіперваріабельними ділянками, і можуть мати різний нуклеотидний склад і довжину, навіть у межах однієї таксономічної одиниці.…”