A novel marker technique : using miniature inverted-repeat transposable elements (MITEs) in combination with resistant gene analogues (RGAs)

[摘要] ENGLISH ABSTRACT: Given the organisation of the maize genome as well as demands placed on thesaturation of molecular linkage maps it would be desirable to identify informativemolecular markers that is located or linked to genic rich areas.Sequences of gene products from different gene classes were investigated. Proteinscontaining a nucleotide binding site (NBS) and leucine-rich repeat (LRR) regioncomprise the largest class of disease resistance proteins. Resistant gene analogue(RGA) primers belonging to this specific class were derived from previous publishedliterature studies. By means of similarity studies of short stretches of conservedamino acid and DNA sequences, primers were developed that belonged to theperoxidase and reductase gene classes. A novel class of transposable element wasidentified, that occurred in the gene rich areas of a diverse range of grass genomes.Of all the MITE families described so far, the Heartbreaker (Hbr) and Hb2 familyelements were of particular interest.The unique properties of MITEs, especially their high copy number, polymorphism,stability and preference for genic areas together with the RGA primers, wereexploited to develop a new marker technique for the isolation of a class of molecularmarker with a strong preference for genic areas.Using the publicly available recombinant inbred population, Tx303 x C0159, 196MITE/RGA markers were added to the existing recombinant inbred linkage mapconsisting of ±1033 already established markers. It became apparent that just likeloci for disease resistance, the 196 MITE/RGA fragments were not randomlydistributed across the maize genome but occurred in clusters spread across the tenmaize chromosomes. Ninety-two (92) of the MITE/RGA fragments showedsignificant correlation to previously mapped maize resistance genes. To establishthe conservation and specificity of both the Hbr and Hb2 elements, sequences of 19MITE/RGA fragments were ascertained. When comparing the partial MITE elementsequences from these fragments, a high degree of element conservation was observed. One fragment showed good sequence correlation to a NADPH He Toxinreductase protein product and mapped to the same chromosomal location as thehm1 gene locus in maize. This fragment can be considered a candidate gene forresistance against the pathogen, Helminthosporium carbonum. The Hbr primer usedproved to be very specific for the Heartbreaker MITE element, this was in contrast tothe non-specificity of the Hb2 primer.The applicability of this technique was tested on two maize diseases that causeimmense damage in the maize production industries in South Africa. FourteenMITE/RGA markers were used to fine map the putative chromosomal locations forthe HtN1, Ht1, Ht2 and Ht3 genes that confer resistance. against Setosphaeriaturcica, the northern corn leaf blight (NelS) pathogen in maize. Three MITE/RGAfragments were identified that aided in the saturation of the linkage map forquantitative trait resistance (QTl) against gray leaf spot (GlS) in maize.This novel MITE/RGA technique presented a unique opportunity to search foradditional candidate genes by using polymerase chain reaction (peR) analysis.When compared to the conventional amplified fragment length polymorphism (AFLP)technique, the MITE/RGA technique proved to be just as efficient but was more costeffective and less time consuming.

[发布机构] Stellenbosch University