Some AFLP amplicons are highly conserved DNA sequences mapping to the same linkage groups in two F2 populations of carrot

Abstract

Amplified fragment length polymorphism (AFLP) is a fast and reliable tool to generate a large number of DNA markers. In two unrelated F 2 populations of carrot (Daucus carota L.), Brasilia x HCM and B493 x QAL (wild carrot), it was hypothesized that DNA 1) digested with the same restriction endonuclease enzymes and amplified with the same primer combination and 2) sharing the same position in polyacrylamide gels should be conserved sequences. To test this hypothesis AFLP fragments from polyacrylamide gels were eluted, reamplified, separated in agarose gels, purified, cloned and sequenced. Among thirty-one paired fragments from each F 2 population, twenty-six had identity greater than 91% and five presented identity of 24% to 44%. Among the twenty-six conserved AFLPs only one mapped to different linkage groups in the two populations while four of the five less-conserved bands mapped to different linkage groups. Of eight SCAR (sequence characterized amplified regions) primers tested, one conserved AFLP resulted in co-dominant markers in both populations. Screening among 14 carrot inbreds or cultivars with three AFLP-SCAR primers revealed clear and polymorphic PCR products, with similar molecular sizes on agarose gels. The development of co-dominant markers based on conserved AFLP fragments will be useful to detect seed mixtures among hybrids, to improve and to merge linkage maps and to study diversity and phylogenetic relationships.

Development of a diagnostic DNA marker for the geographic origin of Shorea leprosula

“…One study based on the AFLP method for linkage analysis found that readily identifiable co-dominant markers ranged from 6% to 12.6% among all polymorphic AFLP markers (Waugh et al 1997). Santos and Simon (2002) reported that only one out of eight (12.5%) SCAR markers developed from AFLP markers can be converted into a co-dominant marker, whereas Paran and Michelmore (1993), who introduced SCAR markers, developed three co-dominant markers from nine RAPD markers.…”

Development of SCAR Markers for Species Identification in the Genus Shorea (Dipterocarpaceae)

“…In the present study markers were developed from anonymous DNA (AFLP) and combined with the chloroplast marker to discriminate between two closely related species. SCAR markers are generally used for different identification purposes in sample sizes of less than 100 (BRADEEN and SIMON, 1998;NEGI et al, 2000;SANTOS and SIMON, 2002;HUARACHA et al, 2004). More often, markers for species identification are tested in only a few samples per species (BEISMANN et al, 1997;ISHIYAMA et al, 2003;KRESS et al, 2005;ZIEGENHAGEN et al, 2005).…”

Merging Carrot Linkage Groups based on Conserved Dominant AFLP Markers in F2 Populations

“…Each polymorphic AFLP fragment was identifi ed by 1) six letters corresponding to the primer combination, followed by 2) the estimated number of nucleotides of the DNA fragment, and 3) a letter indicating the parental origin of the fragment: B, H, 4 and Q, respectively for Brasilia, HCM, B493 and QAL. Polymorphic AFLP markers in both populations, which were digested with the same endonuclease enzyme, amplifi ed with the same primer combination and shared the same position in a polyacrylamide gel, were selected for elution and re-amplifi cation and further sequencing (Santos and Simon, 2002a). Those proved to be identical in nucleotide sequence were used as anchor to merge both linkage maps in order to identify conserved AFLP fragments.…”

“…Maps consisted mostly of amplifi ed fragment length polymorphisms (AFLPs) and a few randomly amplifi ed polymorphic DNAs (RAPDs), sequence characterized amplifi ed regions (SCARs) and microsatellites, and they were merged based on two codominant markers they shared and on 28 sequenceconserved dominant AFLP markers. AFLPs generated from the same primer pairs and of the same size have been used to join maps (e.g., Alonso-Blanco et al, 1998) and for this study we sequenced putative shared bands and used those with high sequence homology (>91%) since we have determined that these sequence-conserved bands are much more likely to share map location in carrot (Santos and Simon, 2002a). The goal of the present study was to merge both maps as part of a larger goal of constructing a cross-validated consensus map of carrot.…”