A new type of fingerprinting technique is presented, based on amplified fragment length polymorphism (AFLP). Rather than two endonucleases as in AFLP, we propose the use of three enzymes, hence the method is called three endonuclease (TE)-AFLP. Genomic DNA is digested and two sets of adapters are selectively ligated onto the restriction fragments in a single reaction volume. No adapters complementary to the ends generated by a frequent cutter are added. Due to the addition of a third endonuclease, the TE-AFLP method provides a high discriminatory power and a reduction in the number of bands. The latter makes it especially suitable for the analysis of complex genomes. TE-AFLP fingerprints are suitable for detection by automatic fluorescent sequencers and are obtained in less than half the time and at reduced costs compared to a typical AFLP. The reliability of this method was investigated by determining the influence of varying digestion, ligation and PCR components on the fingerprint. Moreover, cross-experiments to study inheritance of loci were performed with a primitive insect and with tomato strains. The features of TE-AFLP are discussed in comparison with conventional AFLP.

TE-AFLP: combining rapidity and robustness in DNA fingerprinting.

The construction of a tetraploid cotton genome wide comprehensive reference map.

Amplified fragment length polymorphism (AFLP) is a powerful fingerprinting technique that is widely applied in ecological and population genetic studies. However, its routine use has been limited by high costs associated with the optimization of fluorescently labelled markers, especially for individual study systems. Here we develop a low-cost AFLP protocol that can be easily transferred between distantly related plant taxa. Three fluorescently labelled EcoRI-primers with anchors that target interspecifically conserved genomic regions were used in combination with a single non-labelled primer in our AFLP protocol. The protocol was used to genotype one gymnosperm, two monocot and three eudicot plant genera representing four invasive and four native angiosperm species (Pinus pinaster (Pinaceae), Pennisetum setaceum and Poa annua (Poaceae), Lantana camara (Verbenaceae), Bassia diffusa (Chenopodiaceae), Salvia lanceolata, Salvia africana-lutea, and Salvia africana-caerulea (Lamiaceae)). Highly polymorphic and reproducible genotypic fingerprints (between 37-144 polymorphic loci per species tested) were obtained for all taxa tested. Our single protocol was easily transferred between distantly related taxa. Measures of expected heterozygosity ranged from 0.139 to 0.196 for P. annua and from 0.168 to 0.272 for L. camara which compared well with previously published reports. In addition to ease of transferability of a single AFLP protocol, our protocol reduces costs associated with commercial kits by almost half. The use of highly conserved but abundant anchor sequences reduces the need for laborious screening for usable primers that result in polymorphic fingerprints, and appears to be the main reason for ease of transferability of our protocol between distantly related taxa.

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Towards a transferable and cost-effective plant AFLP protocol

The dawdling development in genetic improvement of cotton with conventional breeding program is chiefly due to lack of complete knowledge on and precise manipulation of fiber productivity and quality. Naturally available cotton continues to be a resource for the upcoming breeding program, and contemporary technologies to exploit the available natural variation are outlined in this paper for further improvement of fiber. Particularly emphasis is given to application, obstacles, and perspectives of marker-assisted breeding since it appears to be more promising in manipulating novel genes that are available in the cotton germplasm. Deployment of system quantitative genetics in marker-assisted breeding program would be essential to realize its role in cotton. At the same time, role of genetic engineering and in vitro mutagenesis cannot be ruled out in genetic improvement of cotton.

/pdf/marker-assisted-breeding-as-next-generation-strategy-for-o9qege183o.pdf

Marker-assisted breeding as next-generation strategy for genetic improvement of productivity and quality: can it be realized in cotton?

The lack of genetic diversity within cultivated upland cotton (Gossypium hirsutum L.) has hindered the construction of genomewide linkage maps and their applications in genetics and breeding. The objective of this investigation was to develop candidate gene markers for fi ber quality and yield on the basis of approximately 90 genes implicated in fi ber development. Polymorphisms using sequence-tagged site (STS) and single nucleotide polymorphism (SNP) markers based on single strand conformation polymorphism (SSCP) and cleaved amplifi ed polymorphism (CAP) were evaluated among three upland and fi ve Pima cotton (G. barbadense L.) genotypes. Of the 90 primer pairs, 75 resulted in polymerase chain reaction amplifi cations, including 11 that yielded polymorphic STS markers. Of the 48 primer pairs that produced polymorphic SSCP markers, 27 yielded interspecifi c polymorphism, while 15 yielded both inter- and intraspecifi c polymorphisms. Six pairs yielded only intraspecifi c polymorphisms. A total of 18 SNPs, including four indels, were identifi ed in seven of the 15 fi ber gene fragments on the basis of direct DNA sequencing, and the average length was 350 bp, with a mean of 1.3 SNPs per fragment. The average rate of SNPs per nucleotide was 0.34%, and 0.31% and 0.41% in coding and noncoding regions, respectively. Eight of the 15 SNPs were interspecifi c and 78% were nucleotide substitutions, with the four indels contributing to interspecifi c polymorphism. Six selected SNPs were confi rmed by restriction enzyme digestion. The high level of SSCP polymorphism observed within a selected set of agronomically improved lines of upland cotton suggests that the use of SSCP will greatly facilitate genomewide mapping in upland cotton.

DNA Polymorphisms of Genes Involved in Fiber Development in a Selected Set of Cultivated Tetraploid Cotton

Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system. To take advantages of the traditional AFLP techniques, we propose substitution of a restriction enzyme that recognizes a restriction site containing ATG, called ATG-anchored AFLP (ATG-AFLP) analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI in combination with MseI to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for the NsiI site were designed for ligation and PCR. Six NsiI and eight MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%) were polymorphic among four genotypes from two cultivated cotton species (Upland cotton, Gossypium hirsutum and Pima cotton, G. barbadense). The number of ATG-AFLP markers was sufficient to separate the four genotypes into two groups, consistent with their evolutionary and breeding history. Our results also showed that ATG-AFLP generated less number of total and polymorphic fragments per primer combination (2–3 vs. 4–5) than conventional AFLP within Upland cotton. Using a recombination inbred line (RIL) population, 62 polymorphic ATG-AFLP markers were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the nine ATG-AFLP fragments randomly chosen, three were found to be highly homologous to cotton cDNA sequences. An in-silico analysis of cotton and Arabidopsis cDNA confirmed that the ATG-anchored enzyme combination NsiI/MseI did generate more fragments than the EcoRI/MseI combination.

Danielle Miranda

Papers

ATG-anchored AFLP (ATG-AFLP) analysis in cotton