Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information. Such techniques have the potential to generate phenotypically linked functional markers, especially when fingerprints are generated from the transcribed or expressed region of the genome. It is to be expected that these recently developed techniques will generate larger datasets, but their shortcomings should also be acknowledged and carefully investigated.

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Advances in plant gene-targeted and functional markers: a review

The narrow genetic base of elite Upland cotton (Gossypium hirsutum L.) germplasm has been a significant impediment to sustained progress in the development of cotton cultivars to meet the needs of growers and industry in recent years. The prospect of widening the genetic base of Upland cotton by accessing the genetic diversity and fiber quality of Pima cotton (Gossypium barbadense L.) has encouraged interspecific hybridization and introgression efforts for the past century. However, success is limited due mainly to genetic barriers between the two species in the forms of divergent gene regulatory systems, accumulated gene mutations, gene order rearrangements and cryptic chromosomal structure differences that have resulted in hybrid breakdown, hybrid sterility and selective elimination of genes. The objective of this paper is to provide a mini-review in interspecific hybridization between Upland and Pima cotton relevant to breeding under the following sections: (1) qualitative genetics; (2) cytogenetic stocks; (3) quantitative genetics; (4) heterosis, and (5) introgression breeding. Case studies of successful examples are provided.

Introgression genetics and breeding between Upland and Pima cotton: a review

Genetic relationships were estimated among 24 accessions belonging to 11 species of Capsicum, using 2,760 RAPD markers based on touch-down polymerase chain reactions (Td-RAPD-PCR). These markers were implemented in analyses of principal coordinates, unweighted pair group mean average, and 2,000 bootstrap replications. The accessions were divided into four groups, corresponding to previously described Capsicum complexes: C. annuum complex (CA), C. baccatum complex (CB), C. pubescens complex (CP), and C. chacoense accessions (CA/B). Their overall mean genetic similarity index was 0.487 ± 0.082, ranging from 0.88 to 0.32, based on Jaccard’s coefficient. The highest genetic variation was observed among the accessions in CP; the accessions in CB had a low level of variation as judged from the standard deviations of the genetic similarity indices. Based on the Td-RAPD-PCR markers, the 24 accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Accessions of C. chacoense were found to be equally related to complexes CA, CB, and CP.

Genetic relationships within and between Capsicum species.

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 availability of a simple, reproducible and cost-effective molecular marker is a prerequisite for plant genetic analysis. We have developed a novel promoter-targeted marker, CAAT box- derived polymorphism (CBDP) using the nucleotide sequence of CAAT box of plant promoters. CBDP, like random amplified polymorphic DNA (RAPD), uses single primer in polymerase chain reaction (PCR) for generating markers. However unlike RAPD, the CBDP primers are 18 nucleotides long and consist of a central CCAAT nucleotides core flanked by the filler sequence towards the 5′ end and di- or trinucleotides towards the 3′ end. In this study, a small set of 25 CBDP primer was designed and initially tested in a representative set of eight cultivars of jute for generation of polymorphic markers. Further, to achieve high reproducibility, a touchdown PCR was employed with an annealing temperature of 50oC. All the CBDP primers generated polymorphic markers in jute cultivars, and an UPGMA dendrogram based on Jaccard’s similarity grouped them into two clusters represented by Corchorus capsularis and C. olitorius, respectively. Interestingly, such grouping of jute cultivars was consistent with genetic relationships established earlier for these cultivars using other DNA markers. Moreover, these CBDP primers also generated polymorphic markers in representative sets of cotton (Gossypium species) and linseed (Linum usitatissimum ) cultivars. Given the high success rate of CBDP primers in generating markers in the tested species and advantages like ease in marker development and assay with reproducible profiles, they could potentially be exploited in other species as well for assessing genetic diversity, cultivar identification, construction of linkage map and marker- assisted selection.

CAAT box- derived polymorphism (CBDP): a novel promoter -targeted molecular marker for plants

Non-coding sequences account for a majority of the higher plant genome, some of which have important effects in gene regulation and plant development. In an effort to develop molecular marker systems to search for polymorphisms associated with high fiber yield and quality in cotton, we have developed a methodology that could specifically target the regulatory regions of the cotton genome. In this study we designed 10-nucleotide degenerate promoter primers based on conserved core promoter sequences and tested their applicability in PCR amplifications in combination with 10-mer random amplified polymorphic DNA (RAPD) primers. The amplified markers are called promoter anchored amplified polymorphism based on RAPD (PAAP-RAPD). Forty cotton genotypes with diverse genetic and geographical backgrounds were used to test the PAAP-RAPD system using polyacrylamide gel electrophoresis. Based on PAAP-RAPD markers amplified from 12 primer combinations, the 40 genotypes were classified into five distinctive groups: two Upland cotton (Gossypium hirsutum) groups from China, another two Upland cotton groups from the USA, and one group from American Pima cotton (G. barbadense). The groupings are in general consistent with their genetic and geographical origins. Thirty-six PAAP-RAPD and RAPD fragments were cloned and four of them were further subjected to sequence analysis. Signal scanning using software PLACE confirmed that they contained an array of cis-regulatory sequences in addition to the core promoter sequences. The results demonstrate the potential application of PAAP-RAPD as a new marker system specifically targeting regulatory regions of the plant genome.

Promoter anchored amplified polymorphism based on random amplified polymorphic DNA (PAAP-RAPD) in 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.

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

Information on gene expression during cotton fiber development for high fiber quality genotypes including Pima (Gossypium barbadense L.) and Acala cotton (G. hirsutum L.) is lacking, impeding the discovery of novel genes and DNA markers for fiber quality traits. In the present study, massively parallel sequencing technology was used to generate a total of 394,301 expressed sequence tag (EST) sequence reads, totaling over 42 million bp, from immature ovaries at 10 days post-anthesis of Pima Phy 76 and Acala 1517-99. For Pima Phy 76, 1,900 EST contigs were tentatively assembled from 21.8% (50,876) ESTs which resulted in 1,541 tentative consensus sequences (TCs). 1,005 and 1,064 EST contigs were homologous to TCs from the A2 and D5 genomes, respectively, while 738 of the EST contigs were shared by both genomes. For Acala 1517-99, 2,151 EST contigs were tentatively assembled from 24.5% (39,368) ESTs and resulted in 1,787 TCs. 1,220 and 1,206 EST contigs were homologous to known transcripts from A2 and D5 genomes and only 452 of the EST contigs were shared by both genomes. 691 EST contigs are also homologous (E-value ≤ 1 × 10−10) between Pima Phy 76 and Acala 1517-99, of which 38 contigs pairs were highly homologous (E-value ≤ 1 × 10−60) with sequence variations, predominantly single nucleotide polymorphisms (SNPs). A Blast search of a sample of contigs to Upland cotton ESTs in GenBank indicated that more than 75% EST contigs may contain SNPs. This report represents the first research in cotton using 454 pyrosequencing technology to enrich ESTs for high fiber quality cotton.

Ed. Hughs

Papers

Promoter anchored amplified polymorphism based on random amplified polymorphic DNA (PAAP-RAPD) in cotton

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

Comparative transcriptome analysis of Pima and Acala cotton during boll development using 454 pyrosequencing technology