Target region amplification polymorphism (TRAP) is a fairly new PCR-based molecular marker technique which uses gene-based information for primer design. The objective of this study was to evaluate the utility of TRAP markers for assessing genetic diversity and interrelationships in sugarcane germplasm collections. Thirty genotypes from the genera Saccharum, Miscanthus, and Erianthus were used in the study. Among the genus Saccharum were the species, S. officinarum L., S. barberi Jesw., S. sinense Roxb., S. spontaneum L., S. robustum Brandes and Jeswiet ex Grassl, cultivars, cultivar-derived mutants and interspecific hybrids between S. officinarum and S. spontaneum. Six fixed primers, designed from sucrose-and cold tolerance-related EST (expressed sequence tags) sequences, paired with three arbitrary primers, were used to characterize the germplasm. Both the cluster and principal coordinate (PCoA) analyses placed the Erianthus spp. and Miscanthus spp. genotypes distinctly from each other and from the Saccharum species, thus, supporting their taxonomic classification as separate genera. Genotypes of the low sucrose and cold tolerant species, S. spontaneum, formed one distinct group, while the rest of the Saccharum species formed one interrelated cluster with no distinct subgroups. Sequence analysis of TRAP bands derived from a S. spontaneum genotype revealed homology with known gene sequences from other grass species including Sorghum. A BLASTn search using the homologous sequences from Sorghum matched with the S. officinarum GenBank accession from which the fixed TRAP primer was designed. These results ratify TRAP as a potentially useful marker technique for genetic diversity studies in sugarcane.
Framework genetic linkage maps of two progenitor species of cultivated sugarcane, Saccharum officinarum 'La Striped' (2n = 80) and S. spontaneum 'SES 147B' (2n = 64) were constructed using amplified fragment length polymorphism (AFLP), sequence related amplified polymorphism (SRAP), and target region amplification polymorphism (TRAP) markers. The mapping population was comprised of 100 F 1 progeny derived from the interspecific cross. A total of 344 polymorphic markers were generated from the female (S. officinarum) parent, out of which 247 (72%) were single-dose (segregating in a 1:1 ratio) and 33 (9%) were double-dose (segregating in a 3.3:1 ratio) markers. Sixty-four (19%) markers deviated from Mendelian segregation ratios. In the S. spontaneum genome, out of a total of 306 markers, 221 (72%) were single-dose, 43 (14%) were double-dose, and 42 markers (14%) deviated from Mendelian segregation ratios. Linkage maps with Kosambi map distances were constructed using a LOD score C5.0 and a recombination threshold of 0.45. In Saccharum officinarum, 146 markers were linked to form 49 linkage groups (LG) spanning 1732 cM whereas, in S. spontaneum, 121 markers were linked to form 45 LG spanning 1491 cM. The estimated genome size of S. officinarum 'La Striped' was 2448 cM whereas that of S. spontaneum 'SES 147B' was 3232 cM. Based on the two maps, genome coverage was 69% in S. officinarum and 46% in S. spontaneum. The S. officinarum parent 'La Striped' behaved like an auto-allopolyploid whereas S. spontaneum 'SES 147B' behaved like a true autopolyploid. Although a large disparity exists between the two genomes, the existence of simple duplex markers, which are heterozygous in both parents and segregate 3:1 in the progeny, indicates that pairing and recombination can occur between the two genomes. The study also revealed that, compared with AFLP, the SRAP and TRAP markers appear less effective at generating a large number of genome-wide markers for linkage mapping in sugarcane. However, SRAP and TRAP markers can be useful for QTL mapping because of their ability to target gene-rich regions of the genome, which is a focus of our future research.
Aflatoxin contamination of maize {Zea mays L.) grain caused by Aspergillus flavus is a serious health hazard to animals and humans. Resistance to infection by A. flavus is poorly understood. The objectives of this investigation were to identify potential candidate markers associated with resistance in maize kernels and pollen grains to A. flavus using a mapping population derived from a cross between Mp313E (resistant) and SC212m (susceptible) inbred lines. The parents, F,, and F2 plants, were planted in the field in 2005. Each Fj plant was self-pollinated to produce F2.3 seed. Fresh pollen collected from parental lines, F,, and each Fj plant was germinated on a growth medium in the presence of A. flavus conidia. Selfed seeds from parents, F,, and Fj plants, were challenged with A. flavus conidial suspension and incubated using a medium-free method. Percent kernels uninfected (PKU) and number of pollen grains germinated (NPG) were recorded. A linkage map was constructed with JoinMap 3.0 using DNA profiles of all F2 individuals produced from amplified fragment length polymorphism (AFLP) and target region amplification polymorphism (TRAP) markers. Interval mapping and multiple-QTL model (MQM) mapping analyses were performed using MapQTL 4.0 software. Three marker-QTL associations were observed for log-transformed PKU. Potential markers associated with this trait were also identified via discriminant analysis (DA). The markers identified via DA pointed to the same genomic regions as identified via the QTL-mapping strategy. For log-transformed NPG, five marker-QTL associations were detected. One QTL was associated with a TRAP marker. The DA confirmed the existence of three QTL. The QTL detected for NPG were different from the QTL detected for PKU. Thus, resistances of pollen and kernels to A. flavus appeared to be controlled by different genetic systems/mechanisms. Correlation between pollen germination and percent kernel infection was negligible (r -0.067), suggesting that the two traits can be improved independently. KEYWORDS.Amplified fragment length polymorphism (AFLP), Aspergillus flavus, discriminant analysis (DA), maize (Zea mays L.), quantitative trait loci (QTL), target region amplification polymorphism (TRAP)
The cultivated sugarcane (Saccharum spp. hybrids, 2n = 100-130) is one crop for which interspeciWc hybridization involving wild germplasm has provided a major breakthrough in its improvement. Few clones were used in the initial hybridization event leading to a narrow genetic base for continued cultivar development. Molecular breeding would facilitate the identiWcation and introgression of novel alleles/genes from the wild germplasm into cultivated sugarcane. We report the identiWcation of molecular markers associated with sugar-related traits using an F 1 population derived from a cross between S. oYcinarum 'Louisiana Striped' £ S. spontaneum 'SES 147B', the two major progenitor species of cultivated sugarcane. Genetic linkage maps of the S. oYcinarum and S. spontaneum parents were produced using the AFLP, SRAP and TRAP molecular marker techniques. The mapping population was evaluated for sugar-related traits namely, Brix (B) and pol (P) at the early (E) and late (L) plant growing season in the plant cane (04) and Wrst ratoon (05) crops (04EB, 04LB, 04LP, 05EB and 05EP). For S. oYcinarum, combined across all the traits, a total of 30 putative QTLs was observed with LOD scores ranging from 2.51 to 7.48. The phenotypic variation (adj. R 2 ) explained by all QTLs per trait ranged from 22.1% (04LP) to 48.4% (04EB). For S. spontaneum, a total of 11 putative QTLs was observed with LOD scores ranging from 2.62 to 4.70 and adj. R 2 ranging from 9.3% (04LP) to 43.0% (04LB). Nine digenic interactions (iQTL) were observed in S. oYcinarum whereas only three were observed in S. spontaneum. About half of the QTLs contributed by both progenitor species were associated with eVects on the trait that was contrary to expectations based on the phenotype of the parent contributing the allele. Quantitative trait loci and their associated eVects were consistent across crop-years and growing seasons with very few QTLs being unique to the early season. When the data were reanalyzed using the non-parametric discriminant analysis (DA) approach, signiWcant marker-trait associations were detected for markers that were either identical to or in the vicinity of markers previously identiWed using the traditional QTL approach. Discriminant analysis also pointed to previously
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