Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.

Physical mapping of the minimal region of loss in 5q- chromosome.

Acquired interstitial loss of all or part of the long arm of human chromosome 5 (5q-) is an anomaly that is seen frequently in patients with preleukemic myelodysplasia and acute myelogenous leukemia. Loss of a critical region of overlap at band 5q31.1 in all of these cases, with various cytogenetic breaks, signifies the existence of a key negative regulator of leukemogenesis. Previous studies have defined the proximal and distal ends of the critical region to reside between the genes for IL9 and EGR1, respectively. In this report, we describe a yeast artificial chromosome contig spanning this myeloid tumor suppressor locus. The combined order of the polymorphic loci is centromere-IL9-(D5S525-D5S558-D5S89-D5S526 -D5S393)-D5S399-D5S396-D5S414-EGR1 and telomere. The physical distance between the IL9 and EGR1 genes is estimated to be < 2.4 Mb. Here we report the utility of these polymorphic loci by detecting a submicroscopic deletion of 5q31; an acute myelogenous leukemia patient with a three-way translocation, t(5;18;17)(q31;p11;q11), as the sole anomaly revealed allele loss of the D5S399 and D5S396 loci.

Temporal control of epigenetic centromere specification

All living organisms require accurate mechanisms to faithfully inherit their genetic material during cell division. The centromere is a unique locus on each chromosome that supports a multiprotein structure called the kinetochore. During mitosis, the kinetochore is responsible for connecting chromosomes to spindle microtubules, allowing faithful segregation of the duplicated genome. In most organisms, centromere position and function is not defined by the local DNA sequence context but rather by an epigenetic chromatin-based mechanism. Centromere protein A (CENP-A) is central to this process, as chromatin assembled from this histone H3 variant is essential for assembly of the centromere complex, as well as for its epigenetic maintenance. As a major determinant of centromere function, CENP-A assembly requires tight control, both in its specificity for the centromere and in timing of assembly. In the last few years, there have been several new insights into the molecular mechanism that allow this process to occur. We will review these here and discuss the general implications of the mechanism of cell cycle coupling of centromere inheritance.

The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map

A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the crimap program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups assigned to each of the 31 automosomes and a single linkage group to all but three chromosomes.

Isolation and characterization of microsatellite loci from Macadamia

Thirty-three microsatellite loci were isolated for the Australian rainforest tree Macadamia integrifolia. Genotyping across a test panel of 43 commercial cultivars generated an average polymorphic information content of 0.480. Five loci showed no polymorphism across cultivars. Significant linkage disequilibrium was detected in 10 pairwise comparisons, including two pairs of loci identified from the same clone sequence. The 33 microsatellite loci represent a significant tool for genome mapping and population genetic studies.

Characterization of 27 microsatellite loci in the European flat oyster Ostrea edulis

The flat oyster Ostrea edulis is native to Europe and populations have been severely depleted by the parasite Bonamia ostreae since the 1980s. Additional genetic markers are required to improve population genetics study and linkage map development for selection for B. ostrea-resistance in this species. Here, we characterized 27 novel microsatellite loci for O. edulis. Number of alleles per locus ranged from 6 to 25 and observed heterozygosity between 0.375 and 1. Null alleles were suggested at a few loci but most loci were in Hardy-Weinberg agreement enabling their reliable use in further population and mapping genetics approaches.

Characterization Of Microsatellite Markers Developed From Prosopis Rubriflora And Prosopis Ruscifolia (leguminosae - Mimosoideae), Legume Species That Are Used As Models For Genetic Diversity Studies In Chaquenian Areas Under Anthropization In South America.

Prosopis rubriflora and Prosopis ruscifolia are important species in the Chaquenian regions of Brazil. Because of the restriction and frequency of their physiognomy, they are excellent models for conservation genetics studies. The use of microsatellite markers (Simple Sequence Repeats, SSRs) has become increasingly important in recent years and has proven to be a powerful tool for both ecological and molecular studies. In this study, we present the development and characterization of 10 new markers for P. rubriflora and 13 new markers for P. ruscifolia. The genotyping was performed using 40 P. rubriflora samples and 48 P. ruscifolia samples from the Chaquenian remnants in Brazil. The polymorphism information content (PIC) of the P. rubriflora markers ranged from 0.073 to 0.791, and no null alleles or deviation from Hardy-Weinberg equilibrium (HW) were detected. The PIC values for the P. ruscifolia markers ranged from 0.289 to 0.883, but a departure from HW and null alleles were detected for certain loci; however, this departure may have resulted from anthropic activities, such as the presence of livestock, which is very common in the remnant areas. In this study, we describe novel SSR polymorphic markers that may be helpful in future genetic studies of P. rubriflora and P. ruscifolia.

Development And Characterization Of Microsatellite Loci For Ocotea Species (lauraceae) Threatened With Extinction.

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 x 10-24 to 7.7 x 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes.

Development And Characterization Of Microsatellite Loci For Tabebuia Cassinoides (bignoniaceae).

Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon.

New Microsatellite Markers For Wild And Commercial Species Of Passiflora (passifloraceae) And Cross-amplification.

• We developed the first microsatellites for Passiflora setacea and characterized new sets of markers for P. edulis and P. cincinnata, enabling further genetic diversity studies to support the conservation and breeding of passion fruit species. • We developed 69 microsatellite markers and, in conjunction with assessments of cross-amplification using primers available from the literature, present 43 new polymorphic microsatellite loci for three species of Passiflora. The mean number of alleles per locus was 3.1, and the mean values of the expected and observed levels of heterozygosity were 0.406 and 0.322, respectively. • These microsatellite markers will be valuable tools for investigating the genetic diversity and population structure of wild and commercial species of passion fruit (Passiflora spp.) and may be useful for developing conservation and improvement strategies by contributing to the understanding of the mating system and hybridization within the genus.

Microsatellite Loci For Orthophytum Ophiuroides (bromelioideae, Bromeliaceae) Species Adapted To Neotropical Rock Outcrops.

• Microsatellite primers were developed for Orthophytum ophiuroides, a rupicolous bromeliad species endemic to neotropical rocky fields. These microsatellite loci will be used to investigate population differentiation and species cohesion in such fragmented environments. The loci were tested for cross-amplification in related bromeliad species. • Eleven polymorphic microsatellite markers were isolated and characterized from an enriched library of O. ophiuroides. The loci were tested on 42 individuals from two populations of this species. The number of alleles per locus ranged from three to nine and the expected and observed heterozygosities ranged from 0.167 to 0.870 and from 0.369 to 0.958, respectively. Seven loci successfully amplified in other related bromeliad species. • Our results suggest that the microsatellite loci developed here will be useful to assess genetic diversity and gene flow in O. ophiuroides for the investigation of population differentiation and species cohesion in neotropical mountainous habitats.

Development And Characterization Of 32 Microsatellite Loci In Genipa Americana (rubiaceae).

• Microsatellite primers were developed for the tree species Genipa americana (Rubiaceae) for further population genetic studies. • We identified 144 clones containing 65 repeat motifs from a genomic library enriched for (CT)8 and (GT)8 motifs. Primer pairs were developed for 32 microsatellite loci and validated in 40 individuals of two natural G. americana populations. Seventeen loci were polymorphic, revealing from three to seven alleles per locus. The observed and expected heterozygosities ranged from 0.24 to 1.00 and from 0.22 to 0.78, respectively. • The 17 primers identified as polymorphic loci are suitable to study the genetic diversity and structure, mating system, and gene flow in G. americana.

Leaf-, Panel- And Latex-expressed Sequenced Tags From The Rubber Tree (hevea Brasiliensis) Under Cold-stressed And Suboptimal Growing Conditions: The Development Of Gene-targeted Functional Markers For Stress Response.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.

Microsatellite In Aeschynomene Falcata (leguminosae): Diversity, Cross-amplification, And Chromosome Localization.

Aeschynomene falcata is an important forage species; however, because of low seed production, it is underutilized as forage species. Aeschynomene is a polyphyletic genus with a challenging taxonomic position. Two subgenera have been proposed, and it is suggested that Aeschynomene can be split in 2 genera. Thus, new markers, such as microsatellite sequences, are desirable for improving breeding programs for A. falcata. Based on transferability and in situ localization, these microsatellite sequences can be applied as chromosome markers in the genus Aeschynomene and closely related genera. Here, we report the first microsatellite library developed for this genus; 11 microsatellites were characterized, with observed and expected heterozygosities ranging from 0.0000 to 0.7143 and from 0.1287 to 0.8360, respectively. Polymorphic information content varied from 0.1167 to 0.7786. The departure from Hardy-Weinberg equilibrium may have resulted from frequent autogamy, which is characteristic of A. falcata. Of the 11 microsatellites, 9 loci were cross-amplified in A. brevipes and A. paniculata and 7 in Dalbergia nigra and Machaerium vestitum. Five of these 7 cross-amplified microsatellites were applied as probes during the in situ hybridization assay and 2 showed clear signals on A. falcata chromosomes, ensuring their viability as chromosome markers.

Preference Mapping Of Dulce De Leche Commercialized In Brazilian Markets.

Dulce de leche samples available in the Brazilian market were submitted to sensory profiling by quantitative descriptive analysis and acceptance test, as well sensory evaluation using the just-about-right scale and purchase intent. External preference mapping and the ideal sensory characteristics of dulce de leche were determined. The results were also evaluated by principal component analysis, hierarchical cluster analysis, partial least squares regression, artificial neural networks, and logistic regression. Overall, significant product acceptance was related to intermediate scores of the sensory attributes in the descriptive test, and this trend was observed even after consumer segmentation. The results obtained by sensometric techniques showed that optimizing an ideal dulce de leche from the sensory standpoint is a multidimensional process, with necessary adjustments on the appearance, aroma, taste, and texture attributes of the product for better consumer acceptance and purchase. The optimum dulce de leche was characterized by high scores for the attributes sweet taste, caramel taste, brightness, color, and caramel aroma in accordance with the preference mapping findings. In industrial terms, this means changing the parameters used in the thermal treatment and quantitative changes in the ingredients used in formulations.