On the structural differences between markers and genomic AC microsatellites

AC microsatellites have proved particularly useful as genetic markers. For some purposes, such as in population biology, the inferences drawn depend on the quantitative values of their mutation rates. This, together with intrinsic biological interest, has led to widespread study of microsatellite mutational mechanisms. Now, however, inconsistencies are appearing in the results of marker-based versus non-marker-based studies of mutational mechanisms. The reasons for this have not been investigated, but one possibility, pursued here, is that the differences result from structural differences between markers and genomic microsatellites. Here we report a comparison between the CEPH AC marker microsatellites and the global population of AC microsatellites in the human genome. AC marker microsatellites are longer than the global average. Controlling for length, marker microsatellites contain on average fewer interruptions, and have longer segments, than their genomic counterparts. Related to this, marker microsatellites show a greater tendency to concentrate the majority of their repeats into one segment. These differences plausibly result from scientists selecting markers for their high polymorphism. In addition to the structural differences, there are differences in the base composition of flanking sequences, marker flanking regions being richer in C and G and poorer in A and T. Our results indicate that there are profound differences between marker and genomic microsatellites that almost certainly affect their mutation rates. There is a need for a unified model of mutational mechanisms that accounts for both marker-derived and genomic observations. A suggestion is made as to how this might be done.

The structure of interrupted human AC microsatellites

Microsatellite lengths change over evolutionary time through a process of replication slippage. A recently proposed model of this process holds that the expansionary tendencies of slippage mutation are balanced by point mutations breaking longer microsatellites into smaller units and that this process gives rise to the observed frequency distributions of uninterrupted microsatellite lengths. We refer to this as the slippage/point-mutation theory. Here we derive the theory's predictions for interrupted microsatellites comprising regions of perfect repeats, labeled segments, separated by dinucleotide interruptions containing point mutations. These predictions are tested by reference to the frequency distributions of segments of AC microsatellite in the human genome, and several predictions are shown not to be supported by the data, as follows. The estimated slippage rates are relatively low for the first four repeats, and then rise initially linearly with length, in accordance with previous work. However, contrary to expectation and the experimental evidence, the inferred slippage rates decline in segments above 10 repeats. Point mutation rates are also found to be higher within microsatellites than elsewhere. The theory provides an excellent fit to the frequency distribution of peripheral segment lengths but fails to explain why internal segments are shorter. Furthermore, there are fewer microsatellites with many segments than predicted. The frequencies of interrupted microsatellites decline geometrically with microsatellite size measured in number of segments, so that for each additional segment, the number of microsatellites is 33.6% less. Overall we conclude that the detailed structure of interrupted microsatellites cannot be reconciled with the existing slippage/point-mutation theory of microsatellite evolution, and we suggest that microsatellites are stabilized by processes acting on interior rather than on peripheral segments.

Characterization of microsatellite markers for Sycoscapter nonpollinating fig wasps

We isolated 18 microsatellites from Sycoscapter australis, a nonpollinating fig wasp that develops in figs of Ficus macrophylla, and assessed their variability in 20 wasps. We further optimized nine of these loci for use in three other Sycoscapter species that develop in Ficus rubiginosa figs and assessed their variability in 47-140 wasps per species. These are the first microsatellites developed for nonpollinating fig wasps and show sufficient polymorphism to become important tools in evolutionary and genetical studies of Sycoscapter wasps.

Characterization of microsatellite loci for the endangered cactus Echinocactus grusonii, and their cross-species utilization

Echinocactus grusonii is common in trade but critically endangered in its natural habitat. With the ultimate aim of developing a certification scheme to aid in the conservation of this species, we have isolated E. grusonii microsatellites from a nonenriched library. Fifty-seven sequences contained a microsatellite array, of which 12 were polymorphic among 30 individuals from a single wild population. All 12 microsatellite primer pairs amplified product in one or more species in a screen of 27 other cactus species.

Characterization of microsatellite loci for the critically endangered cactus Ariocarpus bravoanus

Ariocarpus bravoanus is common in trade but critically endangered in its natural habitat. With the ultimate aim of developing a certification scheme to aid in the conservation of this species, we have isolated A. bravoanus microsatellites from a nonenriched library. Fifty-four sequences contained a microsatellite array, of which eight were polymorphic among 23 individuals, 20 from one population and three plants from trade.

Characterization and PCR multiplexing of polymorphic microsatellite loci in cashew (Anacardium occidentale L.) and their cross-species utilization

Cashew (Anacardium occidentale L.) is the most economically important tropical nut crop in the world, and yet there are no sequence tagged site (STS) markers available for its study. Here we use an automated, high-throughput system to isolate cashew microsatellites from a non-enriched genomic library blotted onto membranes at high density for screening. Sixty-five sequences contained a microsatellite array, of which 21 proved polymorphic among a closely related seed garden population of 49 genotypes. Twelve markers were suitable for multiplex analysis. Of these, 10 amplified in all three related tropical tree species tested: Anacardium microcarpum, Anacardium pumilum and Anacardium nanum.

An enhanced microsatellite map of diploid Fragaria

A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. × ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F2 mapping population (FV×FN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.

A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens × F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

Likelihood-based estimation of microsatellite mutation rates

Microsatellites are widely used in genetic analyses, many of which require reliable estimates of microsatellite mutation rates, yet the factors determining mutation rates are uncertain. The most straightforward and conclusive method by which to study mutation is direct observation of allele transmissions in parent-child pairs, and studies of this type suggest a positive, possibly exponential, relationship between mutation rate and allele size, together with a bias toward length increase. Except for microsatellites on the Y chromosome, however, previous analyses have not made full use of available data and may have introduced bias: mutations have been identified only where child genotypes could not be generated by transmission from parents' genotypes, so that the probability that a mutation is detected depends on the distribution of allele lengths and varies with allele length. We introduce a likelihood-based approach that has two key advantages over existing methods. First, we can make formal comparisons between competing models of microsatellite evolution; second, we obtain asymptotically unbiased and efficient parameter estimates. Application to data composed of 118,866 parent-offspring transmissions of AC microsatellites supports the hypothesis that mutation rate increases exponentially with microsatellite length, with a suggestion that contractions become more likely than expansions as length increases. This would lead to a stationary distribution for allele length maintained by mutational balance. There is no evidence that contractions and expansions differ in their step size distributions.

A novel satellite mission concept for upper air water vapour, aerosol and cloud observations using integrated path differential absorption LiDAR limb sounding

We propose a new satellite mission to deliver high quality measurements of upper air water vapour. The concept centres around a LiDAR in limb sounding by occultation geometry, designed to operate as a very long path system for differential absorption measurements. We present a preliminary performance analysis with a system sized to send 75 mJ pulses at 25 Hz at four wavelengths close to 935 nm, to up to 5 microsatellites in a counter-rotating orbit, carrying retroreflectors characterized by a reflected beam divergence of roughly twice the emitted laser beam divergence of 15 µrad. This provides water vapour profiles with a vertical sampling of 110 m; preliminary calculations suggest that the system could detect concentrations of less than 5 ppm. A secondary payload of a fairly conventional medium resolution multispectral radiometer allows wide-swath cloud and aerosol imaging. The total weight and power of the system are estimated at 3 tons and 2,700 W respectively. This novel concept presents significant challenges, including the performance of the lasers in space, the tracking between the main spacecraft and the retroreflectors, the refractive effects of turbulence, and the design of the telescopes to achieve a high signal-to-noise ratio for the high precision measurements. The mission concept was conceived at the Alpbach Summer School 2010.

Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array

Background A whole-genome genotyping array has previously been developed for Malus using SNP data from 28 Malus genotypes. This array offers the prospect of high throughput genotyping and linkage map development for any given Malus progeny. To test the applicability of the array for mapping in diverse Malus genotypes, we applied the array to the construction of a SNPbased linkage map of an apple rootstock progeny. Results Of the 7,867 Malus SNP markers on the array, 1,823 (23.2 %) were heterozygous in one of the two parents of the progeny, 1,007 (12.8 %) were heterozygous in both parental genotypes, whilst just 2.8 % of the 921 Pyrus SNPs were heterozygous. A linkage map spanning 1,282.2 cM was produced comprising 2,272 SNP markers, 306 SSR markers and the S-locus. The length of the M432 linkage map was increased by 52.7 cM with the addition of the SNP markers, whilst marker density increased from 3.8 cM/marker to 0.5 cM/marker. Just three regions in excess of 10 cM remain where no markers were mapped. We compared the positions of the mapped SNP markers on the M432 map with their predicted positions on the ‘Golden Delicious’ genome sequence. A total of 311 markers (13.7 % of all mapped markers) mapped to positions that conflicted with their predicted positions on the ‘Golden Delicious’ pseudo-chromosomes, indicating the presence of paralogous genomic regions or misassignments of genome sequence contigs during the assembly and anchoring of the genome sequence. Conclusions We incorporated data for the 2,272 SNP markers onto the map of the M432 progeny and have presented the most complete and saturated map of the full 17 linkage groups of M. pumila to date. The data were generated rapidly in a high-throughput semi-automated pipeline, permitting significant savings in time and cost over linkage map construction using microsatellites. The application of the array will permit linkage maps to be developed for QTL analyses in a cost-effective manner, and the identification of SNPs that have been assigned erroneous positions on the ‘Golden Delicious’ reference sequence will assist in the continued improvement of the genome sequence assembly for that variety.

Fighting in fig wasps: do males avoid killing brothers or do they never meet them?

1. In many fig wasp species, armoured wingless males regularly engage in lethal fights for access to females inside figs, which act as discrete mating patches. 2. Kin selection generally opposes killing brothers, because their reproductive success provides indirect genetic benefits (inclusive fitness). However, siblicide may be avoided if (i) brothers do not occur in the same figs, or (ii) males avoid fighting brothers in the same fig. Alternatively, (iii) siblicide may occur because intense mate competition between brothers at the local scale overcomes kin selection effects, or (iv) males do not recognise kin. 3. A fig may also contain wasps from other closely related species and it is not known if males also fight with these individuals. 4. Nine microsatellite loci were used in the first genetic analysis of fighting in fig wasps. We assigned species and sibling identities to males and tested alternative fighting scenarios for three Sycoscapter wasp species in figs of Ficus rubiginosa. 5. Approximately 60% of figs contained males frommore than one Sycoscapter species and approximately 80% of fights were between conspecifics, but a surprising 20% were between heterospecific males. 6.Within species, fewfigs contained brothers, suggesting that females typically lay one son per fig. Overall, most males do not compete with brothers and all fights observed were between unrelated males. Key words:Competition, fighting, genetics, kin selection, microsatellites, relatedness.

Microsatellite markers for hoop-petticoat daffodils (Narcissus sect. Bulbocodii; Amaryllidaceae)

• Premise of the study: Microsatellite markers were developed using hoop-petticoat daffodils ( Narcissus sect. Bulbocodii ; Amaryllidaceae) to aid in the taxonomic revision of the section, and further to evaluate their broad applicability for daffodil cultivar identification. • Methods and Results: Three hundred fifty-one primer pairs were developed using a commercial service. Nineteen polymorphic and repeatable markers were developed by screening 67 of these primer pairs. Of these, 11 chosen markers were used to screen 317 samples; the number of alleles per locus ranged from four to 21, and the observed heterozygosity ranged from 0.101 to 0.297. There were null genotypes in some samples for six of the markers. All the microsatellites were transferable to other Narcissus sections. • Conclusions: The results indicate that these new markers have sufficient potential variation to be used for taxonomic revision of the genus and to distinguish many commercial daffodil cultivars.