Development and Characterization of Genic Microsatellites for the Ornamental Plant Green and Gold (Chrysogonum virginianum)

The genus Chrysogonum is native to the eastern United States. Three entities have been recognized—either as three varieties of Chrysogonum virginianum or as two species, one of them with two varieties. The current study suggests that a fourth entity should be recognized. Several forms of the complex are in commercial trade as ornamentals. As very limited molecular information on Chrysogonum is available, we developed a set of genic simple sequence repeat markers (eSSRs) from de novo transcriptome sequencing. We tested a set of 17 eSSRs on a collection of C. virginianum genomic DNA samples from the three botanical varieties, and a new putative type observed in Tennessee, dubbed “Ocoee-type” for its geographic origin. The polymerase chain reaction and capillary electrophoresis analyses with downstream population genetics tools verified the usefulness of the eSSRs. By applying this approach, we showed recognizable variation within Chrysogonum, although it did not correspond exactly to previous infraspecific classifications. Finally, as demonstrated for the commercial cultivar Pierre included in the study, the eSSRs can be used for enhancing the future breeding or hybridization efforts of this ornamental plant.

Identification and characterization of genic microsatellites in Cunninghamia lanceolata (Lamb.) Hook (Taxodiaceae)

Genomic resources for conventional breeding programs are extremely limited for coniferous trees, and existing simple sequence repeat markers are usually identified through the laborious process of hybridization screening. Therefore, this study aimed to identify gene-based microsatellites in the Chinese fir, Cunninghamia lanceolata (Lamb.) Hook by screening transcript data. We identified 5200 microsatellites. Trinucleotide motifs were most common (47.94%) and were followed by tetranucleotide motifs (24.92%). The AG/CT motif (43.93%) was the most abundant dinucleotide repeat, whereas AAG/CTT (25.07%) was the most common trinucleotide repeat. A total of 411 microsatellite primer pairs were designed and 97 polymorphic loci were identified by 8 genotypes. The number of alleles per locus (Na) in these polymorphic loci ranged from 2 to 5 (mean, 2.640), the Ho values were 0.000-1.000 (mean, 0.479), and the HE values were 0.125-0.775 (mean, 0.462). The polymorphic information content (PIC) values were 0.110-0.715 (mean, 0.383). Seventy-two of the 97 polymorphic markers (74.23%) were present within genes with predicted functions. In addition, in genetic diversity and segregation analyses of 16 genotypes, only 5.88% of the polymorphic loci displayed segregation distortion at the p<0.05 level. Transferable amplification of a randomly selected set of 30 genic microsatellites showed that transferability decreased with increasing evolutionary distance between C. lanceolata and target conifers. Thus, these 97 genic markers will be useful for genetic diversity analysis, germplasm characterization, genome mapping and marker-assisted breeding in C. lanceolata, and evolutionary genetic analysis in Taxodiaceae.

Genetic and Physical Mapping of Genic Microsatellites in Barley (Hordeum vulgare L.)

Due to the availability of sequence data from large-scale EST (expressed sequence tag) projects, it has become feasible to develop microsatellite or simple sequence repeat (SSR) markers from genes. A set of 111&nbsp;090 barley ESTs (corresponding to 55.9 Mb of sequence) was employed for the identification of microsatellites with the help of a PERL5 script called MISA. As a result, a total of 9 564 microsatellites were identified in 8 766 ESTs (SSR-ESTs). Cluster analysis revealed the presence of 2 823 non-redundant SSR-ESTs in this set. From these 754&nbsp;primer pairs were designed and analysed in a set of seven genotypes including the parents of three mapping populations. Finally, 185 microsatellite (EST-SSRs) loci were placed onto the barley genetic map. These markers show a uniform distribution on all the linkage groups ranging from 21 markers (on 7H) to 35 markers (3H). The polymorphism information content (PIC) for the developed markers ranged from 0.24 to 0.78 with an average of 0.48. For the assignment of these markers to BAC clones, a PCR-based strategy was established to screen the &ldquo;Morex&rdquo;-BAC library. By using this strategy BAC addresses were obtained for a total of 127 mapped EST-SSRs, which may provide at least two markers located on a single BAC. This observation is indicative of an uneven distribution of genes and may lead to the identification of gene-rich regions in the barley genome. &nbsp;