SNP-Based Analysis Reveals Authenticity and Genetic Similarity of Russian Indigenous V. vinifera Grape Cultivars

9 Russian Vitis vinifera grape varieties and the European variety Muscat Hamburg were sequenced and genotyped using 527 SNPs (single nucleotide polymorphisms) with high minor allele frequency for the first time. The data were coupled with previously identified genotypes of 783 varieties and subjected to parentage and population analysis. As a result, contrary to the historical and ampelographic data published in many sources from 1800 to 2012, only two of the nine Russian varieties (Pukhlyakovskiy Belyi and Sibirkovyi) were related to foreign ones and were obviously imported from Europe to the Russian Empire. The remaining seven varieties, led by Krasnostop Zolotovskiy, are not directly related either in the Caucasus or in Europe, they form separate clusters on the genetic distance-based dendrogram and the world parentage network of V. vinifera. The resulting pedigree of Muscat Hamburg and its descendants is in accordance with SSR-based (simple sequence repeats) studies and the described pedigree of this variety which confirms the use of the reduced SNP set for further studies.

Post-Transplant Monitoring of Erythroid Lineage Specific Chimerism.

Hematopoietic stem cell transplantation (HSCT) has been increasingly used for treatment of non-malignant hematologic disease, including red blood cell diseases. Critical to the development of novel stem cell therapies is the ability to rapidly evaluate the kinetics of donor stem cell engraftment, lineage specific differentiation and the functional impact of transplantation. Here we report the development of a generalizable method to rapidly assess lineage specific chimerism through the technique of RT-PCR combined with pyrosequencing. To apply this RNA-based method for assessment of RBC lineage engraftment, we used the HapMap and Ensembl databases and direct high-throughput genotyping to identify a panel of expressed single nucleotide polymorphisms (SNPs) of high minor allele frequency (2pq>0.15) in diverse ethnic populations, encoded by genes uniquely expressed on erythroid cells. This panel consisted of 10 SNPs from 7 genes, encoding red cell antigens and structural proteins (Kidd, Duffy, Diego and Lutheran antigens, spectrin, beta-globin, FUT1). Following optimization of PCR amplification and pyrosequencing, mixing studies with samples with defined alleles were used to validate that each SNP could provide a quantitative measure of donor/recipient DNA and RNA. The high minor allele frequency of each of the 10 SNPs was confirmed by direct genotyping of 10 North American normal volunteers and 80 Iranian individuals with thalassemia trait or thalassemia major. This expressed RBC SNP panel was further confirmed to be informative for transplant patient-donor pairs through direct genotyping of 9 North American and 13 Iranian patients who underwent allogeneic HSCT for malignant (CLL, Hodgkin’s, NHL) and non-malignant hematologic diseases (sickle cell, polycythemia vera, Blackfan-Diamond Anemia, thalassemia) and their HLA-matched donors (19 related, 3 unrelated). At least 1 informative RBC SNP was identified for each patient-donor pair. A median of 3 and 4 SNPs were informative for matched related and unrelated pairs, respectively. Thirteen of the 22 patients developed mixed hematopoietic chimerism following HSCT. In patient-donor pairs with more than 1 informative SNP, high concordance in serial post-transplant chimerism measurements between individual SNPs was observed. Serial analysis of overall and erythroid lineage specific chimerism among 5 patients with malignant diseases revealed equivalent levels of erythroid and leukocyte donor engraftment. In contrast, differential WBC and RBC engraftment was detected in most of the patients with non-malignant diseases affecting erythropoiesis, in which increased donor representation was observed. RNA pyrosequencing provides rapid measurement of erythroid lineage chimerism, without requiring the laborious process of cell isolation. We report the generation of a RBC-specific SNP panel that is informative across ethnically diverse populations and can provide valuable functional information for diseases in which RBC engraftment is critically important. Similar methods can be applied to generate panels of expressed SNPs informative for other cell lineages to comprehensively assess the impact of multi-lineage engraftment following treatment with novel transplant regimens.