Graduate Thesis Or Dissertation
 

Simple sequence repeat marker development and use in European hazelnut (Corylus avellana L.)

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  • New highly polymorphic simple sequence repeat (SSR) markers for European hazelnut (Corylus avellana L.). were developed from several sources. The markers were characterized, and segregation in a mapping population allowed to assign them to linkage groups. Their transferability across genera and species in the Betulaceae family was investigated. SSR markers were used to fingerprint several hazelnut accessions, including those from recent collections in northern Spain, Turkey, Georgia and Azerbaijan. A total of 170 new single-locus polymorphic SSR primers were developed in hazelnut: 76 from a library enriched for GA repeats, 13 from a library enriched for CA repeats, 23 from internal repeats in cloned inter simple sequence repeats (ISSR) fragments, 52 from repeats at the ends of ISSR fragments, and 6 from Betulaceae sequences in GenBank. These polymorphic loci were characterized using 50 hazelnut accessions, including the parents of a mapping population and 48 accessions that represented the high level of genetic diversity in European hazelnut. One hundred forty three of the new polymorphic SSR loci were mapped in a full-sib population. The map of the resistant parent consists of 344 markers (175 random amplified polymorphic DNA (RAPD) markers and 169 SSRs) spanning 837 cM with an average of 2.43 cM between markers, while the map of the susceptible parent consists of 366 markers (183 RAPDs and 183 SSRs) spanning 926 cM with an average of 2.53 cM between markers. SRR locus A601 co-segregated with the S-locus that controls pollen-stigma incompatibility, and four loci (B774, B720, B760, and SSR62) were tightly linked to the S-locus (≤6 cM). Five loci (B716, B776, SSR9, SSR28, and SSR 100) were placed on linkage group 6 with the 'Gasaway' gene for resistance to eastern filbert blight. The Betula genes BpMADS2 and BetV1 and a Corylus lipoxygenase (LOX) gene were also placed on the hazelnut genetic map. A high percentage of loci (26.5%) gave poor fit to Mendelian expectation. Transferability of 66 Corylus and 52 Betula SSR primer pairs was studied by amplifying 69 accessions representing diverse taxa in the family Betulaceae. The transferability of 157 additional SSR loci developed from Corylus was investigated using 32 Betulaceae accessions. Transferability across species was 92.31%, while across genera in the Betulaceae it was 40.90%. The highest transferability across genera was 58.72% between Betula and Alnus. For the Corylus loci, transferability across species averaged 93.95% and across genera averaged 42.16%. The highest cross-genera amplification was 58.72% in Carpinus species. Using 12 microsatellite loci, genetic diversity was investigated in hazelnut accessions from Turkey, Georgia and Azerbaijan and compared to cultivars in the Italian-Spanish cluster. A high level of genetic diversity (H₀ = 0.69) in the cultivars from Turkey, Georgia and Azerbaijan was observed. A moderate shift in allelic frequencies (FST = 0.11 to 0.13) was seen between accessions from these three countries and the Italian-Spanish accessions, with accessions from the Black Sea and Caucasus tending to form subgroups by country of origin. The same 12 microsatellite loci were used to investigate genetic diversity in 69 hazelnut accessions collected in northern Spain. Of these, 50 had fingerprints identical to 'Casina', while the remaining 19 were unique and highly diverse. Genotyping profiles were produced for 160 additional accessions using the twelve dozen SSR loci.
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