Graduate Thesis Or Dissertation
 

Fingerprinting and genetic stability of Rubus using molecular markers

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  • Two studies used DNA markers to assess their usefulness in germplasm identification and evaluation of genetic stability in four cryopreserved Rubus accessions that were stored for over 10 years in liquid nitrogen. In the first study, 12 robust Simple Sequence Repeat (SSR) markers were developed from two microsatellite-enriched libraries of ‘Meeker’ raspberry and ‘Marion’ blackberry. Of the 70 and 78 SSR-containing sequences identified, four SSR markers were obtained from the ‘Meeker’ library and eight from that of ‘Marion’. These twelve genomic SSRs and one Expressed Sequence Tag- (EST)-SSR designed from an (AT)6- containing R. idaeus sequence (AF292369) from Genbank were used for fingerprinting 48 raspberry and 48 blackberry cultivars stored at the Repository. The SSR markers developed in Rubus were highly polymorphic. Twelve SSRs generated 96 alleles in raspberries and 177 in blackberries. These markers distinguished between the raspberries and blackberries except in ‘Logan’ and ‘Boysen’ clones. Neighbor Joining cluster analysis based on the proportion of shared allele distance using 13 SSRs separated the cultivars into two main groups: the raspberries and the blackberries. Hybrid berries and cultivars with uncommon ancestry grouped separately from the two major groups. The raspberry and blackberry groups were further divided according to their pedigrees. In the second study, two types of markers, SSRs and Amplified Fragment Length Polymorphisms (AFLP) were used to evaluate genetic fidelity of regrown cryopreserved Rubus shoot tips stored for 12 years in liquid nitrogen. Analyses were done on two groups of plants separated based on the length of time they were subcultured after storage. Group one plants were analyzed after subculturing for seven months using Platinum Taq polymerase in the PCR reactions while Group two plants were analyzed immediately after recovery from cryopreservation using AmpliTaq Gold polymerase in the PCR reactions. No polymorphism was detected in either group of plants based on SSR analysis using 10 loci. Ten AFLP primer pairs amplified 547 fragments in R. grabowskii, 400 in ‘Mandarin’, 530 in ‘Silvan’, and 521 in ‘Hillemeyer’ Group one plants. An appreciably lower number of PCR products were amplified in Group two plants: 331 fragments in ‘Hillemeyer’, and 379 in ‘Silvan’. Differences in number of AFLP markers between Groups one and two were caused by use of different polymerases during the analysis. AFLP markers, with a high marker index, revealed polymorphism in three of four Rubus genotypes in Group one. However, no polymorphism was detected in Group two plants based on AFLP analysis. Recovery of plants from cryopreservation was low in the three accessions that exhibited AFLP polymorphisms (R. grabowskii, ‘Silvan’ and ‘Mandarin’). ‘Hillemeyer’ regrowth was 80% while R. grabowskii was 40%, ‘Silvan’, 20% and ‘Mandarin’, 10%, indicating less than ideal regrowth for the three genotypes. Such polymorphism might have been generated through somaclonal variants regenerated from callus tissue. Genotypic influence on stability may explain why those three genotypes were prone to variation while ‘Hillemeyer’ remained genetically stable despite long culture periods. High recovery rates and careful treatment and monitoring of regrown plants should therefore be employed to ensure maintenance of genetic fidelity of cryopreserved plants. The variation detected may also be transient and requires further morphological and molecular analysis of adult regrown cryopreserved plants that were transplanted and are growing in the greenhouse.
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