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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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