Genetic Fidelity Testing of Tissue Culture Raised Plants
Objective: Development, standardization and validation of DNA markers for genetic fidelity
testing of tissue culture raised plants.
Details of progress made up to March 31, 2008:
Ten varieties each of banana and sugarcane, seven varieties of black pepper, twenty three
varieties of potato and one variety of vanilla plants were obtained from different authentic
sources. DNA was isolated from all these varieties by modified CTAB method.
Isolation of Microsatellite Markers:
Microsatellites or simple sequence repeats (SSRs) are tandemly repeated motifs of 1-6
bases and are widely distributed throughout the eukaryotic genomes, making them the preferred
marker for very-high resolution genetic mapping. They are characterized by a high degree of
length polymorphism and have proven to be an extremely valuable tool for genome mapping in
many organisms. A major challenge for utilizing SSR markers for genetic fidelity testing is the
requirement of prior information of genomic DNA sequence for designing primers to the
flanking regions of the repeat motifs. Since genomic DNA sequences are not yet known for
potato, banana, sugarcane, black pepper and vanilla crops plants, SSRs would be isolated from
them based on the selective hybridization procedure (Glenn and Schable, Methods in
Enzymology, 2005, 395: 202-222).
In brief, the protocol consists of isolating genomic DNA from the plant source and
restriction digestion with a 4-base cutter (like Sau3A and RsaI) followed by dephosphorylation
of the digested genomic fragments to prevent self-ligation in the subsequent ligation step. The
dephorphorylated DNA digests were size fractionated to select fragments in the range of 1001000 base pairs, which was accomplished by gel elution. SuperSNX linkers were then ligated to
both ends of the DNA fragments and amplification of the linker ligated DNA using SuperSNX
forward primer.
Microsatellite enrichment strategy- SSR library construction:
This procedure involved a method of enrichment of microsatellite containing DNA
fragments by hybridization of amplified linker ligated DNA with biotinylated SSR oligos that
are captured by streptavidin coated para-magnetic beads. Repeat enrichment of genomic DNA
fragments was done using the following biotinylated repeat Oligos: [Mix 1: (AT)14, (AAAC)6,
(AATC)6, Mix 2:(AAC)8, (AAG)8, (ATC)8 and Mix 3:(ACT)10, (ACG)7, (AGG)7, (ACAG)6].
The hybrids of SSR containing fragments with biotinylated probe bound to streptavidin coated
para-magnetic beads were separated by magnetic particle concentrator. After extensive washing
to remove non-specific binding DNA fragments, repeat-containing DNA fragments were eluted
employing a magnetic stand.
Such repeat-enriched DNA fragments were used as templates for PCR amplification
employing primers complementary to the linker region and the amplicons were then ligated with
TOPO cloning vector. The ligated mixture was used to transform chemically competent
Escherichia coli cells to obtain a sub-genomic library which is expected to be enriched for
SSRs. Following blue/white and colony PCR screening, the positive clones were sequenced and
screened for microsatellites using the software MICAS. Sequences containing microsatellites
identified on at least one strand were processed further. Both strands were then contiged and
edited to ensure accuracy of the sequence. So far, sub-genomic libraries were constructed for
banana, black pepper and potato crop plants. The microsatellite enrichment and screening
strategy has till now yielded nine microsatellites for black pepper and three for potato. The
screening for additional microsatellite markers is in progress. The microsatellites obtained above
have to be validated for polymorphism in different varieties of each species before they could be
used as tools for testing the genetic fidelity of tissue cultured plants. Since it is desirous for
genetic fidelity testing, to isolate multiple SSR markers, which are polymorphic and informative,
similar strategy would be adopted in future to isolate more number of microsatellites in these
crop plants from the sub-genomic libraries constructed as above. SSR-enriched sub-genomic
library construction and microsatellite enrichment strategies would also be carried out in
sugarcane and vanilla plants in future.