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Molecular Plant Breeding 2012, Vol.3, No.3, 26
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Figure 2 Agarose gel electrophoresis of ds cDNA from
V.
amurensis
Note: 1: ds cDNA; M: DL2000 Marker
Figure 3 Agarose gel electrophoresis of
V. amurensis
ds cDNA
after fractionation
Note: 1
-
16: ds cDNA separated by CHROMA SPIN-400; M:
DL2000 Marker
with the ratio of 1:1.5 could produce high numbers
of clones that would be pooled to form the original,
unamplified library. After the drip procedure by
the CHROMA SPIN
-
400 Column, the 7~10 fractions
containing cDNA with lengther no longer than 500 bp
was collected (Figure 3). To obtain a library of the
desired complexity, three separate ligations were
performed for an optimal ratio of cDNA to vector. The
results showed that the ligation with the ratio of 1:1.5
could produce high numbers of clones that would be
pooled to form the original, unamplified library.
The library qualification evaluation showed that the
titer of primary cDNA library was 1.12×10
6
pfu/mL
and the titer of amplified library was 2.82×10
9
pfu/mL.
Furthermore, the percentage of recombination was
about 100%, and the fragment size of insert was
0.5~2.0 kb, with the average insert size of 0.80 kb
(Figure 4). The results made clear that the cDNA
library of
V. amurensis
veraison pericarp constructed
by SMART
TM
was successful and had high quality.
1.3 cDNA library characterization
The size-distribution of 1 009 individual clones derived
from the library constructed at veraison stage was
analyzed. The average total length was 550.14 bp
without any treatment. After vector sequence supperssion,
Figure 4 PCR detection of inserts size from
V. amurensis
cDNA
library on agarose gel
Note: 1
-
16: cDNA insert fragment; M: DL2000 Marker
the EST sequence was 407.17 bp long on average,
in which there were 11 vector sequences. The Poly
A tails were cut off, and then the ESTs with lengths
less than 100 bp, 35 sequences in total were
removed. Finally, 974 high quality ESTs were obtained,
representing as much as 96.53% of the total sequences.
The percentage of vector sequences and low quality
sequences was 3.74%. The total valid length ranged
between 100 bp to 595 bp, and the mean valid length
was 403.15 bp. The EST sequences described here
have been deposited into GenBank dbEST database
under accession numbers GW666520~GW667493.
710 unigenes were obtained using the phrap software,
including 97 contigs and 613 singletons. The valid
lengths of unigene sequences ranged from 102 bp to
1 024 bp with the average length of 431.35 bp.
Redundancy of individual clones in the veraison
library was 27.10%, which turned out to increase
during development (Table 1). Davies and Robinson
(2000) identified a few genes known as Grape
Ripening Induced Proteins (GRIP), the over-expression
of which led to the increased redundancy.
Table 1 General characteristics of
Vitis amurensis
veraison
pericarp ESTs
Library representation
Total ESTs
1 009
Vector ESTs
11
Low Quality ESTs
24
High Quality ESTs
974
Total valid length (bp)
392 672
Mean valid length (bp)
403.15
Contigs
97
singletons
613
Unigenes
710
Novelty (%)
72.90
Redundancy (%)
27.10
GC (%)
44.09