Page 10 - BM 2010 Vol.1 No.1

Bioscience Methods
BM 2010, Vol.1, No.1 http://bm.sophiapublisher.com
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locus Tyr1 with the newly found NBS-LRR class
genetic markers (Deng et al., 2000), BAC clones
were separately screened with the probe of Pt8a and
Pt9a fragment. The BAC library used was
constructed from BamH Ⅰ -partially digested
high-molecular weight genomic DNA of USDA
17-47 and consisted of more than 24 000 clones
with an average insert size of 115 kb. Sixteen
high-density colony filters of citrus BAC clones
were screened as described previously (Deng et al.,
2001). BAC DNA of positive clones was prepared
using a modified alkaline procedure described by
Zhang et al. (1996). DNA preps were digested with
Hind Ⅲ and BamHⅠ at 37℃ for 4 h, and run on
1% agarose gels in 1×TAE Buffer. The gels were
stained with ethidium bromide and documented.
DNA fragments in gels were transferred onto nylon
membranes under alkaline conditions. Southern
hybridization was conducted by using DIG
(digoxigenin)-labeled probes. Manufacturer's
recommendations were followed in probe labeling,
hybridization, and detection (Roche Diagnostics
Corporation, Indianapolis, IN, USA).
3.4 Primer design, PCR amplification, and
restriction digestion
Both ends of the inserts from the selected clones
were sequenced by the pair of primers (T7 and
BAC4). New primers were designed from the end
sequence using OLIGO 6. Amplifications were
performed on a GeneAmp PCR System 9700
thermal cycler (PE Applied Biosystem) in a 15μL
reaction volume; each reaction contained 10
mmol/L Tris-HCl (pH 8.3), 2.0 mmol/L MgCl2, 0.2
mmol/L dNTPs, 10 pmol of each forward and
reverse primers, 0.6 unit of Taq polymerase,
100~150 ng genomic DNA. The initial denaturation
was at 93℃ for 2 min, followed by 42 cycles of 1
min at 92 ℃ , 1 min at 50~60℃ (depend on the
primers combination), and 2 min at 72℃. When
restriction digestions were required to reveal
polymorphisms between the two parents, 12 μL of
each PCR reaction was incubated at 37℃ 3~16 h
with 3~5 units of restriction enzymes in a 18 μL
volume. PCR products or their digests were
separated on 1.6% agarose gels. The gels were
mixed up with ethidium bromide at 500 ng/mL
before electrophoresis and then visualized with
ultraviolet image system. Markers defined by a pair
of specific primers that based on the sequencing of
the two ends of genomic clones were referred as
sequence characterized amplified regions (SCARs)
markers (Williams et al., 1991). Markers defined by
a pair of specific primers and restriction digestion
enzymes were referred as CAPS markers
(Konieczny and Ausubel, 1993). Fragments
associated with citrus nematode resistance were
identified using the BSA approach as described
above, firstly by BSA and secondly as in
accordance with the individuals' nematode
resistance phenotype.
3.5 Primer walking-based sequencing of BACs
BAC DNA was prepared from 1 000 mL of cell
culture, using the Qiagen plasmid maxi kit (Qiagen,
Valencia, CA, USA). Sequencing was performed by
the UF-DSCL, as described previously. Initial
sequence was obtained with primer Pt8(F+R) and
Pt9(F+R). From this and subsequent new sequences,
primers were designed with the assistance of
computer program OLIGO 6 and used to walk on
BACs for sequencing in both directions. After
primer walking, numerous new primers were
designed from the newly obtained sequence to
screen the‘9145 family’ and plus BSA analysis to
develop the better, more specific marker with length
of over 1 000 bases.
3.6 Map construction with the Join-Map 2.0
A total of nineteen molecular markers closely linked
to Tyr1, including four RAPD markers (Gmitter et
al., 1996) and four SCARs markers(Deng et al.,
1997) were used to screen the 9145 family, and
eight of them to screen ‘9401 family’. Based on the
segregation data of the above markers separately
in‘9145 family’ and ‘9401 family’, marker orders
and genetic distances were calculated with JoinMap
version 2.0, using a LOD value threshold of 3.0,
recombination threshold value of 0.45, jump
threshold of 1.0 and triplet threshold of 7.0. The
Kosambi function was used to convert
recombination units into genetic distance.