Page 5 - BM 2010 Vol.1 No.1

Bioscience Methods
BM 2010, Vol.1, No.1 http://bm.sophiapublisher.com
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(designated as 18P33a, Pt9a and pt8a) were
associated with Ctv, and one fragment (Pt8a) was
linked closely with the Tyr1.
In this study, we selected the Pt8a and Pt9a positive
BAC clones from USDA 17-47 BamH Ⅰ BAC
library, sequencing and redesigning primers to
screen the ‘ 9145 family’ and plus analysis by
BSA, newly developed markers to integrate into the
genetic map of citrus nematode resistance loci of
Tyr1, and three NBS-LRR class gene sequences
were obtained by Pt9 fragment based BAC clone
primer-walking, from which more specific markers
closely linked with Tyr1 were developed and its
improved genetic map was constructed.
1 Results
1.1 Identification of BAC clones using Pt8a and
Pt9a as probes
Over 200 positive BAC clones were screened out of
the BamHⅠ BAC library, using Pt8a and Pt9a as
probes, under moderate stringency hybridization
condition. From which, 23 clones were selected by
PCR amplification with ‘Pt9F+R’ according to
the length of the products and 28 clones were
selected with ‘Pt8F+R’. Over ten clones were
finally chosen to sequence their insert's ends after
amplification of the Pt8a and Pt9a fragments from
these clones and polymorphisms were revealed
between the nematode resistant and susceptible
plants by restriction digestion of the amplified
product with specific cutter.
1.2 Identification of SCARs and CAPS markers
linked to CN resistant locus “TyrI”
The new primers designed from the insert end
sequences were used in the bulked segregation
analysis of the‘9145 family’. Fourteen pairs of primers
(Table 1) from seven clones (3p21, 45b9, 4L17, 34d12,
40b15, 7A4 and 63F7) were found the kind of
polymorphisms existed in CN family by BSA at
first. But, after further screening among the‘9145
family’, several pairs of primers from clone 34d12,
63F7 were taken out, which are not matching up
with the phenotypes of known resistant or
susceptible individuals. Some kind of departure
from the‘ Tyr1’locus were observed in the bulked
segregant analysis of ‘4L17L/HindⅢ’(Figure 1C),
indicating that the marker was not tightly linked with
the CN resistance gene.
After southern hybridization, Pt8a and Pt9a
fragment were present as single or two copies in
4L17, 29F20 and 7A4 BAC clones and they were
chosen to go sequencing by primer walking. From
the 7A4 clone, a 6 319 bp length sequence
(GenBank accession number: AY336943) was
obtained from the both sides of the ‘Pt9F+R’
fragment. BLAST searches of the GenBank
database revealed high sequence similarities
between the ORF domains of the 6 319 bp length
sequence and known plant disease resistance genes.
A series of new, more specific primers were
designed based on the new sequence, mainly from
the ORF domains. Several pairs of primers have
been selected as the more specific markers to the TyrI
locus after doing BSA, screening the ‘9145 family’
and other known- phenotype populations or
individuals, among which the ‘7A4(1407)/BfaⅠ’
and ‘ 7A4(2168)/BfaⅠ’are the better candidates.
As shown in Figure 1B, the polymorphic band from
‘ 7A4(1407)/BfaⅠ’ was clearly revealed and the
difference were easier to be judge from, and their
reliability and repeatability were also confirmed
under the standard protocol and ordinary lab condition.
1.3 Linkage-map construction
A genetic linkage map of citrus nematode resistance
with 9 previously obtained markers and 10 newly
developed markers (Table 1) were constructed by
JoinMap 2.0 (Figure 2C). The nine previously
developed markers were selected out by BSA of
‘9145 family’ and their segregation data were
obtained by screening all the individuals of the
family. All the previously developed markers were
mainly served as anchoring markers in the mapping
population. Among these markers, OPX10, OPO04,
OPO07, OPW14 were RAPD markers, SCO07,
SCAD08, SCAm02, SCT08 were SCAR markers
origionally from RAPD markers, Pt8a was locus
co-segregating with Tyr1 (Ling et al., 2000, Deng et
al., 2001). Y65F+R marker was newly developed
from Ctv candidate genomic sequence.