Page 9 - BM 2010 Vol.1 No.1

Bioscience Methods
BM 2010, Vol.1, No.1 http://bm.sophiapublisher.com
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false-positive/negatives will be produced in the
screening process.
The fairly small size of CN population has been the
bottle-neck which limit the progress and hinder the
whole process for the not large enough population
for mapping. It is of great difficulty to score for
phenotype, not only for the time-consuming and
labor-intensive process of the CN resistance
evaluation, but also for the difficulty to distinguish
among the individuals by present evaluation
methods. If some new techniques were developed to
quicken up the screening for CN resistance, it
would be a great progress. In banana, a biochemical
method ‘butanol/HCl’ assay of condensed tannin' was
suggested for screening of resistance to R.similes
(Collingborn et al., 2000). This would be a brand-new
way for future efforts to solve the problem of CN
resistance evaluation.
2.4 The potential for applications of these
specific markers
Much works confirmed that the NBS-LRR class
resistance genes inherited from Poncirus trifoliate
were heterozygous alleles in the progenitor and the
F1 progeny from it were segregating. However, only
a few F1 hybrids from P. trifoliata have inherited the
CN resistance. Among the citrumelo hybrids (C.
paradisi Macf.×P. trifoliata) only Swingle
citrumelo was reported to be highly resistant to T.
semipenetrans (O'Bannon and Ford, 1977). The
hybrid Carrizo citrange, having been utilized in
Florida for its tolerance to the burrowing nematode
(Radopholus similes Cobb), is susceptible to the
citrus nematode. Therefore, the genes conferring
resistance to citrus nematode by the Tyr1 locus is
completely different from the genes conferring
resistance the burrowing nematode. P. trifoliata had
been and has been the only genetic donator to the
resistance to citrus nematode. Lately, several new
selections identified to be highly resistant to T.
semipenetrans were also from F1 progeny of
Poncirus trifoliata (Galeano et al., 2003). Although
the present linkage map is not well saturated with
molecular markers, a few of the RGC-derived
specific markers actually provide enough
information for us to go MAS. As presented in
much of the species and rootstocks screening results,
the reliability and repeatability of the RGC-derived
markers had been confirmed, especially the more
specific marker 7A4(1407) and 7A4(2168), derived
from the NBS-LRR sequences of BAC clone insert,
proved to be highly reproducible in the rootstock
resistance screening evaluation, are the better
candidates for making MAS feasible in citrus
rootstock breeding.
3 Materials and Methods
3.1 Plant materials
Intergeneric hybrids including 62 individuals,
designated‘9145 family’, from LB6-2 [Clementine
mandarin ( C. reticulata)×Hamlin orange (C.
sinensis)]×Swingle citrumelo (C. paradisi×P.
trifoliata ) was used as the linkage group of the
major gene Tyr1 with molecular markers for their
well defined characterization of nematode
resistance (Ling et al., 2000). 225 individuals out of
687 offspring from a larger population backcross
between DPI 4-5 [‘Nakon’pummelo ( Citrus grandis
L.)] and USDA 17-47 [‘Thong Dee’pummelo
( Citrus grandis L.) × Pomeroy trifoliate orange (P.
trifoliata )], designated “9401 family”, was also
utilized here to screen the segregation of the newly
developed markers and to determine the genetic
distance of Ctv and Tyr1.
3.2 DNA extraction and bulked segregant
analysis
Genomic DNAs of each individual were extracted
from fully expanded tender leaves, following a
procedure described previously (Durham et al.,
1992). Two pairs of DNA bulks were constructed
based on the analysis results of Ling et al. (2000).
Each pair of DNA bulks was generated by randomly
pooling the extracted DNAs from the 6 to 12
individuals comprising each extreme of the
phenotype distribution. For the CN resistant bulk
(R1 and R2) and susceptible bulk (S1 and S2), DNA
samples were pooled at equal ratios and diluted to
10 ng/μL.
3.3 High-density colony screening and characteri-
zation of Pt8 and Pt9 positive BAC clones
To set up the relationship of nematode resistance