U.S. patent application number 13/792248 was filed with the patent office on 2013-07-11 for nucleotide sequence for columbidae gender and nucleotide primer pair for columbidae gender.
This patent application is currently assigned to KAOHSIUNG MEDICAL UNIVERSITY. The applicant listed for this patent is Kaohsiung Medical University. Invention is credited to Hsueh-Wei Chang, Chien-Chung Cheng, Yu-Chen Hung, Ying-Fang Su.
Application Number | 20130177917 13/792248 |
Document ID | / |
Family ID | 44710106 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177917 |
Kind Code |
A1 |
Chang; Hsueh-Wei ; et
al. |
July 11, 2013 |
NUCLEOTIDE SEQUENCE FOR COLUMBIDAE GENDER AND NUCLEOTIDE PRIMER
PAIR FOR COLUMBIDAE GENDER
Abstract
The invention provides a nucleotide sequence for Columbidae
gender identification, including SEQ ID NO: 9 or a complementary
sequence thereof, and also provides a nucleotide primer pair for
Columbidae gender identification, including a first primer pair of
a first primer designed within the region of the SEQ ID NO: 9 or a
complementary sequence thereof, and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof.
Inventors: |
Chang; Hsueh-Wei;
(Kaohsiung, TW) ; Cheng; Chien-Chung; (Taipei,
TW) ; Su; Ying-Fang; (Kaohsiung, TW) ; Hung;
Yu-Chen; (Tainan, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kaohsiung Medical University; |
Kaohsiung |
|
TW |
|
|
Assignee: |
KAOHSIUNG MEDICAL
UNIVERSITY
Kaohsiung
TW
|
Family ID: |
44710106 |
Appl. No.: |
13/792248 |
Filed: |
March 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12854864 |
Aug 11, 2010 |
8420316 |
|
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13792248 |
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Current U.S.
Class: |
435/6.12 ;
536/23.5 |
Current CPC
Class: |
C12Q 1/689 20130101;
C12Q 1/6879 20130101 |
Class at
Publication: |
435/6.12 ;
536/23.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2010 |
TW |
99109810 |
Claims
1. A nucleotide sequence for Columbidae gender identification,
comprising SEQ ID NO: 9 or the complementary sequence thereof.
2. The nucleotide sequence for Columbidae gender identification as
claimed in claim 1, further comprising SEQ ID NO: 10 or the
complementary sequence thereof.
3. A nucleotide primer pair for Columbidae gender identification,
comprising a first primer pair of a first primer designed within
the region of the SEQ ID NO: 9 or the complementary sequence
thereof, and a P2 primer (SEQ ID NO: 1) or the complementary
sequence thereof.
4. The nucleotide primer pair for Columbidae gender identification
as claimed in claim 3, further comprising a second primer pair of a
second primer designed within the region of the SEQ ID NO: 10 or
the complementary sequence thereof, and a P2 primer (SEQ ID NO: 1)
or the complementary sequence thereof.
5. The nucleotide primer pair for Columbidae gender identification
as claimed in claim 3, wherein the first primer pair comprises the
complementary sequence of the SEQ ID NO: 9 the P2 primer (SEQ ID
NO: 1), or SEQ ID NO: 9 and the complementary sequence of the P2
primer (SEQ ID NO: 1).
6. The nucleotide primer pair for Columbidae gender identification
as claimed in claim 4, wherein the first primer pair comprises the
complementary sequence of the SEQ ID NO: 10 and the P2 primer (SEQ
ID NO: 1), or SEQ ID NO: 10 and the complementary sequence of the
P2 primer (SEQ ID NO: 1).
7. The nucleotide primer pair for Columbidae gender identification
as claimed in claim 3, wherein the first primer pair comprises the
complementary sequence of the SEQ ID NO: 11 and the P2 primer (SEQ
ID NO: 1), or SEQ ID NO: 11 and the complementary sequence of the
P2 primer (SEQ ID NO: 1).
8. The nucleotide primer pair for Columbidae gender identification
as claimed in claim 4, wherein the first primer pair comprises the
complementary sequence of the SEQ ID NO: 12 and the P2 primer (SEQ
ID NO: 1), or SEQ ID NO: 12 and the complementary sequence of the
P2 primer (SEQ ID NO: 1).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of pending U.S. patent
application Ser. No. 12/854,864, filed Aug. 11, 2010 and entitled
"Method for Columbidae gender identification, nucleotide sequence
for Columbidae gender and nucleotide primer pair for Columbidae
gender" which claims priority to Taiwan Patent Application No.
099109810, filed Mar. 31, 2010, both of which are disclosed herein
in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] A sequence listing submitted as a text file via EFS-Web is
incorporated herein by reference. The text file containing the
sequence listing is named "0911-A51805-DUS_Seq_Listing.txt"; its
date of creation was Jan. 24, 2013; and its size is 5,229
bytes.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to bird gender identification,
and in particular relates to Columbidae gender molecular
identification.
[0005] 2. Description of the Related Art
[0006] Gender identification is especially important to monitor the
population size in areas that have decreased population trends. The
sex ratio is an important factor in breeding birds, monitoring
population growth, and detecting global changes in relationships.
However, determining the sex of a bird belonging to the Columbidae
family is difficult because most are monomorphic.
[0007] Traditionally, molecular gender identification of birds is
based on the differences in length of intron between the
chromo-helicase-DNA binding protein (CHD)-Z and CHD-W genes
amplified by the Griffiths P2/P8 primer set (Griffiths R, Double M
C, On K, Dawson R J. (1998) A DNA test to sex most birds. Molecular
Ecology 7:1071-5), i.e., males contain a single CHD-Z band (ZZ) and
females contain two bands (CHD-Z and CHD-W; ZW), after
electrophoresis. Currently, other methods such as re-designed
non-P2/P8 primers for polymerase chain reaction (Fridolfsson, A.
and Ellegren, H. (1999) A simple and universal method for molecular
sexing of non-ratite birds. Journal of Avian Biology, 30, 116-121.;
Chang, H. W., Chou, T. C., Gu, D. L., Cheng, C. A., Chang, C. C.,
Yao, C. T., Chuang, L. Y., Wen, C. H., Chou, Y. C., Tan, K. Y. et
al. (2008) An improved polymerase chain reaction method for gender
identification of eagles. Molecular and cellular probes, 22,
184-188.), polymerase chain reaction-RFLP (Sacchi, P., Soglia, D.,
Maione, S., Meneguz, G., Campora, M. and Rasero, R. (2004) A
non-invasive test for sex identification in short-toed Eagle
(Circaetus gallicus). Molecular and cellular probes, 18, 193-196.;
Reddy, A., Prakash, V. and Shivaji, S. (2007) A rapid,
non-invasive, polymerase chain reaction-based method for
identification of sex of the endangered Old World vultures
(white-backed and long-billed vultures)--Implications for captive
breeding programmes. Current Science 2007; 92:659-62),
RAPD-polymerase chain reaction (Wu, C. P., Horng, Y. M., Wang, R.
T., Yang, K. T. and Huang, M. C. (2007) A novel sex-specific DNA
marker in Columbidae birds. Theriogenology, 67, 328-333.) and
AFLP-polymerase chain reaction (Huang, C. W., Cheng, Y. S.,
Rouvier, R., Yang, K. T., Wu, C. P. and Huang, M. C. (2007) AFLP
fingerprinting for paternity testing in ducks. Br Poult Sci, 48,
323-330.) have been developed for the gender identification of
birds. However, all require the gel electrophoresis step after the
polymerase chain reaction and are not robust for gender
identification of birds in a high-throughput format.
[0008] Alternatively, melting curve analysis is capable of
simultaneously measuring the melting temperature (Tm) for many
polymerase chain reaction amplicons. The melting curve analysis
does not require the electrophoresis step; thereby saving time and
having high-throughput. Melting curve analysis is widely used in
many fields such as for the detection of methylation, SNP
genotyping and mutation, genus identification of microbials,
quantification of chromosome conformation capture. However, melting
curve analysis is seldom applied in the gender identification of
birds.
[0009] For melting curve analysis, different Tm values for the
specific polymerase chain reaction amplicons are analogous to the
different shift mobilities for the corresponding electrophoretic
bands. Amplicons with different lengths have different melting
temperatures (Tm), ie. the longer the length is, the greater the Tm
value is, and the shorter the length is, the lower the Tm value is.
Therefore, it is possible that the gender of some Columbidae
species may be identified using melting curve analysis based on the
different Tm values of their CHD-Z and CHD-W amplicons. Although
melting curve analysis may be used to determine Tm values for CHD-Z
and CHD-W amplicons, their differences in length may vary from
species to species. Accordingly, melting curve analysis for species
wherein the difference in length of intron for CHD-Z and CHD-W
amplicons is small, which results in a small Tm value difference
therebetween, is not applicable as the degree of resolution for
real-time polymerase chain reaction machines may be
insufficient.
[0010] Recently, for gender identification of three species
belonging to the Columbidae family, a female-specific primer
generated from an RAPD method has been disclosed (Wu, C. P., Horng,
Y. M., Wang, R. T., Yang, K. T. and Huang, M. C. (2007) A novel
sex-specific DNA marker in Columbidae birds. Theriogenology, 67,
328-333.). The polymerase chain reaction product for the lengths of
777-, 778-, and 770-bp, for three species belonging to the
Columbidae family such as Streptopelia orientals, Streptopelia
chinensis and Columba livia, have been respectively generated.
Also, 16S rRNA (256-bp) has been used as a polymerase chain
reaction control for dove gender identification using the
female-specific primer. However, if DNA is degraded or the quality
of DNA from samples collected during field research is poor, it may
be difficult to amplify a long polymerase chain reaction product
that required by the female-specific primer. Therefore, it is
possible that some degraded and shorter female DNA samples may not
be correctly detected using the female-specific primer.
Specifically, if there is only one copy for nucleus genes, the
number of mitochondrial may be 1000. Thus, the amount of copies of
mitochondrial genes may be about 1000 times that of the nucleus
genes. Therefore, as long as one of the 1000 mitochondrials is
complete, amplification of the 16S rRNA thereof is possible, i.e.,
a female may be regarded as a male as their 16S rRNA would be
positive and the female-specific polymerase chain reaction would be
negative.
[0011] Therefore, a high-throughput, and precisely accurate method
for Columbidae gender identification is currently needed.
BRIEF SUMMARY OF THE INVENTION
[0012] The invention provides a method for Columbidae gender
identification comprising: providing a DNA sample of a bird
belonging to the Columbidae family; performing a polymerase chain
reaction to the DNA sample with a first primer pair of a first
primer designed within the region of the SEQ ID NO: 9 or a
complementary sequence thereof and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof, and a second primer pair of a
second primer designed within the region of the SEQ ID NO: 10 or a
complementary sequence thereof and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof; and determining gender by
performing a melting curve analysis to a product from the
polymerase chain reaction, wherein the result showing two peaks
indicate a female gender and showing one peak indicate a male
gender.
[0013] The invention provides another method for Columbidae gender
identification comprising: providing a DNA sample of a bird
belonging to the Columbidae family; performing a polymerase chain
reaction to the DNA sample with a first primer pair of a first
primer designed within the region of the SEQ ID NO: 9 or a
complementary sequence thereof and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof, and a second primer pair of a
second primer designed within the region of the SEQ ID NO.: 10 or a
complementary sequence thereof and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof; and determining gender by
performing an electrophoresis analysis to a product from the
polymerase chain reaction, wherein the result showing two bands
indicate a female gender and showing one band indicate a male
gender.
[0014] The invention further provides a nucleotide sequence for
Columbidae gender identification, comprising SEQ ID NO: 9 or a
complementary sequence thereof.
[0015] The invention further provides a nucleotide primer pair for
Columbidae gender identification, comprising a first primer pair of
a first primer designed within the region of the SEQ ID NO: 9 or a
complementary sequence thereof, and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof.
[0016] A detailed description is given in the following embodiments
with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present invention can be more fully understood by
reading the subsequent detailed description and examples with
references made to the accompanying drawings, wherein:
[0018] FIGS. 1A-1C show the 3% gel electrophoresis results for
products obtained by performing polymerase chain reaction to the
DNA samples of Columba livia, Columba pulchricollis and
Streptopelia tranquebarica with P2/P8 primers, respectively;
[0019] FIGS. 1D-1F show the melting curve analysis results for
products obtained by performing polymerase chain reaction to the
DNA samples of Columba livia, Columba pulchricollis and
Streptopelia tranquebarica with P2/P8 primers, respectively;
[0020] FIGS. 2A-2C show the comparison for CHD-Z and CHD-W gene
sequences of Columba livia, Columba pulchricollis and Streptopelia
tranquebarica. The sequences of C. livia-Z, C. pulchricollis-Z and
S. tranquebarica-Z are SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7,
respectively, while the sequences of C. livia-W, C. pulchricollis-W
and S. tranquebarica-W are SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID
NO: 8, respectively. In addition, the sequence of P2 primer is SEQ
ID NO: 1 while the sequence of P8 primer [anti-sense] is SEQ ID NO:
13;
[0021] FIGS. 3A-3B show the 1.5% gel electrophoresis results for
products obtained by performing polymerase chain reaction to the
DNA samples of Columba livia, Columba pulchricollis and
Streptopelia tranquebarica with the re-designed primers of the
invention, respectively; and
[0022] FIGS. 3C-3D show the melting curve analysis results for
products obtained by performing polymerase chain reaction to the
DNA samples of Columba livia, Columba pulchricollis and
Streptopelia tranquebarica with the re-designed primers of the
invention, respectively
DETAILED DESCRIPTION OF THE INVENTION
[0023] The following description is of the best-contemplated mode
of carrying out the invention. This description is made for the
purpose of illustrating the general principles of the invention and
should not be taken in a limiting sense. The scope of the invention
is best determined by reference to the appended claims.
[0024] In the invention, polymerase chain reactions are performed
to DNA samples of Columba livia, Columba pulchricollis and
Streptopelia tranquebarica with P2 (5'-TCTGCATCGCTAAATCCTTT-3')
(SEQ ID NO: 1) (forward)/P8 (5'-CTCCCAAGGATGAGRAAYTG-3') (SEQ ID
NO: 2) (reverse) (Griffiths R, Double M C, On K, Dawson R J. (1998)
A DNA test to sex most birds. Molecular Ecology 7:1071-5),
respectively, and products from the polymerase chain reactions are
purified by electrophoresis and DNA sequenced.
[0025] In the polymerase chain reaction for the DNA samples of
Columba livia, SEQ ID NO: 3 with higher molecular weight and SEQ ID
NO: 4 with lower molecular weight are obtained. In the polymerase
chain reaction for the DNA samples of Columba pulchricollis, SEQ ID
NO: 5 with higher molecular weight and SEQ ID NO: 6 with lower
molecular weight are obtained. In the polymerase chain reaction for
the DNA samples of Streptopelia tranquebarica, SEQ ID NO: 7 with
higher molecular weight and SEQ ID NO: 8 with lower molecular
weight are obtained. After BLAST analysis, SEQ ID NOs: 3, 5 and 7
are identified to be very similar to CHD-Z gene of other birds,
while SEQ ID NOs: 4, 6 and 8 are identified to be very similar to
the CHD-W gene of other birds, and thus SEQ ID NOs: 3, 5 and 7 are
identified as the CHD-Z gene sequences of Columba livia, Columba
pulchricollis and Streptopelia tranquebarica, respectively, and SEQ
ID NOs: 4, 6 and 8 are identified as the CHD-W gene sequences of
Columba livia, Columba pulchricollis and Streptopelia
tranquebarica, respectively.
[0026] The conditions for the polymerase chain reaction may be
optionally modulated according to different circumstance; however,
preferably, the conditions may be: 95.degree. C. (4 min);
95.degree. C. (30 sec, 5 cycles); 47.degree. C. (30 sec);
72.degree. C. (30 sec); 95.degree. C. for (30 sec, 49 cycles);
46.degree. C. (20 sec); 72.degree. C. (20 sec); and 72.degree. C.
(5 min).
[0027] CHD-Z genes and CHD-W genes of different species of
Columbidae are aligned with the aid of a bioinformatics tool,
SDSC-Biology Workbench, and then a CHD-ZW common region and a CHD-W
specific region are obtained. In one embodiment, the different
species of Columbidae may comprise Columba livia, Columba
pulchricollis and Streptopelia tranquebarica. In one embodiment,
the CHD-W specific region may be the SEQ ID NO: 9 or a
complementary sequence thereof, preferably, and the CHD-ZW common
region may be the SEQ ID NO: 10 or a complementary sequence
thereof, preferably.
[0028] Next, according to the CHD-W specific region, or the CHD-W
specific region and CHD-ZW common region, the invention provides a
method for Columbidae gender identification.
[0029] In one aspect of the invention, Columbidae gender
identification is performed by a polymerase chain reaction based
melting curve analysis.
[0030] In one embodiment, first, a DNA sample of a bird belonging
to the Columbidae family may be provided, and a polymerase chain
reaction is performed to the DNA sample with a first primer pair of
a first primer designed within the region of the SEQ ID NO: 9 or a
complementary sequence thereof and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof. After that, a melting curve
analysis is performed to a product from the polymerase chain
reaction, wherein the result shows one peak indicating a female
gender and no peak indicating a male gender or that the DNA quality
is poor and undeterminable. At this time, in order to confirm that
the gender of the Columbidae is male, another embodiment in the
following may be adopted.
[0031] In another embodiment, first, a DNA sample of a bird
belonging to the Columbidae family may be provided, and a
polymerase chain reaction is performed to the DNA sample with a
first primer pair of a first primer designed within the region of
the SEQ ID NO: 9 or a complementary sequence thereof and a P2
primer (SEQ ID NO: 1) or a complementary sequence thereof, and a
second primer pair of a second primer designed within the region of
the SEQ ID NO.: 10 or a complementary sequence thereof and a P2
primer (SEQ ID NO: 1) or a complementary sequence thereof. After
that, a melting curve analysis is performed to a product from the
polymerase chain reaction, wherein the result shows the two peaks
indicating a female gender and the one peak indicating a male
gender.
[0032] Examples of the first primer pair may comprise the
complementary sequence of the SEQ ID NO: 9 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 9 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
Moreover, examples of the first primer pair may also comprise a
complementary sequence of the SEQ ID NO: 11 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 11 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
In addition, examples of the second primer pair may comprise the
complementary sequence of the SEQ ID NO: 10 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 10 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
Moreover, examples of the second primer pair may also comprise a
complementary sequence of the SEQ ID NO: 12 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 12 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1)
(reverse).
[0033] Because the female gender of a bird belonging to the
Columbidae family has both the CHD-Z gene and CHD-W gene, when the
sample is of a female gender and the first primer pair of the first
primer (within the CHD-W specific region)/P2 primer or the
complementary sequence thereof is used to perform the polymerase
chain reaction, the female DNA sample will generate one polymerase
chain reaction product segment, which is a polymerase chain
reaction product generated from the first primer pair of the first
primer (within the CHD-W specific region)/P2 primer or the
complementary sequence thereof. Therefore, when a melting curve
analysis is performed to the polymerase chain reaction product, a
melting curve will be generated with one peak. Furthermore, when
the sample is obtained from a female gender and the first primer
pair of the first primer (within the CHD-W specific region)/P2
primer or the complementary sequence thereof and the second primer
pair of the second primer (within the CHD-ZW common region)/P2
primer or the complementary sequence thereof are used
simultaneously to perform the polymerase chain reaction, the female
DNA sample will generate two polymerase chain reaction product
segments, which is a polymerase chain reaction product generated
from the first primer pair of the first primer (within the CHD-W
specific region)/P2 primer or the complementary sequence thereof
and the second primer pair of the second primer (within the CHD-ZW
common region)/P2 primer or the complementary sequence thereof,
respectively. Therefore, when a melting curve analysis is performed
to the polymerase chain reaction product, the two polymerase chain
reaction product segments will generate two melting curves (each
polymerase chain reaction product segment has one melting curve)
and have two peaks (each melting curve has one peak). In one
embodiment, the product generated from the first primer pair of the
first primer (the complementary sequence of the SEQ ID NO: 11,
within the CHD-W specific region) (reverse)/P2 primer (forward) is
about 252-b.p. and the product generated from the second primer
pair of the second primer (the complementary sequence of the SEQ ID
NO: 12, within the CHD-ZW common region) (reverse)/P2 primer
(forward) is about 104-b.p.
[0034] The male gender of a bird belonging to the Columbidae family
only has a CHD-Z gene, and thus when the first primer pair of the
first primer (within the CHD-W specific region)/P2 primer or the
complementary sequence thereof is used to perform the polymerase
chain reaction, the male DNA sample is not able to generate a
polymerase chain reaction product. Therefore, when a melting curve
analysis is performed, no melting curve will be generated with no
peaks. Furthermore, when the sample is of a male and the first
primer pair of the first primer (within the CHD-W specific
region)/P2 primer or the complementary sequence thereof and the
second primer pair of the second primer (within the CHD-ZW common
region)/P2 primer or the complementary sequence thereof are used
simultaneously to perform the polymerase chain reaction, the male
DNA sample will only generate one polymerase chain reaction product
segment, which is a polymerase chain reaction product generated
from the second primer pair of the second primer (within the CHD-ZW
common region)/P2 primer or the complementary sequence thereof.
Therefore, when a melting curve analysis is performed to the
polymerase chain reaction product, only one melting curve will be
generated with only one peak. In one embodiment, the product
generated from the second primer pair of the second primer (the
complementary sequence of the SEQ ID NO: 12, within the CHD-ZW
common region) (reverse)/P2 primer (forward) is about 104-b.p.
[0035] Since DNA of a female or a male may be used to perform a
polymerase chain reaction with the second primer pair of the second
primer (within the CHD-ZW common region)/P2 primer or the
complementary sequence thereof to generate a product, the
polymerase chain reaction performed with the second primer pair of
the second primer (within the CHD-ZW common region)/P2 primer or
the complementary sequence thereof may be used as a positive
control.
[0036] According to the differences of the primer used and
diversity of the Columbidae to be identified, the differences in
length between products from the first primer/P2 primer or the
complementary sequence thereof and the second primer/P2 primer or
the complementary sequence thereof is at least greater than abut
100-b.p. Therefore, when the two primer pairs are used to perform a
polymerase chain reaction simultaneously and then melting curve
analysis is performed, the two peaks are able to be clearly
separated. In one embodiment, the differences in length between the
product from the first primer/P2 primer or the complementary
sequence thereof (252-b.p.) and product from the second primer/P2
primer or the complementary sequence thereof (104-b.p.) is abut
148-b.p. Moreover, in one embodiment, the melting temperature
(peak) of the product from the first primer/P2 primer or the
complementary sequence thereof is about 78.5-79.5.degree. C., and
the melting temperature (peak) of the product from the second
primer/P2 primer or the complementary sequence thereof is about
77-78.degree. C. The melting temperature difference of the two
peaks may be about 0.5-2.5.degree. C., and in one embodiment is
about 1.5.degree. C.
[0037] All birds belonging to the Columbidae family may be used by
the method to identify gender thereamong. In one embodiment, a bird
belonging to the Columbidae family may comprise Columba livia,
Columba pulchricollis or Streptopelia tranquebarica.
[0038] The polymerase chain reaction may be modulated according to
the circumstances and have no particular limitations. Furthermore,
the polymerase chain reaction mentioned above may be a typical
polymerase chain reaction or a real-time polymerase chain reaction.
If a typical polymerase chain reaction is preformed, the step
needed for performing the melting curve analysis may be performed
after the polymerase chain reaction is completed. If a real-time
polymerase chain reaction is preformed, a melting curve analysis
may be performed immediately in the same machine that the
polymerase chain reaction has been preformed after the polymerase
chain reaction is completed. Compared with the typical polymerase
chain reaction method, a polymerase chain reaction based melting
curve analysis, only an additional fluorescent reagent is needed to
be added therein, such as am SYBR green I.
[0039] The method using the melting curve analysis of the invention
does not require electrophoresis analysis and has advantages of
high-throughput (96-or 384 well polymerase chain reaction may be
selected) and time savings.
[0040] In another aspect of the invention, Columbidae gender
identification is performed by a polymerase chain reaction based
electrophoresis analysis.
[0041] In one embodiment, first, a DNA sample of a bird belonging
to the Columbidae family may be provided, and a polymerase chain
reaction is performed to the DNA sample with a first primer pair of
a first primer designed within the region of the SEQ ID NO: 9 or a
complementary sequence thereof and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof. After that, an electrophoresis
analysis is performed to a product from the polymerase chain
reaction, wherein the result shows the one band indicating a female
gender and no band indicating a male gender or that the DNA quality
is poor and undeterminable. At this time, in order to confirm that
the gender of the Columbidae is male, another embodiment in the
following may be adopted.
[0042] In another embodiment, first, a DNA sample of a bird
belonging to the Columbidae family may be provided, and a
polymerase chain reaction is performed to the DNA sample with a
first primer pair of a first primer designed within the region of
the SEQ ID NO: 9 or a complementary sequence thereof and a P2
primer (SEQ ID NO: 1) or a complementary sequence thereof, and a
second primer pair of a second primer designed within the region of
the SEQ ID NO.: 10 or a complementary sequence thereof and a P2
primer (SEQ ID NO: 1) or a complementary sequence thereof. After
that, an electrophoresis analysis is performed to a product from
the polymerase chain reaction, wherein the result shows the two
bands indicating a female gender and the one band indicating a male
gender.
[0043] Examples of the first primer pair may comprise the
complementary sequence of the SEQ ID NO: 9 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 9 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
Moreover, examples of the first primer pair may also comprise a
complementary sequence of the SEQ ID NO: 11 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 11 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
In addition, examples of the second primer pair may comprise the
complementary sequence of the SEQ ID NO: 10 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 10 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
Moreover, examples of the second primer pair may also comprise a
complementary sequence of the SEQ ID NO: 12 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 12 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1)
(reverse).
[0044] Because the female gender of a bird belonging to the
Columbidae family has both the CHD-Z gene and CHD-W gene, when the
sample is of a female gender and the first primer pair of the first
primer (within the CHD-W specific region)/P2 primer or the
complementary sequence thereof is used to perform the polymerase
chain reaction, the female DNA sample will generate one polymerase
chain reaction product segment, which is a polymerase chain
reaction product generated from the first primer pair of the first
primer (within the CHD-W specific region)/P2 primer or the
complementary sequence thereof. Therefore, when an electrophoresis
analysis is performed to the polymerase chain reaction product, one
product will be generated and have one band. Furthermore, when the
sample is obtained from a female gender and the first primer pair
of the first primer (within the CHD-W specific region)/P2 primer or
the complementary sequence thereof and the second primer pair of
the second primer (within the CHD-ZW common region)/P2 primer or
the complementary sequence thereof are used simultaneously to
perform the polymerase chain reaction, the female DNA sample will
generate two polymerase chain reaction product segments, which is a
polymerase chain reaction products generated from the first primer
pair of the first primer (within the CHD-W specific region)/P2
primer or the complementary sequence thereof and the second primer
pair of the second primer (within the CHD-ZW common region)/P2
primer or the complementary sequence thereof, respectively.
Therefore, when an electrophoresis analysis is performed to the
polymerase chain reaction product, two bands will be generated. In
one embodiment, the product generated from the first primer pair of
the first primer (the complementary sequence of the SEQ ID NO: 11,
within the CHD-W specific region) (reverse)/P2 primer (forward) is
about 252-b.p. and the product generated from the second primer
pair of the second primer (the complementary sequence of the SEQ ID
NO: 12, within the CHD-ZW common region) (reverse)/P2 primer
(forward) bout 104-b.p.
[0045] The male gender of a bird belonging to the Columbidae family
only has a CHD-Z gene, and thus when the first primer pair of the
first primer (within the CHD-W specific region)/P2 primer or the
complementary sequence thereof is used to perform the polymerase
chain reaction, the male DNA sample is not able to generate a
polymerase chain reaction product. Therefore, when an
electrophoresis analysis is performed, no band will appear.
Furthermore, when the sample is of a male gender and the first
primer pair of the first primer (within the CHD-W specific
region)/P2 primer or the complementary sequence thereof and the
second primer pair of the second primer (within the CHD-ZW common
region)/P2 primer or the complementary sequence thereof are used
simultaneously to perform the polymerase chain reaction, the male
DNA sample will only generate one polymerase chain reaction product
segment, which is a polymerase chain reaction products generated
from the second primer pair of the second primer (within the CHD-ZW
common region)/P2 primer or the complementary sequence thereof.
Therefore, when an electrophoresis analysis is performed to the
polymerase chain reaction product, only one band will appear. In
one embodiment, the product generated from the second primer pair
of the second primer (the complementary sequence of the SEQ ID NO:
12, within the CHD-ZW common region) (reverse)/P2 primer (forward)
is about 104-b.p.
[0046] On the other hand, since the DNA of a female or male gender
both, are able to be used to perform a polymerase chain reaction
with the second primer pair of the second primer (within the CHD-ZW
common region)/P2 primer or the complementary sequence thereof to
generate a product, the polymerase chain reaction performed with
the second primer pair of the second primer (within the CHD-ZW
common region)/P2 primer or the complementary sequence thereof may
be used as a positive control.
[0047] According to the differences of the primer used and
diversity of the Columbidae to be identified, the differences in
length between products from the first primer/P2 primer or the
complementary sequence thereof and the second primer/P2 primer or
the complementary sequence thereof is at least greater than abut
100-b.p. Therefore, when the two primer pairs are used to perform a
polymerase chain reaction simultaneously and then electrophoresis
analysis is performed, the two bands are able to be clearly
separated. In one embodiment, the differences in length between the
product from the first primer/P2 primer or the complementary
sequence thereof (252-b.p.) and product from the second primer/P2
primer or the complementary sequence thereof (104-b.p.) is abut
148-b.p.
[0048] All birds belonging to the Columbidae family may be used by
the method to identify gender thereamong. In one embodiment, a bird
belonging to the Columbidae family may comprise Columba livia,
Columba pulchricollis or Streptopelia tranquebarica.
[0049] The polymerase chain reaction may be modulated according to
the circumstances and have no particular limitations.
[0050] According to the foregoing, it is known that the gender of a
bird belonging to the Columbidae family may be identified by using
the CHD-W specific region, or CHD-W specific region and CHD-ZW
common region. Accordingly, the invention may further provide a
nucleotide sequence for Columbidae gender identification which may
comprise a CHD-W specific region and CHD-ZW common region CHD-W
specific region, or CHD-W specific region and CHD-ZW common region.
In one embodiment, a nucleotide sequence for Columbidae gender
identification provided by the invention may comprise SEQ ID NO: 9
or a complementary sequence thereof, or may further comprise SEQ ID
NO: 10 or a complementary sequence thereof.
[0051] According to the foregoing, the invention may further
provide a nucleotide primer pair for Columbidae gender
identification, which may comprise a first primer pair of a first
primer designed within the region of the SEQ ID NO: 9 or a
complementary sequence thereof, and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof, or may further comprise a second
primer designed within the region of the SEQ ID NO: 10 or a
complementary sequence thereof, and a P2 primer (SEQ ID NO: 1) or a
complementary sequence thereof.
[0052] Examples of the first primer pair may comprise the
complementary sequence of the SEQ ID NO: 9 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 9 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
Moreover, examples of the first primer pair may also comprise a
complementary sequence of the SEQ ID NO: 11 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 11 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
In addition, examples of the second primer pair may comprise the
complementary sequence of the SEQ ID NO: 10 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 10 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1) (reverse).
Moreover, examples of the second primer pair may also comprise a
complementary sequence of the SEQ ID NO: 12 (reverse) and the P2
primer (SEQ ID NO: 1) (forward), or SEQ ID NO: 12 (forward) and the
complementary sequence of the P2 primer (SEQ ID NO: 1)
(reverse).
EXAMPLE
[0053] 1. Sample Source and DNA Extraction
[0054] Muscle tissue samples for male-female paired Columba livia
(females (Bd90 and Bd91) and males (Bd92 and Bd93)), Columba
pulchricollis (female (Bd5137) and males (Bd3314, Bd4970, and
Bd5417)), and Streptopelia tranquebarica (female (Bd101) and male
(Bd102)), which died due to collision, were collected from the
Taiwan Endemic Species Research Institute. Following, the genders
of the samples were determined by anatomical inspection. Next,
tissue DNAs were extracted by a DNeasy tissue kit (Qiagen,
Valencia, Calif., USA) according to instructions.
[0055] 2. Primary Molecular Gender Identification by Griffiths
P2/P8 Primer Set Polymerase Chain Reaction
[0056] The P2 (SEQ ID NO: 1) (forward)/P8 (SEQ ID NO: 2) (reverse)
primers (Griffiths) for molecular gender identification of birds
were used for the three species belonging to the Columbidae family.
A polymerase chain reaction cocktail contained a 1.times.
polymerase chain reaction buffer, 0.16 .mu.M primers, 0.2 mM dNTPs,
0.7U Platinum-Taq enzyme (Invitrogen), 1.5 mM MgCl.sub.2, SYBGreen
I (1:2000; Invitrogen), and 10-20 ng DNA in a total volume of 10
.mu.l. The polymerase chain reaction program was as described:
95.degree. C. (4 min); 95.degree. C. (30 sec, 5 cycles); 47.degree.
C. (30 sec); 72.degree. C. (30 sec); 95.degree. C. for (30 sec, 49
cycles); 46.degree. C. (20 sec); 72.degree. C. (20 sec); and
72.degree. C. (5 min).
[0057] (1) Electrophoresis Analysis
[0058] The polymerase chain reaction products (CHD-Z and CHW
amplicons) were used to perform an electrophoresis analysis, and
the results are shown as FIGS. 1A-1C. In FIGS. 1A to 1C, the three
species belonging to the Columbidae family (Columba livia, Columba
pulchricollis and Streptopelia tranquebarica. C. livia) in 3% gel
electrophoresis, display that those of a female gender had two
specific bands representing the CHD-Z (top) and CHD-W (bottom) and
those of a male gender only had one specific band representing the
CHD-Z.
[0059] (2) Melting Curve Analysis
[0060] Following completion of the polymerase chain reaction, a
melting curve was analyzed with a default program of the iQ.TM.5
real-time polymerase chain reaction machine (Bio-Rad Laboratories,
Hercules, Calif.), i.e., 55.degree. C. to 95.degree. C. with a
heating rate of 0.5.degree. C./s for 80 repeated counts. Melting
curve data were represented as -dF/dT vs. T (F and T are regarded
as the fluorescence and temperature). The melting temperature (Tm;
the temperature corresponding to the peak for -dF/dT) for each
polymerase chain reaction product was determined by a Bio-Rad iQ5
default software. RFU represents relative fluorescence unit.
[0061] For the melting curve analysis of the P2/P8 polymerase chain
reaction amplicons (CHD-Z and/or CHD-W genes) of the species of the
Columbidae, there was only one Tm peak at about 80.degree. C.,
which was undistinguishable for both the female and male gender of
the birds belonging to the Columbidae. The results are shown as
FIGS. 1D to 1F. FIGS. 1D to 1F are the results for Columba livia,
Columba pulchricollis and Streptopelia tranquebarica, respectively
and show Tm values of P2/P8 amplicons of both the female gender
(Bds 91, 5137 and 101) and male gender (Bds 93 and 5417) for C.
livia, C. pulchricollis, and S. tranquebarica were similar (80.0 to
80.5.degree. C.). Therefore, melting curve analysis could not be
performed when using the P2/P8 primer set when the gender of the
Columba livia, Columba pulchricollis and Streptopelia tranquebarica
were tested.
[0062] 3. Sequencing of the CHD-Z and CHD-W Amplicons
[0063] For sequencing, DNA in the gel mentioned above was extracted
by a gel extraction kit (Qiagen). Then the sequencing was
performed. The results for the sequencing are shown in FIGS. 2A-2C.
Referring to FIGS. 2A-2C, SEQ ID NO: 3 (C. livia-Z) with higher
molecular weight and SEQ ID NO: 4 (C. livia-W) with lower molecular
weight for Columba livia were obtained, SEQ ID NO: 5 (C.
pulchricollis-Z) with higher molecular weight and SEQ ID NO: 6 (C.
pulchricollis-W) with lower molecular weight for Columba
pulchricollis were obtained, SEQ ID NO: 7 (S. tranquebarica-Z) with
higher molecular weight and SEQ ID NO: 8 (S. tranquebarica-W) with
lower molecular weight for Streptopelia tranquebarica were
obtained. Moreover, the sequence of P2 primer in FIG. 2A is SEQ ID
NO: 1, the sequence of P8 primer [anti-sense] in FIG. 2C is SEQ ID
NO: 13.
[0064] 4. Calculation of the Differences in Length of Intron
between CHD-Z and CHD-W Amplicons
[0065] Sequences were aligned with the aid of a bioinformatics
tool, SDSC-Biology Workbench. The results are shown in FIGS. 2A-2C.
The differences in length of intron between the CHD-Z and CHD-W
amplicons using a Griffiths P2/P8 primer set for each species was
calculated as described in Chang, H. W., Gu, D. L., Su, S. H.,
Chang, C. C., Cheng, C. A., Huang, H. W., Yao, C. T., Chou, T. C.,
Chuang, L. Y. and Cheng, C. C. (2008) High-throughput gender
identification of Accipitridae eagles with real-time polymerase
chain reaction using TaqMan probes. Theriogenology, 70, 83-90. In
brief, the deleted regions (indicated by dash lines in FIGS. 2A-2C)
within the sequences for the CHD-Z and CHD-W amplicons were
calculated from the pre-aligned sequences of the same species.
[0066] CHD-Z and CHD-W genes of Columba livia, Columba
pulchricollis and Streptopelia tranquebarica are represented as
CHD-ZICHD-W (the differences in length between the two genes).
Columba livia: SEQ ID NO: 3/SEQ ID NO: 4 (20-bp), Columba
pulchricollis: SEQ ID NO: 5/SEQ ID NO: 6 (21-bp), and Streptopelia
tranquebarica: SEQ ID NO: 7/SEQ ID NO: 8 (18-bp).
[0067] After comparing every gene sequences, differences in length
of polymerase chain reaction products for CHD-Z and CHD-W gene
obtained by using the P2/P8 primers was only about 18-21 b.p.,
which indicated that the P2/P8 primers were not suitable for
Columbidae gender identification.
[0068] In addition, according to the sequence alignment mentioned
above, a CHD-W specific sequence was obtained and named as a CHD-W
specific region which is SEQ ID NO: 9 as shown as a black box
region 1 of FIG. 2B, a CHD-ZW common sequence was obtained and
named as a CHD-ZW common region which is SEQ ID NO: 10 as shown as
a black box region 2 of FIG. 2A.
[0069] 5. Secondary Molecular Gender Identification Using
Re-Designed Primers
[0070] To solve the intrinsic problems of performing melting curve
analysis (FIG. 1) in gender identification using a P2/P8 primer
set, a polymerase chain reaction reverse primer for the CHD-W
specific region and the CHD-ZW common region for the three species
belonging to the Columbidae family were re-designed within the
CHD-W specific region (SEQ ID NO: 9) and CHD-ZW common region (SEQ
ID NO: 10), respectively to increase differences in length between
the polymerase chain reaction products. The reverse primer for the
CHD-W specific region was SEQ ID NO: 11, and the reverse primer for
the CHD-ZW common region was SEQ ID NO: 12. The primer designed
within the CHD-W specific region/P2 primer were designed to be
female-specific and the primer designed within the CHD-ZW common
region/P2 primer were regarded as the positive polymerase chain
reaction control for both female and male gender identification.
The polymerase chain reaction products amplified by SEQ ID NO: 11
(within the CHD-W specific region) primer (reverse)/P2 (forward)
were 252-bp, and SEQ ID NO: 12 (within the CHD-ZW common region)
primer (reverse)/P2 (forward) were 104-bp.
[0071] Polymerase Chain Reaction
[0072] The polymerase chain reaction program was slightly modified
in comparison to the primary molecular gender identification
(mentioned above) as follows: denaturation (95.degree. C., 3 min),
denaturation (95.degree. C., 30 sec, 50 cycles), annealing
(58.degree. C., 30 sec), and extension (72.degree. C., 15 sec).
[0073] (1) Electrophoresis Analysis
[0074] To test the performance of re-designed sex-specific primers,
a polymerase chain reaction was amplified by the primer designed
within the CHD-W specific region/P2 primer and the primer designed
within the CHD-ZW common region/P2 primer in different polymerase
chain reaction-wells and the polymerase chain reaction amplicons
were examined in 1.5% agarose gel. The results are shown as FIGS.
3A and 3B, respectively. The female birds (CHD-ZICHD-W type), such
as Bds 90, 91, 5137, and 101, were correctly amplified for both
polymerase chain reaction reactions using the primer designed
within the CHD-W specific region/P2 primer and the primer designed
within the CHD-ZW common region/P2 primer. However, the male birds
(CHD-Z/CHD-Z type) were only correctly amplified for the primer
designed within the CHD-ZW common region/P2 primer but not for
polymerase chain reaction using the primer designed within the
CHD-W specific region/P2 primer. Although some of the male genders
(Bds 4970 and 5417) showed some non-specific bands with incorrect
sizes using the primer designed within the CHD-W specific region/P2
primer (indicated by star in FIG. 3B), the non-specific polymerase
chain reaction products were sequenced and performed BLAST for
identification that they were not homologous to CHD genes (data not
shown).
[0075] (2) High-Throughput Molecular Gender Identification of the
Three Species Belonging to the Columbidae family--Melting Curve
Analysis
[0076] Following completion of the polymerase chain reaction, a
melting curve was analyzed with the default program of the iQ.TM.5
real-time polymerase chain reaction machine (Bio-Rad Laboratories,
Hercules, Calif.), i.e., 55.degree. C. to 95.degree. C. with a
heating rate at 0.5.degree. C./s for 80 repeated counts. Melting
curve data were represented as -dF/dT vs. T (F and T are regarded
as the fluorescence and temperature). The melting temperature (Tm;
the temperature corresponding to the peak for -dF/dT) for each
polymerase chain reaction product was determined by a Bio-Rad iQ5
default software. RFU represents relative fluorescence unit.
Melting curve analysis data were duplicately experimented. The
results are shown in FIGS. 3C and 3D.
[0077] In FIGS. 3C and 3D, the melting curve analysis for the same
polymerase chain reaction (FIGS. 3A and 3B) was performed. In FIG.
3C, four females (Bds 90, 91, 5137 and 101) were used to perform a
melting curve analysis and the result showed that the four
individual females of the three species belonging to the Columbidae
family were all determined to be of a female gender because they
displayed both peaks for the primer designed within CHD-W specific
region/P2 primer and the primer designed within the CHD-ZW common
region/P2 primer (W:79.0-79.5.degree. C.; ZW: 77.5.degree. C). In
contrast, six males (Bds 92, 93, 3314, 4970, 5417 and 102)) were
used to perform a melting curve analysis and the result showed that
the six individual females of the three species belonging to the
Columbidae family were all determined to be of a male gender
because they displayed a single peak for the primer designed within
the CHD-ZW common region/P2 primer (ZW: 77.5.degree. C.).
[0078] For certain males (Bds 4970 and 5417), non-specific
polymerase chain reaction amplification using the primer designed
within CHD-W specific region/P2 primer were found but were
discriminated to the primer designed within CHD-W specific
region/P2 primer and the primer designed within the CHD-ZW common
region/P2 primer products by their different Tm values (77.5 and
79.0-79.5 vs. 82.0.degree. C. (FIG. 3D).
[0079] Using the re-designed sex-specific primers, the primer
designed within CHD-W specific region/P2 primer and primer designed
within the CHD-ZW common region/P2 primer, the differences in
length between two polymerase chain reaction amplicons for the
species belonging to the Columbidae family were extended to
148-b.p. (FIGS. 2A-2C) to resolve the different curves resulted
from the melting curve analysis (FIG. 3).
[0080] Although nonspecific amplification was observed in the
primer designed within the CHD-W specific region/P2 primer for some
male samples (FIG. 3), it was still easy to distinguish the
nonspecific amplicon with the target polymerase chain reaction
amplicons based on the Tm values.
[0081] Accordingly, the re-designed sex-specific primers of the
invention may be used to perform a method for Columbidae gender
high-throughput identification using melting curve analysis.
[0082] While the invention has been described by way of example and
in terms of the preferred embodiments, it is to be understood that
the invention is not limited to the disclosed embodiments. To the
contrary, it is intended to cover various modifications and similar
arrangements (as would be apparent to those skilled in the art).
Therefore, the scope of the appended claims should be accorded the
broadest interpretation so as to encompass all such modifications
and similar arrangements.
Sequence CWU 1
1
13120DNAArtificial SequenceP2 Primer (forward primer) 1tctgcatcgc
taaatccttt 20220DNAArtificial SequenceP8 Primer (reverse primer)
2ctcccaagga tgagraaytg 203370DNAColumba livia 3tctgcatcgc
taaatccttt aatattttct cgaggaatgg ttcgtggtct tccacgtttt 60tttggccgtt
ttctttctga tatggagtca ctatcagatc cagagtatct tctgctccta
120ctgcgccttc cttcacttcc attaaagctg atctggaatt tcagaataag
tagttcaaag 180ctatgcgatt gacaaacaca ggtcaagttt tgcctaacct
gtcaaaaata cgtgttcaga 240aaacggaaaa aaccctaaaa aaacaaaacc
caacaacaat ccccaacaaa ctaaaccaac 300agcaacacaa aagcacaagt
caatcagaac caagacacac ctgttttgca cagttcctca 360tccttgggag
3704350DNAColumba livia 4tctgcatcgc taaatccttt aatattttct
cgaggaatag ttcgtggtcg tccacgtttt 60tttggtcgtt ttctttctga catggagtca
ctatcagatc cagaatatct tctgcttcta 120ctgcatttcc cttcacttcc
attaaagctg atctggaatt tcagattaag tagttcaaag 180ctatgtgact
aaaacatttt aataatgtgc tatctagcct gtcaaaaatg gggggtgaaa
240agtacaagcc aaaaacaaca gtaatgaaaa aacaaagaaa cacaacaaca
agaagttagt 300tggtcaaaac ccagagatac ctgttttgca cagttcctca
tccttgggag 3505370DNAColumba pulchricollis 5tctgcatcgc taaatccttt
aatattttct cgaggaatgg ttcgtggtct tccacgtttt 60tttggccgtt ttctttctga
tatggagtca ctatcagatc cagagtatct tctgctccta 120ctgcgccttc
cttcacttcc attaaagctg atctggaatt tcagaataag tagttcaaag
180ctacgcgatt gacaaacaca ggtcaagttt tgcctaacct gtcaaaaata
cgtgttcaga 240aaacggaaaa aaccctaaaa aaccaaaacc caacaacaat
ccccaacaaa ttaaaccaac 300agcaacacaa aagcacaagt caatcagaac
caagagacac ctgttttgca cagttcctca 360tccttgggag 3706349DNAColumba
pulchricollis 6tctgcatcgc taaatccttt aatattttct cgaggaatag
ttcgtggtcg tccacgtttt 60tttggtcgtt ttctttctga gatggagtca ctatcagatc
cagaatatct tctgcttcta 120ctgcatttcc cttcgcttcc attaaagctg
atctggaatt tcagattaag tagttcaaag 180atatgtgact aaaacatttt
aataatgtgc tatctagcct gtcaaaaatg gggggtgaaa 240agtacaagcc
aaaacaacag taatgaaaaa acaaacaaac acaacaacaa gaagttagtt
300ggtcaaaacc cagagatacc tgttttgcac aatttctcat ccttgggag
3497367DNAStreptopelia tranquebarica 7tctgcatcgc taaatccttt
aatattttct cgaggaatgg ttcgtggtct tccacgtttt 60tttggccgtt ttctttctga
tatggagtca ctatcagatc cagagtatct tctgctccta 120ctgcgccttc
cttcacttcc attaaagctg atctggaatt tcagaataag tagttcaaag
180ctacgcgatt gacaaacaca ggtcaagttt tgcctaacct gtcaaaaata
tgtgttcaga 240aaacggaaaa aaaactaaaa aaacaaaacc caacaacccc
caacaaacta aaccaacagc 300aacacaaaag cacaagtcaa tcagaaccaa
gagacacctg ttttgcacag tttctcatcc 360ttgggag 3678349DNAStreptopelia
tranquebarica 8tctgcatcgc taaatccttt atgcatttct cgaggaatag
ttcgtggtcg tccacgtttt 60tttggtcgtt ttctttctga gatggagtca ctatcagatc
cagaatatct tctgcttcta 120ctgcatttcc cttcgcttcc attaaagctg
atctggaatt tcagattaag tagttcaaag 180ctatgtgact aaaacatttt
aataatgtgc tatctagcct gtcaaaaatg gggggtgaaa 240agtacaagcc
aaaacaacag taatgaaaaa acaaacaaac acaacaacaa gaagttagtt
300ggtcaaaacc caaagatacc tgttttgcac agttcctcat ccttgggag
349938DNAArtificial SequenceCHD-W specific region or primer for
CHD-W specific region 9tggggggtga aaagtacaag ccaaaaacaa cagtaatg
381051DNAArtificial SequenceCHD-ZW common region or primer for
CHD-ZW common region 10cgttttcttt ctgabatgga gtcactatca gatccagarg
tatcttctgc t 511120DNAArtificial SequencePrimer for CHD-W specific
region 11gggtgaaaag tacaagccaa 201223DNAArtificial SequencePrimer
for CHD-ZW common region 12atggagtcac tatcagatcc aga
231320DNAArtificial SequenceP8 Primer (anti-sense) 13carttyctca
tccttgggag 20
* * * * *