Download Sequence Variation and Gene Duplication at

ª The American Genetic Association. 2005. All rights reserved.
For Permissions, please email: [email protected].
Journal of Heredity 2005:96(4):310–317
doi:10.1093/jhered/esi055
Advance Access publication April 20, 2005
Sequence Variation and Gene
Duplication at MHC DQB Loci of Baiji
(Lipotes vexillifer), a Chinese River
Dolphin
G. YANG, J. YAN, K. ZHOU,
AND
F. WEI
From the Jiangsu Key Laboratory for Bioresource Technology, College of Life Sciences, Nanjing Normal University, Nanjing
210097, China (Yang, Yan, and Zhou); Institute of Genetic Resources, Nanjing Normal University, Nanjing 210097, China
(Yang, Yan, and Zhou); and Institute of Zoology, Chinese Academy of Science, Beijing 100081, China (Wei).
Address correspondence to G. Yang at the address above, or email: [email protected].
Abstract
The major histocompatibility complex (MHC) is a fundamental part of the vertebrate immune system, and the high
variability in many MHC genes is thought to play an important role in the recognition of parasites. Baiji (Lipotes vexillifer) is
one of the most endangered species in the world. Its wild population has declined to fewer than 100 individuals and has
a very high risk of becoming extinct in the near future. In this study we present a first step in the molecular characterization
of a DQB-like locus of baiji by nucleotide sequence analysis of the polymorphic exon 2 segments. In the examined 172 bp
sequences from a group of 18 incidentally captured or stranded individuals, 48 variable sites were determined and 43 alleles
were identified, many of which were represented by only one clone. Three to seven alleles were found in each individual,
suggesting gene duplications. No deletion, insertion, or exceptional stop codon was detected, suggesting these alleles
function in vivo. Phylogenetic reconstruction using neighbor joining grouped the 43 alleles into two distinct lineages,
differing by seven nucleotides and four amino acids. Substitutions of amino acids tend to be clustered around sites postulated
to be responsible for selective peptide recognition. In the peptide-binding region (PBR) of the DQB locus, the average number
of nonsynonymous substitutions per site is greater than that of synonymous substitutions per site (0.1962 versus 0.0256,
respectively). Nucleotide and amino acid sequences both showed a relatively high level of similarity (nucleotides 90.6%; amino
acids 80.6%) to those of beluga whale (Delphinapterus leucas) and narwhal (Monodon monoceros). The high level of baiji MHC
polymorphism revealed in the present study has not been reported in other cetaceans and could be a consequence of the small
baiji population adapting to freshwater with a relatively high level of pathogens.
The baiji (Lipotes vexillifer) is one of five extant river dolphins
and inhabits the middle and lower reaches of the Yangtze
River. Humans have caused the baiji’s population to
dramatically decline to less than 100 individuals in the past
decades by altering and degrading habitat, hunting, polluting
the environment, increasing vessel traffic, and overfishing
(Zhou et al. 1998). This species has been listed as a critically
endangered species in the IUCN Red Data Book (HiltonTaylor, 2000) and categorized as a rank I species in the List of
National Key Protected Animals of China.
One of the primary concerns in the conservation of an
endangered species is how to conserve its genetic diversity,
which if maintained, improves its ability to adapt to future
threats. However, little is known concerning the genetic
310
variability of the baiji. The only relevant study is Yang et al.
(2003), who sequenced a 420 bp segment of mitochondrial
control region and found a very low level of nucleotide and
haplotypic diversity in baiji population.
The major histocompatibility complex (MHC) is one of
the most important genetic systems for infectious disease
resistance in vertebrates (Hedrick and Kim 2000; Hill 1996).
Genes of the MHC are the most polymorphic loci of all
nuclear-encoding genes in the vertebrate genome (Yuhki and
O’Brien 1990). It has been suggested that species and/or
populations with a low level of MHC diversity might be
particularly vulnerable to infectious diseases (O’Brien and
Yuhki 1999) because the encoded proteins of the MHC
genes play an important role in the immune response against
Yang et al. Baiji MHC DQB Exon 2
Table 1.
List of samples analyzed in the present study
Serial number
Sampling year(s)
Sample location
Tissue
NJNU0001
NJNU0002
NJNU0005
NJNU0007
NJNU0010
NJNU0011
NJNU0017
NJNU0022
NJNU0023
NJNU0024
NJNU0214
NJNU0266
NJNU0359
NJNU-0359f
NJNU0381
NJNU0388
NJNU-N86
NJNU-An2
1956
1957
1974
1978
1979
1979
1981
1984
1984
1986
1987
1987
1992
1992
1996
1998
Unknown
Unknown
Nanjing, Jiangsu Province
Nanjing, Jiangsu Province
Nanjing, Jiangsu Province
Guichi, Anhui Province
Guichi, Anhui Province
Zhangjiagang, Jiangsu Province
Taicang, Jiangsu Province
Tongling, Anhui Province
Wangjang, Anhui Province
Wuhu, Anhui Province
Nanjing, Jiangsu Province
Zhongyang, Anhui Province
Yizheng, Jiangsu Province
Yizheng, Jiangsu Province
Nanjing, Jiangsu Province
Chongming Island, Shanghaia
Unknownb
Unknownc
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Skeleton
Muscle
Muscle
Muscle
Muscle
Skeleton
Skeleton
No. of clones sequenced
4
5
5
5
5
5
4
5
5
5
5
4
14
5
16
15
5
5
No. of alleles
4
5
5
3
5
5
3
5
5
5
4
3
7
3
6
6
5
3
Total: 117
a
Presented by the Shanghai Natural History Museum, Shanghai, China.
b
Presented by Nanjing University, Jiangsu Province, China.
c
Presented by Anhui Normal University, Anhui Province, China.
pathogens. The examination of MHC variability has been the
focus of many genetic studies of endangered species in
recent years (Haig 1998; Hedrick 1994; Yang et al. 2002).
Therefore, in the present study we characterize the genetic
variation at exon 2 of the baiji MHC DQB locus through
polymerase chain reaction (PCR), cloning, and direct
nucleotide sequencing. This DQB locus was chosen because
of its highly polymorphic nature and its important role in the
immune response (Murray et al. 1995). Our objective was to
investigate the evolutionary and adaptive potential of the
most endangered cetacean species based on an analysis of
a functional genomic region.
Materials and Methods
A total of 18 baiji samples, 4 myologic and 14 skeletal, were
available for the present study. These samples were gathered
from stranded or incidentally killed individuals from the
middle and lower reaches of the Yangtze River. Voucher
specimens are preserved in the Institute of Genetic
Resources, Nanjing Normal University (NJNU). Sample
information, including serial number and sampling location,
is shown in Table 1.
The extraction of total genomic DNA from muscles
followed the procedures described in Yang et al. (2002). For
extracting DNA from skeletal samples, the skeleton was first
washed with 2% hydrogen peroxide (H2O2), thoroughly
rinsed with double distilled water, dried in an oven, and then
irradiated under ultraviolet (UV) light for at least 15 min to
damage or crosslink contaminated DNA on the sample
surface. The sample was then ground into a powder and
mixed with 5 ml 0.5 M ethylenediaminetetraacetic acid
(EDTA) solution (pH 8.0) in a 20 ml centrifuge tube. The
mixed solution was incubated at 608C for approximately 72 h.
We used two primers to amplify exon 2 of the MHC
DQB gene, as described in Murray et al. (1995): 59-CTGGTAGTTGTGTCTGCACAC-39 (forward) and 59-CATGTGCTACTTCACCAACGG-39 (reverse). All amplification
reactions were conducted on a Perkin Elmer Cetus Thermal
Cycler 2400 or an MJ Research Thermal Cycler 200 in a total
volume of 100 ll containing 10–100 ng of extracted DNA
template, 10 mM of Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM
MgCl2, 150 lM of each dNTP, 0.01% gelatin, 3 units of Taq
DNA polymerase (Promega), and 0.3 lM of each primer.
Amplifications were performed with the following temperature profiles: 35 cycles at 958C for 30 s, 558C for 30 s, and
728C for 50 s, followed by a 7-min extension at 728C. PCR
products were purified using Wizard PCR Preps DNA
Purification Kit (Promega) according to the manufacturer’s
protocol and then cloned into a pMD-18T vector (Takara)
using a TA cloning kit. Four to 16 recombinant plasmids for
each individual (Table 1) were selected, isolated, purified, and
then sequenced and analyzed employing an ABI PRISM 310
Automated Genetic Analyzer (Applied Biosystems, Foster
City, CA) in the forward direction using M13 forward primer
59-CATGTGCTACTTCACCAACGG-39, under the instruction of the BigDye Terminator Cycle Sequencing Ready
Reaction Kit (Applied Biosystems).
To determine the variable nucleotide sites and unique
alleles, the resultant sequences were aligned by the computer
software ClustalX 1.8 (Jeanmougin et al. 1998) and corrected
311
Journal of Heredity 2005:96(4)
by hand. Phylogenetic analysis was performed using the
Molecular Evolutionary Genetics Analysis (MEGA) version
2.0 computer program (Kumar et al. 2001). A phylogenetic
tree, including present baiji alleles and alleles from the white
whale (Delphinapterus leucas), narwhal (Monodon monoceros), and
some ungulate species, was reconstructed using the neighborjoining (NJ) algorithm (Saitou and Nei 1987), with genetic
distance determined by the Kimura two-parameter model.
Bootstrap confidence intervals were obtained from 1000
replicates. Relative frequencies of nonsynonymous (dN) and
synonymous (dS) substitutions were calculated for the peptide
binding region (PBR) and non-PBR following Nei and
Gojobori (1986) and using the Jukes and Cantor (1969)
correction incorporated in MEGA 2.0 (Kumar et al. 2001).
Results
A total of 117 clones from 18 individuals were sequenced
and 172 bp sequences were obtained; 48 variable sites were
found in the sequences and a total of 43 distinct sequences
(alleles) (Live-DQB*1–43) were identified (Figure 1). Of the
43 alleles, 8 (Live-DQB*4, 5, 8, 11, 13, 16, 28, and 29) were
found from 2 to 15 individuals, whereas the other 35 alleles
were all found from only one individual and one clone
(Table 2). These allelic sequences have been deposited in
GenBank with accession nos. AY177150–177153, AY177283–
177293, and AY333386–333410. Of the 48 variable sites
determined, 18 were found at the first, 20 at the second, and
10 at the third base position of the amino acid codons. The
variable sites included 36 transitions, 11 transversions, and
one site experienced both a transition and transversion.
Forty-one nonsynonymous substitutions, which caused
changes at 31 amino acid sites, were found. No insertions,
deletions, or stop codons were found in these alleles. Three
to seven alleles were found for each individual (Table 2). The
distribution of these alleles in each individual is given in
Table 2. Two alleles were found in two individuals (i.e., LiveDQB*28 and 29), Live-DQB*4 was found in 15 individuals,
and the remaining alleles (i.e., Live-DQB*5, 8, 11, 13, 16)
occurred only once.
The phylogenetic reconstructions based on nucleotide
sequences (Figure 2) and inferred amino acid sequences
(topology not shown here) are nearly identical. The 43 baiji
alleles constituted a clade with a high bootstrap value of 98%
and had a much closer relationship with white whale and
narwhal than with ungulates. The baiji alleles were further
divided into two lineages (i.e., lineage I and II) with bootstrap
values of 65% and 57%, respectively. Lineage I consisted of
alleles Live-DQB*1–7 and Live-DQB*16–30, while lineage II
consisted of alleles Live-DQB*8–15 and Live-DQB*31–43.
The position of Live-DQB*15, however, was unstable. This
allele could be included in lineage I with the modification of
ingroup and/or outgroup species. Seven diagnostic nucleotides (i.e., sites 18–20, 23–25, and 29; Figure 1) and four
diagnostic amino acids (i.e., amino acids 26–28 and 30;
Figure 3) were found divided between two lineages. The
percentage difference among alleles of lineage I ranged from
312
Figure 1. Variable sites of Live-DQB alleles (Live refers to
Lipotes vexillifer). Allele codes are shown on the left. Asterisks (*)
indicate diagnostic sites. Shadows indicate the position of
possible polymorphic motifs.
0.58% to 6.98%, with an average of 2.20%, whereas
differences among lineage II alleles ranged from 0.58% to
6.4%, with an average of 2.08%. Compared to the withinlineage sequence differences, the between-lineage sequence
divergences were much larger, ranging from 4.07% to
11.63%, with an average of 8.90%.
The sequence similarity between the present Live-DQB
alleles and DQB1 genes of the white whale and narwhale
(Murray et al. 1995) varied from 87.8% to 94.2% (average
90.6%) for nucleotides and from 73.7% to 87.7% (average
80.6%) for amino acids. When the present amino acid
sequences were compared with their counterparts in other
animals, it can be seen that Live-DQB alleles do not share the
sequence motif with DQB2 genes of human and other
primates, but share a similar motif with cattle DQB1
sequences (Figure 3).
Yang et al. Baiji MHC DQB Exon 2
Table 2.
Distribution of DQB alleles in 18 baiji individuals
Allele
0001 0002 0005 0007 0010 0011 0017 0022 0023 0024 0214 0266 0359 0381 0388 0359f N86 An2
DQB*1
DQB*2
DQB*3
/
DQB*4
/
DQB*5
DQB*6
DQB*7
/
DQB*8
DQB*9
DQB*10
/
DQB*11
DQB*12
/
DQB*13
DQB*14
DQB*15
/
DQB*16
DQB*17
DQB*18
DQB*19
DQB*20
DQB*21
DQB*22
DQB*23
DQB*24
DQB*25
DQB*26
DQB*27
/
DQB*28
/
DQB*29
DQB*30
DQB*31
DQB*32
DQB*33
DQB*34
DQB*35
DQB*36
DQB*37
DQB*38
DQB*39
DQB*40
DQB*41
DQB*42
DQB*43
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
The columns are alternately shaded to follow the allele distribution.
/ indicates the alleles found from two or more individuals.
The baiji amino acid sequences correspond to sites 21–77
of the beluga DQB1 sequences (Murray et al. 1995). In this
region, 14 sites (i.e., sites 28, 30, 32, 37, 38, 47, 56, 60, 61, 65,
68, 70, 71, and 74) (Brown et al. 1993; see Figure 3) are
responsible for peptide binding. Clearly the amino acid
substitutions tend to have clustered around the peptidebinding region (PBR) (Figure 3). The relative frequency of
nonsynonymous substitutions (dN ¼ 0.1962; SE ¼ 0.0922)
was significantly higher than the frequency of synonymous
substitutions (dS ¼ 0.0256; SE ¼ 0.0286) in the PBR for all
alleles (Table 3). In the non-PBR, however, nonsynonymous
substitutions were found to occur much less frequently
(dN ¼ 0.0245, SE ¼ 0.0085, dS ¼ 0.0805, SE ¼ 0.0381)
(Table 3).
Discussion
In the present study, 43 alleles were identified from exon 2
MHC DQB locus in 18 baiji individuals. The fact that all
individuals had more than two alleles suggests that these
alleles did not come from the same locus, and thus could be
regarded as evidence to support the existence of gene
duplication at the baiji DQB locus. Specifically, the existence
of four or more DQB loci in the baiji genome is indicated by
313
Journal of Heredity 2005:96(4)
Figure 2. Phylogenetic tree of DQB alleles of baiji (Lipotes
vexillifer), beluga whale (Delphinapterus leucas), narwhal (Monodon
monoceros), and some ungulate animals, reconstructed using the
neighbor-joining algorithm with genetic distance of the Kimura
two-parameter model. Bootstrap values from 1000 replicates
were indicated above the tree branches. Sequences of other
cetaceans and ungulates are downloaded from GenBank:
moose (Alces alces): Alal-DQB*2 (AY077785) and Alal-DQB*4
(AY077787); pig (Sus scrofa): SLA-DQB*P06 (AF272715) and
SLA-DQB*36 (AF272723); sheep (Ovis aries): Ovar-DQB*22
(AJ238940) and Ovar-DQB*28 (AJ238946); zebu (Bos indicus):
Bos-DQB*2201 (AJ249718) and Bos-DQB*2401(AJ249897); and
musk ox (Ovibos moschatus): Ovmo-DQB*01 (AY077788) and
Ovmo-DQB*04 (AY077791). The dotted line corresponds to the
unstable position of Live-DQB*15. / indicates the alleles found
from at least two individuals.
the identification of three to seven alleles from each
individual. It is interesting that the DQB genes, which
occupy a single locus in other marine mammals, for example,
beluga, narwhal, elephant seals (Mirounga sp.), and fur seals
(Arctocephalus sp.) (Hoelzel et al. 1999; Murray et al. 1995), are
314
Figure 3. Amino acid sequences of DQB alleles of baiji,
cattle, human, and some other primates. The number above the
sequences represents the amino acid position based on beluga
DQB sequences (Murray et al. 1995). As shown in Figure 1, the
identity of the first allele is indicated by a dot (.). Amino acid
substitutions leading to strong, weak, and no conservation of
physiochemical properties of amino acid are indicated by (:),
(þ), and (^), respectively. An asterisk (*) indicates the fully
conserved residue of the amino acid. The sites postulated to be
responsible for selective peptide recognition are shadowed
(Brown et al. 1993).
apparently duplicated in baiji. Gene duplications have been
revealed in many MHC loci of some other animals, for
example, the owl monkey (Aotus nancymaae) DRB locus
Yang et al. Baiji MHC DQB Exon 2
Table 3. Estimated rate of nonsynonymous (dN) and synonymous (dS) differences (standard error in parentheses) and their ratio
in the PBR and non-PBR for baiji
Region
No. of codons
dN
dS
dN/dS
P
PBR
Non-PBR
Total
14
43
57
0.1962 (0.0922)
0.0245 (0.0085)
0.0574 (0.0175)
0.0256 (0.0286)
0.0805 (0.0381)
0.0666 (0.0291)
7.66
0.30
0.86
P , 0.01
(Nino-Vasquez et al. 2000) and cattle (Bos taurus) DQA and
DQB loci (Elizabeth et al. 2000). For example, in a study of
cattle MHC variation, Elizabeth et al. (2000) found that both
DQA and DQB genes duplicated three times. The presence
of more than two alleles was also found in the Argentine
Creole Horse (Equus caballus) DQB loci (Villegas-Castagnasso
et al. 2003). Gene duplication, in combination with allelic
sequence diversity, effectively increases the diversity of
MHC-encoded proteins and/or protein dimers or polymers,
and thus plays an important role in the immune response to
various pathogens.
The alleles identified in the present study do not show
deletions, insertions, or stop codons, and therefore could be
functional in vivo. Further evidence that these alleles are
functional is the high ratio of nonsynonymous substitution
to synonymous substitution in the PBR (Table 3). This high
ratio of dN/dS indicates that nonsynonymous sites evolved
faster than synonymous sites and implies balancing selection
(or positive Darwinian selection) favored new variants and
increased allelic polymorphism (Bergstrom and Gyllensten
1995). In the majority of proteins, dS/dN is greater than one,
indicating a constraint on the change in amino acid sequence,
that is, negative selection (Li et al. 1985). In contrast,
pseudogenes evolve with no constraints, that is, neutral
evolution. Imanishi and Gojobori (1992) revealed that
variation at one MHC class I pseudogene was similar to
other mammalian pseudogenes and different from the
patterns observed in functional class I sequences.
Of the 14 possible binding sites for foreign peptides, five
(sites 28, 32, 60, 61, and 74) were nonconservative and
modified the physiochemical properties of amino acid
residues, which may indicate that they caused a shift in
selective peptide recognition in the PBR (Figure 3). Of the
three amino acid substitutions that differed between, but not
within, the allelic lineages (i.e., the diagnostic sites 26, 28, and
30), only site 30 is conservative in physiochemical properties.
Site 28 changed from a negatively charged glutamic acid in
lineage I to a polar uncharged threonine in lineage II, which
suggests that alleles in lineages I and II are quite distinct in
antigen recognition. Similarly, at position 61 of allele LiveDQB*10, a nonpolar tryptophan was replaced by a positively
charged polar arginine, and at position 74 a positively charged
polar glutamic acid was replaced by a nonpolar glycine.
There was a relatively high level of similarity between the
baiji sequences and the DQB1 genes of beluga and narwhal
(average 90.6% for nucleotides and 80.6% for amino acids).
When compared with the DQB sequences of human and
other animals, the baiji sequences do not have the DQB2
motif shared by human and other primate species, but do
share the motifs of cattle DQB1 and DQB2. However, it
should be noted that the recognition of cattle DQB2 is less
accurate and uncertain. Haplotypic sequences that differed
from the DQB1 gene were identified as DQB2, which made it
difficult to distinguish cattle DQB1 and DQB2 according to
nucleotide sequences alone (Marello et al. 1995). For this
reason, the present sequences were tentatively identified as
DQB1-like loci. However, because lineage I and II alleles
showed a quite high level of sequence divergences (4.07% to
11.63%, average 8.90%) and seven nucleotide and three
amino acid diagnostic sites, respectively, suggested that they
might be identified in separate loci of MHC DQB. Further
studies are necessary to confirm this.
It has been shown that many marine mammals have
lower levels of MHC diversity than terrestrial mammals
(Murray et al. 1995; Slade 1992). For example, Murray et al.
(1995) found that the genetic variability at the MHC DQB
loci of the beluga and narwhal was much smaller than those
of primates. Currently the only exception is the southern
elephant seal (Mirounga leonina), which had a polymorphism
comparable to those seen in human populations (Hoelzel et al.
1999). Although several hypotheses have been put forward
to explain the reduction of MHC diversity in marine
mammals, the simplest explanation is that the low level of
MHC diversity is caused by a reduced balancing selection
pressure, which in turn is the result of the lower abundance
of parasites in marine waters as compared to terrestrial
environments (Slade 1992). One problem with this hypothesis is that previous studies have revealed many diseases and
epizootic occurrences in marine mammal populations
(Acevedo-Whitehouse et al. 2003; Raga et al. 1997; Van
Bressem et al. 1998, 1999).
Compared with the low level of MHC genetic diversity in
many other marine mammals (Murray et al. 1995; Slade 1992;
Trowsdale et al. 1989), the baiji genome shows evidence of
gene duplication and has unexpectedly high levels of allelic
diversity and sequence variability. It is well known that baiji
has a very limited distribution, is endemic to the Yangtze
River, and its population size has rapidly declined in the past
few decades. Although systematic environmental studies
have yet to be conducted, it is expected that the freshwater
environment has a relatively higher level of pathogens than
marine waters. If so, it is likely that the gene duplication and
relatively high levels of MHC sequence variability in the baiji
discovered in this study are evolutionary adaptations to the
freshwater environment. Furthermore, the high level of
genetic variability at the MHC DQB locus is inconsistent
with a neutral mitochondrial marker (Yang et al. 2003). Such
an inconsistency is also found in some other species, for
315
Journal of Heredity 2005:96(4)
example, the San Nicolas Island fox (Urocyon littoralis dickeyi),
which genetically is the most monomorphic sexually reproducing animal population yet reported (Aguilar et al.
2004). The inconsistency can be explained by the absence of
an association between neutral and fit-related traits in natural
populations (Reed and Frankham 2001).
However, it is worth pointing out that only 8 of the 43
alleles found in the present study were confirmed by
different individuals or clones, whereas the other 35 alleles
were only found once and have very small differences with
some common ones in one or two nucleotide substitutions.
According to the conditions for acceptance of new
sequences as dog MHC gene (Kennedy et al. 2000), only
those sequences confirmed in different clones or individuals
can be considered as new alleles and can be given official
names. Considering that some potential error in Taq
polymerase incorporation, PCR recombination, cloning,
and single direction sequencing, etc., may generate unexpected sequence polymorphism, the present unconfirmed,
one-off alleles urgently need to be confirmed in the future to
clarify the genetic diversity and gene duplication of baiji
revealed in the present study.
Acknowledgments
This research was supported by the National Natural Science Foundation
Commission of China (grants nos. 30270212 and 30070116 to G. Yang),
NSFC project for Distinguished Scholars (grant no. 30125006 to F. Wei),
National ‘‘211’’ Project for the Tenth Five Years (to G. Yang), and
‘‘Qinglan’’ Project of Jiangsu Province (to G. Yang). Dr. Jonathan Geisler,
Georgia Southern Museum and Georgia Southern University, and two other
anonymous referees kindly provided constructive suggestions on the
manuscript.
References
Acevedo-Whitehouse K, Gulland F, Greig D, and Amos W, 2003. Disease
susceptibility in California sea lions. Nature 422:35.
Aguilar A, Roemer G, Debenham S, Binns M, Garcelon D, and Wayne RK,
2004. High MHC diversity maintained by balancing selection in an
otherwise genetically monomorphic mammal. Proc Natl Acad Sci USA
101:3490–3494.
Bergstrom T and Gyllensten U, 1995. Evolution of MHC class II
polymorphism: the rise and fall of class II gene function in primates.
Immunol Rev 143:13–31.
Brown JH, Jardetzhy TS, Gorga JC, Stern LJ, Urban RG, Strominger JL,
and Wiley DC, 1993. Three-dimensional structure of the human class II
histocompatibility antigen HLA-DR1. Nature 332:845–850.
Brunsberg U, Edfors-Lija I, Andersson L, and Gustafsson K, 1996.
Structure and organization of pig MHC class II DRB genes: evidence for
genetic exchange between loci. Immunogenetics 44:1–8.
Denaro M, Hammerling U, Rask L, and Peterson PA, 1984. The Ebb gene
may have acted as a donor gene in a gene conversion-like event generating
the Abm12b mutant. EMBO J 3:3745–3753.
Elizabeth JG, Oliver RA, and Russell GC, 2000. Duplicated DQ haplotypes
increase the complexity of restriction element usage in cattle. J Immunol
165:134–138.
Gorski J and Mach B, 1986. Polymorphism of human Ia antigens: gene
conservation between two Drb loci results in a new HLA-D/DR specificity.
Nature 322:67–69.
316
Gyllensten UB, Sundvall M, and Ehrilich HA, 1991. Allelic diversity is
generated by intraexon sequence exchange at the DRB1 locus of primates.
Proc Natl Acad Sci USA 88:3686–3690.
Haig SH, 1998. Molecular contributions to conservation. Ecology 79:
413–425.
Hedrick PW, 1994. Evolutionary genetics of the major histocompatibility
complex. Am Nat 143:945–964.
Hedrick PW and Kim T, 2000. Genetics of complex polymorphisms:
parasites and maintenance of MHC variation. In: Evolutionary genetics
from molecules to morphology (Singh RS and Krimbas CB, eds). New
York: Cambridge University Press; 204–234.
Hill AVS, 1996. Genetics of infectious disease resistance. Curr Opin Genet
Dev 6:348–353.
Hilton-Taylor C, 2000. 2000 IUCN red list of threatened species. Gland,
Switzerland: IUCN, The World Conservation Union.
Hoelzel AR, Stephens JC, and O’Brien SJ, 1999. Molecular genetic diversity
and evolution at the MHC DQB locus in four species of pinnipeds. Mol
Biol Evol 16:611–618.
Imanishi T and Gojobori T, 1992. Patterns of nucleotide substitution
inferred from the phylogenies of the class I major histocompatibility
complex gene. J Mol Evol 35:196–204.
Jeanmougin F, Thompson JD, Gouy M, Higgins DG, and Gibson TJ, 1998.
Multiple sequence alignment with ClustalX. Trends Biochem Sci 23:403–405.
Jukes TH and Cantor CR, 1969. Evolution of protein molecules. In:
Mammalian protein metabolism (Munro HN, ed). New York: Academic
Press; 21–132.
Kennedy LJ, Altet L, Angles JM, Barnes A, Carter SD, Francino O, Gerlach
JA, Happ GM, Ollier WER, Thomson W, and Wagner JL, 2000.
Nomenclature for factors of the dog major histocompatibility system
(DLA), 1998: first report of the ISAG DLA Nomenclature Committee.
Anim Genet 31:52–61.
Kumar S, Tamura K, Jakobsen IB, and Nei M, 2001. MEGA 2.0: molecular
evolutionary genetics analysis software. Bioinformatics 17:1244–1245.
Li WH, Wu CI, and Lou CC, 1985. A new method for estimating
synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Mol Biol
Evol 2:150–174.
Marello KL, Gallagher A, McKeever DJ, Spooner RL, and Russell GC,
1995. Expression of multiple DQB genes in Bos indicus cattle. Anim Genet
26:345–349.
Mikko S, Spencer M, Morris B, Stabile S, Basu T, Stormont C, and
Andersson L, 1997. A comparative analysis of the DRB3 polymorphism in
the American bison (Bison bison). J Hered 88:499–503.
Murray BW, Malik S, and White BN, 1995. Sequence variation at the major
histocompatibility complex locus DQB in beluga whales (Delphinapterus
leucas). Mol Biol Evol 12:582–593.
Nei M and Gojobori T, 1986. Simple methods for estimating the numbers
of synonymous and nonsynonymous nucleotide substitutions. Mol Biol
Evol 3:418–426.
Nino-Vasquez JJ, Vogel D, Rodriguez R, and Moreno A, 2000. Sequence
and diversity of DRB genes of Aotus nancymaae, a primate model for human
malaria parasites. Immunogenetics 51:219–230.
O’Brien SJ and Yuhki N, 1999. Comparative genome organization of the
major histocompatibility complex: lessons from the Felidae. Immunol Rev
167:133–144.
Raga JA, Balluena JA, Aznar J, and Fernandez M, 1997. The impacts of
parasites on marine mammals: a review. Parassitologia 39:293–296.
Reed DH and Frankham R, 2001. How closely correlated are molecular and
quantitative measures of genetic variation? A meta-analysis. Evolution
55:1095–1103.
Yang et al. Baiji MHC DQB Exon 2
Saitou N and Nei M, 1987. The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425.
Sigurdardottir S, Borsch C, Gustafsson K, and Andersson L, 1992. The
exon encoding the antigen recognition site of class II b-chain is divides into
two subregions with different evolutionary histories. J Immunol 148:
968–973.
Slade RW, 1992. Limited MHC polymorphism in the southern elephant
seal: implications for MHC evolution and marine mammal population
biology. Proc R Soc Lond B 249:163–171.
Wakeland EK, Boehme S, and She JX, 1990. The generation and
maintenance of MHC class II gene polymorphism in rodents. Immunol
Rev 113:207–226.
Wu S, Saunders TL, and Bach FH, 1986. Polymorphism of human Ia
antigens generated by reciprocal intergenic exchange between two Drb loci.
Nature 324:676–679.
Yang G, Chen XY, Ren WH, and Yan J, 2002. MHC and its application in
the population and conservation genetics. Hereditas (Beijing) 24:712–714.
Trowsdale J, Gorves V, and Arnason A, 1989. Limited MHC polymorphism
in whales. Immunogenetics 29:19–24.
Yang G, Liu S, Ren WH, Zhou K, and Wei F, 2003. Mitochondrial control
region variability of baiji and the Yangtze finless porpoises, two sympatric
small cetaceans in the Yangtze River. Acta Theriol 48:469–483.
Van Bressem MF, Jepson P, and Barrett T, 1998. Further insight on the
epidemiology of cetacean morbillivirus in the northeastern Atlantic. Mar
Mamm Sci 14:605–613.
Yuhki N and O’Brien SJ, 1990. DNA variation of the mammalian major
histocompatibility complex reflects genomic diversity and population
history. Proc Natl Acad Sci USA 87:836–840.
Van Bressem MF, Van Waerebeek K, and Raga JA, 1999. A review of virus
infections of cetaceans and the potential impact of morbilliviruses,
poxviruses and papillomaviruses on host population dynamics. Dis Aquat
Organ 38:53–65.
Zhou K, Sun J, Gao A, and Würsig B, 1998. Baiji (Liptoes vexillifer) in the
lower Yangtze River: movements, numbers, threats and conservation needs.
Aquat Mamm 24:123–132.
Villegas-Castagnasso EE, Diaz S, Giovambattista G, Dulout FN, and
Peral-Garcia P, 2003. Analysis of ELA-DQB exon 2 polymorphism in
Argentine Creole horses by PCR-RFLP and PCR-SSCP. J Vet Med A
50:280–285.
Received July 25, 2003
Accepted February 7, 2005
Corresponding Editor: C. Scott Baker
317