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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. 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