HIGH LEVEL OF MHCPOLYMORPHISM IN THE BAIJI AND FINLESS PORPOISE, WITH SPECIAL REFERENCE TO POSSIBLE CONVERGENT ADAPTATION TO THE FRESHWATER YANGTZE RIVER - Biology, Evolution and Conservation of River Dolphins Within South America and Asia - page 398

followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension
at 72°C for 10 min. The PCR products were purified using Wizard PCR Preps DNA
Purification Kit (Promega, USA) according to the manufacturer's instruction.

Table 1. PCR primers used to amplify three MHC loci in the present study.

Size of
amplifications (bp)

Locus Primer sequences Reference

5'-AATCATGTGATCATCCAAGCTGAGTTC-3'
5'-TGTTTGGGGTGTTGTTGGAGCG-3'

DRA 189 This study

5'-CTGGTAGTTGTGTCTGCACAC-3'
5'-CATGTGCTACTTCACCAACGG-3'

Murray et al.
(1995)

DQB 172

5'-TACGTGGMCGACACGSAGTTC-3'
5'-CTCGCTCTGGTTGTAGTAGCS-3'

Flores-Ramirez
et al. (2000)

MHC-I 147

SSCP, Cloning and Sequencing

All the finless porpoise samples were first screened for consistent polymorphism at exon
2 of the MHC-I, DRA and DQB loci using single-strand conformation polymorphism (SSCP).
The selected samples were then characterized at the genetic level by DNA sequence
comparison. For SSCP analysis, 1 μl of the purified PCR product was mixed with 9 μl of
loading dye (95% v/v formamide, 20 mM EDTA, 0.05% w/v Bromophenol Blue, 0.05% w/v
xylene cyanol). After denaturing at 95 o C for 10 min and cooling on ice for 5 min, 5 ul of the
mixture was loaded into a 10% polyacrylamide gel (38:1, acrylamide/bisacrylamide).
Electrophoresis was performed in 1×TBE buffer at 150 V for 16~20 h at room temperature.
After completion of the run, SSCP bands were visualized by silver staining procedures. To
avoid categorizing PCR artifacts as a new allele based on the SSCP bands, the PCR products
were rearranged and separated again on the gel according to assessed similarities. In this
study, each sample was analyzed at least twice following the same procedure. For new
samples, all known alleles were run as references on each SSCP gel.
PCR products of the finless porpoise showing the same SSCP pattern in the replicates
were cloned into the pMD-18T vectors using the TA cloning kit (Takara, Japan). For each
locus, five to six randomly chosen PCR products were cloned from each SSCP genotype.
While for the baiji, all the PCR products were cloned into a pMD-18T vector. Four to six
clones were picked for each cloned PCR product and sequenced in the forward and/or reverse
directions. The sequence reaction was using the BigDye Terminator Cycle Sequencing Ready
Reaction Kit (ABI). Automated DNA sequence analysis was performed on an ABI 3730
automated genetic analyzer.

Data Analysis

Statistical analysis of nucleotide and amino acid sequences were computed in MEGA
version 4 (Tamura et al., 2007). The average rate of nonsynonymous ( d N ) and synonymous
( d S ) substitutions in the overall domain, PBR, and non-PBR were calculated according to the
Nei-Gojobori method (Nei & Gojobori, 1986) with the Jukes-Cantor correction for multiple