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 397

estimated to be much less than before (Hutzler, 2006). More and more conservation biologists
in China have proposed to increase the conservation grade of the finless porpoise from II to I
in the List of Key Nationally Protected Animals.
The baiji and finless porpoise both have low levels of genetic variability at neutral
markers such as at the mitochondrial control region and microsatellites (Yoshida et al., 2001;
Yang et al., 2003 2008; Xia et al., 2005; Zheng et al., 2005). However, no systematic
information on sequence variation in adaptive markers is currently available for the baiji and
finless porpoise. In the present study, sequences of exon 2 of the MHC class I gene and class
II ( DRA and DQB ) gene were determined in both species. It is expected to have an in-depth
understanding on the behavior of these molecules, esp. sequence variability possibly caused
by selection pressure. Findings from this study will provide basic information for studying the
MHC immunogenetics at a population level, and especially to identify a genetic basis for its
adaptation to freshwater by the Yangtze finless porpoise. Moreover, the MHC data sets reveal
a striking interspecific identity and similarity, which suggests that convergent evolution is a
response to the common freshwater environment they have inhabited.
M ATERIALS AND M ETHODS
Samples
Fifteen baiji and 195 finless porpoise samples were available for this study. The baiji
samples were collected from the lower reaches of the Yangtze River, whereas the finless
porpoise samples were collected over a period of more than 20 years from 20 locations along
the coast of China, as well as from the middle and lower reaches of the Yangtze River. The
finless porpoise samples were assigned to different populations a priori , i.e. the Yangtze
River population, the Yellow Sea population, and the South China Sea population, according
to the discriminant features suggested by Gao and Zhou (1995). All these samples were taken
from stranded or incidentally captured/killed individuals. Voucher specimens are preserved in
the Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences,
Nanjing Normal University (NNU), China.
DNA Isolation and PCR
Myologic and skeletal samples were extracted using the DNeasy Tissue Kit (QIAGEN)
and Geneclean for Ancient DNA kit (Q. Biogene), respectively, following the manufacturer's
protocol. The exon 2 fragments for MHC-I, DRA and DQB were amplified using three primer
sets as shown in Table 1. The primers used to amplify the DRA gene were designed against a
conserved region among sheep ( Ovis aries , GenBank Accession, M73983), cattle ( Bos taurus ,
M30120), horses ( Equus caballus , L47174) and humans ( Homo sapiens , M60334) (Sena et
al. 2003). Polymerase chain reactions (PCR) were carried out in a total volume of 50 μl
containing 2.5 mM MgCl 2 , 10 mM Tris- HCl (pH 8.4), 50 mM KCl, 0.2 mM each dNTP, 0.4
μM each primer, 1.0 unit Ex-Taq DNA polymerase (Takara, Japan) and 10-100 ng DNA
template. The PCR cycling scheme included an initial denaturation of 5 min at 94°C,