Isolation Of Polymorphic Microsatellite Loci And Analysis Of Genetic Diversity In Crested Ibis (Nipponia Nippon)

Crested Ibis (Nipponia nippon) is an Endangered species that is endemic to China. All living Crested Ibis are known to descent from two breeding pairs rediscovered in Yangxian county in 1981. Until the end of 2006, the total population has recovered to about 800 individuals, including the wild and the captive populations. In order to provide feasible strategies for its conservation, we collected samples from both the wild and the captive populations including Shaanxi Yangxian Crested Ibis Breeding Centre and Louguantai Saving Center of Rare Wild Animals, isolated polymorphic microsatellite loci, and did comprehensively research on genetic diversity and population structure of this species. Our major results were:1. This study was based on magnetic beads enrichment protocol. Approximately 2000 clones were screened and a total of 120 recombinants that potentially contained microsatellite sequence were obtained. Sixty positive clones were chosen randomly and subject to DNA sequencing. The sequencing results showed that 35 out of 60 clones contained eight repeats or more and had suitable flanking sequences. 30 sets of primers were designed from the 35 candidate microsatellite sequences. 21 primer pairs succeeded in PCR amplification but only 11 primer dyads were polymorphic and consistently yielded their specific PCR products for each sample.2. 11 polymorphic loci and other 2 polymorphic loci developed by other reference were used. One locus from reference appeared heterozygous for all 107 samples and another one locus in our study not only revealed evidence of scoring errors raised from Stuttering but exhibited significant deviations from Hardy-Weinberg equilibrium.3. The lowest value of PIC (polymorphic information content) of the 11 polymorphic loci choosed in this study is 0.196 with average value of 0.323 which is correlation with its experience of a severe but short-lived population bottleneck. Furthermore, value of cumulative discrimination power (CDP) across 11 loci is 99.98%. Therefore, the microsatellite markers could be utilized to genetic pedigree construction for the crested ibis captive population. In addition, it could be helpful in the selecting of proper individuals for the reintroduction program.4. For the wild population (n=9), a total of 27 distinct alleles were observed at eleven loci over the complete data set and 4 private alleles were all observed. The number of alleles per locus was 2.45 and the average heterozygosity across 11 loci was 0.499. A total of 23 alleles were recorded in the Yangxian and Louguantai captive population (n=48 and n=50). The number of alleles per locus was 2.091 and the average heterozygosity across 11 loci was 0.383 and 0.378, respectively. Moreover, mean expected heterozygosity in the wild population was significantly higher than that in the Yangxian and Louguantai populations (p= 0.005 and p= 0.007, respectively; paired t-test), which revealed that the level of genetic variability in the three populations were wild>Yangxian>Louguantai.5. A complete Bayesian method in BAPS (Bayesian Analysis of Population Structure) was chosen to perform individual clustering without prior population information. The results showed that the 107 individuals were grouped into 7 clusters (named as LGT1, LGT2, YX1, YX2, YX-Wild1, YX-Wild2 and Wild). An unrooted NJ tree was constructed at individual and cluster levels, respectively. The NJ tree based on the 107 individuals clearly gave us two unmixed populations, i.e. Louguantai population and Yangxian-Wild population inconsistent with the one population or wild and Louguantai-Yangxian two populations. The assignment tests revealed that mis-assignment only appeared within Louguantai or Yangxian-Wild populations while their birds were allocated to each other with 100% correct assignment rate.6. Genetic admixture analysis revealed that intra population divergence was present (YX1, YX2; LGT1, LGT2). The extent of intra population divergence in LGT population was higher than that in YX population.7. The AMOVA analysis revealed that Louguantai population had significantly differentiated from Yangxian-Wild population. Thus, pre-defined three populations: Louguantai, Yangxian and wild were genetically structured into two parts:(1) Louguantai; (2)Yangxian-Wild.8. In 2002, 30 pairs of birds were diverted to Louguantai Saving Center of Rare Wild Animals and have developed into the biggest breeding population. Louguantai group has been significantly differentiated from its parent Yangxian population after just five years of artificial breeding and intra population divergence was present in two populations. According to the results above, we provided advices for the conservation of Crested Ibis as listed below:1) When founding a new captive population, genetic examination is compulsory in the breeding source population in order to ensure selecting the individuals with abundant genetic variation and with appropriate genetic composition, which could cushion subsequent loss of genetic diversity and make sure its better development in the future.2) Regarding scientific management for the captive populations of the crested ibis. Managers should make full use of the SPARKS studbook and just employ the original friendship-based reproduction strategy as an auxiliary tool. Based on the SPARKS exports, the MateRx gives Mate Suitability Indices while the PM2000 produces Kinship Values. Depending on the recommendations of MateRx and PM2000, managers could select the pairs with good genetic composition and low kinship to reproduce in order to (1) assure homogeneous genetic background among individuals; (2) avoid intra population divergence; (3) minimize the effect of genetic drift; and (4) maximize retention of the founders' genetic diversity from generation to generation.3) In the two captive populations examined here, the Louguantai population showed a significant differentiation from the wild birds but the Yangxian population indicated good genetic compatibility with the wild crested ibis, suggestive of a priority for introducing the Louguantai ibis into the wild population. Even though the captive individuals would be released into their historical distribution, the higher level of genetic variability in the Yangxian population than that in the Louguantai group also justified its reintroduction prior to the Louguantai birds.