Paternity Testing And Correlation Analysis Between MHC And Offspring Mortality Of Deqing Captive Crested Ibises

The crested ibis (Nipponia nippon), a bird under the first-grade state protection, is listed in the four national treasures of China together with the giant panda, golden monkey and takin. The crested ibis was historically distributed in the east and northeast of Asia. However, during the 19th to 20th centuries, the population dramatically declined due to habitat destruction and food resource shortage, which were caused by the loss of wetlands and interference of human activities. The crested ibis was once considered to be extinct in the wild until 1981 during which seven wild birds were discovered in Yangxian, Shaanxi Province, China. Nowadays, the seven wild birds have became the founders of all the captive populations in the world. After three decades of protection, the crested ibis population has increased to more than 2000 individuals, including～1000 wild birds. Nevertheless, the whole population recovered from 7 founders has suffered a severe bottleneck, so that inbreeding was inevitable. Herein, we performed genotyping 11 microsatellite loci along with multi-locus MHC haplotyping/genotyping to evaluate genetic diversity and founder effect of DeQing (DQ) crested ibises. Meanwhile, we constructed a pedigree for this captive population to investigate inbreeding status of the DQ crested ibises. These two pieces of results would lay a solid foundation for designing scientific breeding program in future in order to enhance abilities of disease resistance and environment adaptionof the DQ captive crested ibises. The main results are listed below:(1) We identified 10 polymorphic markers-from 11 microsatellite loci; the remaining one was monomorphic. We detected 23 alleles across the 10 polymorphic microsatellites, giving a range of 2—3 and an average of 2.300 alleles per locus. The average observed heterozygosity (Ho) and the average expected heterozygosity (HE) of the 10 microsatellite loci were 0.459 and 0.444, respectively.(2) The average polymorphism information content (PIC) of the 10 polymorphic microsatellite loci was 0.369 (0.214—0.568), indicating a low level of genetic diversity in the crested ibis. The PIC value was relatively high in NN12 (0.568) and NN18 (0.563), but comparatively low in NN26 (0.214).(3) We identified seven haplotypes in the classical region of the crested ibis MHC, indicating that the adaptive immune system of the endangered bird has a higher level of genetic variation than neutral microsatellites. The seven haplotypes harbor 2 —8 MHC class Ⅱ genes (composed of 1—4 "αβ" units), respectively. The difference in the number of the MHC loci among the seven haplotypes suggests a high death risk in the crested ibises carrying deficient MHC haplotypes, which calls for a scientific reproduction plan and captive breeding management to improve the survival ability of the crested ibis population.(4) The discrimination powers (DPs) of the 10 polymorphic microsatellite loci were between 0.221 and 0.521, while the cumulative DP for the 10 loci went up to 99.1%. The cumulative probability of exclusion (CPE) across the 10 loci was 68.4% in the case of having no genetic information of both parents (parentage identification) and the CPE value went up to 90.1% when having the genetic information of only one parent (paternity/maternity identification). As for the MHC, the DP value was 58.1% and the CPE values for the parentage and paternity/maternity testings were 31.8% and 49.8%, respectively. When the "SSR and MHC loci were combined into one set of markers", the CPE values increased to 78.5% and 95.0%, respectively, showing an enough resolution for performing paternity testing of the DQ captive crested ibises.(5) We performed paternity testing for the DQ birds using a double-genotyping technique of the multi-locus MHC haplotype and 10 polymorphic microsatellite loci. On the basis of paternity identification results, we constructed a pedigree for the DQ crested ibis population and calculated the average inbreeding coefficient for each ibis. The results indicate that the DQ crested ibises have no inbreeding. Nevertheless, we found an increment of the deficient MHC haplotypes in F1 generation (relative to the founders) and in dead individuals (relative to the alive crested ibises), which further reflects the importance of making reasonable captive breeding program.(6) The average HO and HE of the Ⅱ-Ⅰ multi-locus MHC long-fragment genotypes were 0.773 and 0.758 in the founders, respectively, while the average HO and HE of the MHC long-fragment genotypes were 0.694 and 0.717 in their descendants, respectively. Then, we conducted Chi-square analyses on the frequency distributions of the multi-locus MHC genotypes carrying a different number of "αβ" units,9 MHC class Ⅱ long-fragment genotypes, and 13 MHC class Ⅱ-Ⅰ genotypes between the surviving and dead individuals, respectively. The results all reveal a positive correlation between the HT03/HT04 genotype and offspring mortality (all P< 0.05).(7) HT04 is the most deficient haplotype which lacks 3 "αβ" units, while the HT03 is the secondarily deficient haplotype which lacks 2 "αβ" units. We found that the death rate of offspring was closely associated with the heterozygous genotype HT03/HT04, rather than the homozygous HT04/HT04. This result indicates that there are other factors, other than the absence of "αβ" units, causing the death of offspring with HT03, and that the higher death rate in the HT03/HT04 heterozygous than the HT04/HT04 homozygous could be resulted from multiple factors. As a result, this study recommends that when formulating the reproduction program, the production of HT03/HT04 should be avoided if possible and it is better to make the deficient MHC haplotypes hybridized with other qualified haplotypes, in order to improve the survival rate of offspring.