Lack of Structural Variation but Extensive Length Polymorphisms and Heteroplasmic Length Variations in the Mitochondrial DNA Control Region of Highly Inbred Crested Ibis, Nipponia nippon

Abstract

The animal mitochondrial DNA (mtDNA) length polymorphism and heteroplasmy are accepted to be universal. Here we report the lack of structural variation but the presence of length polymorphism as well as heteroplasmy in mtDNA control region of an endangered avian species - the Crested Ibis (Nipponia nippon). The complete control region was directly sequenced while the distribution pattern and inheritance of the length variations were examined using both direct sequencing and genotyping of the PCR fragments from captive birds with pedigrees, wild birds and a historical specimen. Our results demonstrated that there was no structural variation in the control region, however, different numbers of short tandem repeats with an identical motif of CA3CA2CA3 at the 3'-end of the control region determined the length polymorphisms among and heteroplasmy within individual birds. There were one to three predominant fragments in every bird; nevertheless multiple minor fragments coexist in all birds. These extremely high polymorphisms were suggested to have derived from the 'replication slippage' of a perfect microsatellite evolution following the step-wise mutational model. The patterns of heteroplasmy were found to be shifted between generations and among siblings but rather stable between blood and feather samples. This study provides the first evidence of a very extensive mtDNA length polymorphism and heteroplasmy in the highly inbred Crested Ibis which carries an mtDNA genome lack of structural genetic diversity. The analysis of pedigreed samples also sheds light on the transmission of mtDNA length heteroplasmy in birds following the genetic bottleneck theory. Further research focusing on the generation and transmission of particular mtDNA heteroplasmy patterns in single germ line of Crested Ibis is encouraged by this study.

Figure 1. The current distribution range of the wild population, locations of the 43 nests banded in 2011 and 25 nests selected for this study in the wild as well as the Captive Breeding Center of Crested Ibis.

Photo courtesy of Jiao Jingquan.

Figure 2. Structure of mtDNA control region and position of primers designed for this study.

TAS = termination associated sequences, CSB = conserved sequence block. The primer positions are scored based on the complete mtDNA sequence of Crested Ibis deposited in GenBank (Accession no. NC_008132).

Figure 3. Partial sequencing chromatographs and peak distributions of genotyping of representative samples showing length polymorphism and heteroplasmy.

A-27 and A-02 had predominant fragments at 221 bp and 243 bp while their sequencing profiles showed relatively clear chromatograms carrying nine and 11 repeats, respectively. Sample A-30 had at least two predominant peaks at 210 bp and 232 bp whilst its sequencing chromatogram included overlapped signals at an interval of two repeats.

Figure 4. Histograms of the distribution of predominant fragments with different numbers of repeat.

(A) 21 wild birds, (B) 31 captive birds, (C) Ten offspring of the mating groups 3, 5 and 6 with their mother carrying a predominant fragment of nine repeats, (D) 12 offspring of the mating group 1, 2 and 7 their mother carrying a predominant fragment of 11 repeats.

Figure 5. Pedigree chart represents three generations with 17 maternally-related individuals in family No. 1.

G-1, G-2 and G-3 indicate the generations. Squares stand for males and circles for females. Numbers in brackets right to the squares or circles are the number of repeats in every sample.