Abstract
BackgroundThe giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda.ResultsBy screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system.ConclusionThe microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1268-z) contains supplementary material, which is available to authorized users.
Highlights
The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China
Tetranucleotide repeats tend to stutter less than the trinucleotide and dinucleotide repeats and are much more accurate and reliable [22,23], which has become the marker of preferred choice and be widely used in paternity test kits for people [24,25]
We focused on developing microsatellites with high levels of polymorphism, strong stability, good repeatability, and very low genotyping error rate, which would be widely used in the giant panda studies
Summary
The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. Dinucleotide microsatellite is subject to mistyping due to polymerase slippage during polymerase chain reaction (PCR) [17,18] This problem is especially acute when template DNA is of low quality or concentration, as with faecal samples or degraded tissue samples [9,19,20]. Only 15 markers with single motif of (GATA)n were tetranucleotide repeats and nearly no one were used in the wild genetic studies. It was because most of them were unavailable when using the non-invasive samples. We concentrated on the tetranucleotide microsatellites to establish a universal genetic marker system
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