Development of a panel of microhaplotype markers for giant panda

The giant panda is a vulnerable species endemic to China and serves as a flagship species for biodiversity conservation. Accurately assessing population size in small populations has always been the foundation of conservation efforts. Owing to higher dropout rate and instability, the microsatellite marker has always caused the overestimation of the population size. The microhaplotype genetic marker, which is made from several adjacent SNPs, is as stable as the SNP marker and as polymorphic as the microsatellite marker. This study screened a panel of 32 candidate microhaplotype markers with an average polymorphic information content of 0.628 from a whole-genome sequencing dataset of 195 giant pandas. Afterwards, we successfully demonstrated the feasibility and reliability of the microhaplotype markers by genotyping half of them via the Illumina paired-end sequencing, with the markers amplified in four quadruplex PCR reactions, ranging from 142 and 234 bp in length. The cumulative non-exclusion probabilities for the full and half panel of markers are 6.477 × 10⁻²⁷ and 1.444 × 10⁻¹³, respectively. These markers offer a potential tool for individual identification, sex determination, the management and conservation of giant pandas.