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. 2022 Mar 21;12(6):792.
doi: 10.3390/ani12060792.

Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed Sheep TBXT Heterozygotes

Guang Yang  1 , Caiyun Wang  2 , Hong Su  2 , Daqing Wang  2 , Aolie Dou  2 , Lu Chen  2 , Teng Ma  2 , Moning Liu  2 , Jie Su  2 , Xiaojing Xu  2 , Yanyan Yang  3 , Tingyi He  3 , Xihe Li  1 , Yongli Song  1 , Guifang Cao  2
Affiliations

Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed Sheep TBXT Heterozygotes

Guang Yang et al. Animals (Basel). .

Abstract

The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.

Keywords: HRM; TBXT; sheep; short-tailed trait.

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Conflict of interest statement

G.C., G.Y., Y.S., A.D., T.M., C.W., H.S., M.L., D.W., L.C., X.X., T.H., Y.Y., J.S. and X.L. are co-authors of an issued Chinese invention patent (as described above) covering the high-resolution melting curve used in this study. The efficacy of this patented approach has not been demonstrated nor do the authors believe the patent or any future benefits they may receive because of it inappropriately influence any work described in this manuscript. The other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Three genotypes of TBXT in Barag sheep and short-tailed sheep. Blue arrows point to two polymorphic sites. (a) Fragments of chromatograms with genotype C-T/C-T. (b) Fragments of chromatograms for heterozygous genotype C-T/G-G. (c) Fragments of chromatograms with genotype G-G/G-G.
Figure 2
Figure 2
X-ray images of the caudal vertebrae of Barag sheep and short-tailed sheep. (a) Representative X-ray images of the caudal vertebrae of short-tailed sheep. (b) Representative X-ray images of the three types of caudal vertebrae of Barag sheep.
Figure 3
Figure 3
HRM genotyping results. (a) Typical shapes of the melting curves corresponding with the three genotypes (C-T/C-T, C-T/G-G, and G-G/G-G). The color of the curve is automatically assigned by PikoRealTM software. The vertical axis represents the change in fluorescence intensity and the abscissa represents the Tm value. The value of the abscissa corresponding with the main peak of each curve is the Tm value of the amplified fragment. (b) The melting curve image generated during the genotyping of Barag sheep and short-tailed sheep (a total of 32 samples are shown in the image; each sample was run in triplicate).
See this image and copyright information in PMC

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