Variation Of PIS Region And FOXL2 Gene In The Polled Intersex Dairy Goat

Hornlessness and intersex are closely linked in the Polled Intersex Syndrome (PIS), Study showed that it was the missing of one 11.7kb long fragment (PIS region) that induced the Polled Intersex Syndrome. This fragment's missing also affected the expression of Forkhead box L2 gene (FOXL2). FOXL2 is one single exon gene, which encodes one 376aa long protein sequence. This protein possesses one polyAla tract, except for the 110aa DNA-binding domain. Mutations of FOXL2 not only could induce the blepharophimosis syndrome (blepharophimosis syndrome, BPES) type I and type II in humans, but also is a candidate gene for PIS in goats. In order to examine the variations of FOXL2 and the PIS region in the hornless intersex goats, we did comprehensive bioinformatics analysis of the submitted FOXL2 gene sequences and have grasped its basic features on the whole, including the structure of corresponding protein sequences. Meanwhile, variation of FOXL2 was also studied in 34 hornless intersex dairy goat individuals, 34 hornless normal dairy goat individuals and 30 horned normal goat individuals, via amplification, direct sequencing and PCR-RFLP technique. In addition, the polymorphism of PIS region was also studied within and among the above-mentioned three types of goat individuals.Analysis of the 43 FOXL2 gene CDS sequences and corresponding amino acid sequences showed that there was no significant codon bias among them, and relatively higher GC content existed, especially in mammals (70%). TGA is FOXL2's favorite stop codon in mammals. DNA polymorphism analysis indicated that there were 260 variable sites, including 61 singleton variable sites and 199 parsimony informative sites, in the selected region (1–1229bp) of these 43 sequences. There was great sequence length variation between mammals and non-mammals, which was mainly due to, based on the amino acid sequences alignment, the missing of polyAla tract coding region in non-mammals. In mammals exists not only the highly conserved polyAla tract, but also the glycine repeat region and the proline repeat region, which has some variation among different mammals. There are more low complexity regions in mammals than in non-mammals. Ten highly conserved motifs were detected, which could be important functional sites. With the help of direct sequencing and alignment of FOXL2 gene, two SNPs were detected (g.3911C >G and g.4289G >T, based on AY112725). PCR-RFLP analysis and chi-square test showed that there were significant differences in gene frequencies of both the two SNP sites between normal individuals and intersex individuals and between hornless individuals and horned individuals, indicating some correlation between these two SNP sites and hornless and intersex phenotypes.Pairs of primers (PIS1, 26931～39428bp; PIS2, 26931～27667bp; PIS3, 27637～29012bp; PIS4, 38148～39044bp; PIS5, 35532～36425bp) (based on AF404302) were used to amplify the PIS region and screen its variation in dairy goats. Results showed that all those 34 hornless intersex dairy goats missed partial PIS, no complete missing was found. Alignment of PIS5 fragments (35532～36425bp) showed that there was not only partial fragment missing, but also a great deal of base mutations in those intersex individuals; 193 mutation locuses and 18 deletions between normal dairy goat and PIS dairy goat were detected in the region of PIS5 fragment (35607～36198bp). The alignment results of PIS2 and PIS3 fragment showed these base mutations might existed only in some fragments and could affect the regulatory function of the PIS region. It was inferred a great deal of base mutations and partial fragment missing of PIS could cause intersex character.