Classical hybrid sterility genes Prmd9 and Hstx2 realize their hybrid sterility specific phenotypes by interacting, directly or indirectly, with the process of meiotic pairing and synapsis of heterospecific homologs. Here the Hstx2 locus, controlling asymmetry, of hybrid sterility in reciprocal intersubspecific F1 hybrid males was localized to a 4.7 Mb interval on Chr X and three autosomal loci that can abolish that asymmetry were mapped.
To localize the Hstx2 locus consomic F1 female mice (C57BL/6J- Chr X<PWD/Ph/Fore x C57BL/6J) were crossed with PWD/Ph males. All 124 male progeny carryed B6/PWD heterosubspecific autosomal pairs while Chr X loci were either B6 or PWD depending on the recombination breakpoints. Testes weight and sperm count were used as male fertility phenotypes for QTL analysis.
Hybrid sterility locus Hstx2 ( hybrid sterility, chromosome X, 2) was mapped on Chromosome X with a LOD score of 30 and 23 at 34.8 cM for testis weight and sperm count. The 1.5 LOD support interval ranged from 34.6 to 35.7 cM. All males with the PWD/Ph allele at DXMit87 had small testes and little sperm in the ductus epididymus. Hstx2 caused complete sterility on the F1 hybrid intersubspecific background.
To further refine the position of Hstx2 a new partial consomic strain, C57BL/6J-Chr X.1s, was created that extended the proximal interval of the Chr X
PWD/Ph sequence compared to C57BL/6J-Chr X.1
PWD/Ph used in the previous mapping study J:224141.
To localize the region carrying Hstx2 on Chr X
PWD/Ph, females of all four Chr X partial consomic strains were crossed with PWD males and the fertility of the male offspring was analyzed. Only (C57BL/6J-Chr X1.s
PWD/Ph x PWD/Ph/Fore)F1 and (C57BL/6J-Chr X.2
PWD/Ph x PWD/Ph/Fore)F1 males were fully sterile with small testes and no sperm in the ductus epididymus. The position of Hstx2 was localized to a 4.7 Mb interval, X:64,880,641-69,581,094, GRCm38. [Fig 2.]
To further localize Hstx1 all four of the C57BL/6J-Chr X
PWD/Ph/Fore partial consomics were phenotyped and a high percentage of abnormal sperm cells were found in the C57BL/6J-Chr X.1s
PWD/Ph/Fore strain compared to the other 3 partial consomics and B6 males. The analysis independently confirmed the localization of Hstx1 to the same 4.7 Mb interval of Chr X that carries Hstx2. However, because Hstx1 C57BL/6J-Chr X.2
PWD/ph/Fore males did not show high frequency of abnormal sperm in spite of the Hstx1
PWD/Ph allele it was assumed that Hstx1 needed to interact with another genetic factor from the proximal region of Chr X
PWD/Ph.
The 4.7 Mb Chr X candidate region of Hstx1 and Hstx2 lci carried 11 known protein coding genes and 20 miRNA genes [Fig S2.]. Of those, seven protein coding genes show high expression in adult testes: Ctag2, 4930447F0Rik, Slitrk2, 4933436I01Rik, Fmr1, Fmr1nb, and Aff2.
While asymmetric male sterility of (PWD/Ph x C57BL/6J)F1 hybrids depends on the presence of the Hstx2
PWD/Ph allele, another inbred strain derived from M.m. musculus, known as STUS/Fore produces fully sterile F1 hybrid males with B6 mice regardless of the direction of the cross. To map the STUS/PWD autosomal allelic variants that ensure intrameiotic arrest in males carrying Mmm Chr X
B6 84 males from crosses of C57BL/6J females and (PWD/Ph x STUS)F1 or (STUS xPWD/Ph)F1 males were genotyped. QTL analysis of the sperm count revealed QTL on Chrs 3, 9, and 13; while QTL for testes weight mapped to Chrs 3 and 13.
QTL Twq3 (testis weight QTL 3) mapped to Chromosome 3:104,320,699 with a LOD=5.20.
QTL Twq4 (testis weight QTL 4) mapped to Chromosome 13:47,975,634 with a LOD=2.90.
QTL Spcq1 (sperm count QTL 1) mapped to Chromosome 3:JAX00171568 with a LOD=3.70.
QTL Spcq2 (sperm count QTL 2) mapped to Chromosome 9:56,492,601 with a LOD=3.0
QTL Spcq3 (sperm count QTL 3) mapped to Chromosome 13 with a LOD=3.1. Sperm count was evaluated as a binary trait. [Fig 4.A.]
The Chr 3 QTL, Twq3 and Spcq1, 1.5 LOD support interval mapped between 65.0 and 135.3 Mb on GRCm38. Possible candiate genes were Ccna1, Smc4, Mndl, Hormad1, and Sycp1.
The Spcq2 1.5 LOD support interval mapped between 40.5 and 98.8 Mb on GRCm38. Possible candidate genes were H2dafx,Rbm7, 2410076I21Rik, Mns1, Mei4, and Xrn1.
The Chr 13 QTL, Twq4 and Spcq3, 1.5 LOD support interval mapped between 11.4 and 115.9 Mb on GRCm38. Possible candidate genes were Spin1, Cks2, Trip13, Msh3 and Ddx4. [Table S4.]
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