| Silver carp (Hypophthalmichthys molitrix) is one of traditional and main-farmed freshwater fishes, which occupies important status in freshwater aquaculture in China. With the rapid development of molecular biology, the combination of molecular breeding and traditional breeding, which can not only improve the breeding efficiency but also protect the sustainable use of germplasm resources, has become a development trend in fish breeding. In the present study, we constructed linkage maps for silver carp using microsatellite DN A and AFLP markers, and studied the effect of the avoidance of sperm competition by the mixed milt fertilization on the recombination difference between the sexes and the depression of culturing performance. This study provides strong support for promoting the application of molecular genetics and breeding for silver carp.1.Development of microsatellite DNA markers for silver carpA (TG/AC)-containing fragment enriched plasmid library was constructed using FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) method. In total, 863 colonies were picked to sequence after screened by three PCR reactions. Of them, 640 were positive, containing 725 microsatellite repeat sequences. The number of perfect, imperfect and compound microsatellite repeat sequences was 508 (70.0%), 142 (19.6%) and 75 (10.4%), respectively. The most frequent type of repeats is di-nucleotide repeats, the copy numbers of which are mainly distribution between 10 and 19. Among them, GT/AC repeats is 656, accounting for 92.8% of the di-nucleotide repeats (707). Hexa-nucleotide repeats were not found. Subsequently,497 pairs of microsatellite DNA primers were designed. Of them,422 amplified successfully, which can be the candidate markers for construction of linkage maps for silver carp.2. Construction of linkage maps for silver carpA first microsatellite-based sex average linkage map of silver carp was constructed. It consisted of 233microsatellites (159 were developed in this study) assigned to 30 linkage groups (including 3 triplets and 4 doublets) that spanned a length of 812.5cM, covering 77.6% of the estimated genome size (1047.1cM). The maximum and average spaces between 207 loci were 21.7cM and 3.9cM respectively. The length of linkage groups ranged from 1.8cM to 66.1cM with an average of 27.1cM. The number of microsatellite markers per group varied from 2 to 22 with an average of 7.7.To improve the map quality, AFLP markers were also used. A combination of microsatellite and AFLP sex average linkage map and two parental maps were constructed. The sex average map contained 33 linkage groups (including 2 triplets and 4 doublets), to which 483 markers (245microsatellites and 238AFLPs) were assigned. The map spanned a length of 1352.2cM, covering 86.4% of the estimated genome size (1565.8cM). The maximum and average spaces between 420 loci were 21.5cM and 3.2cM respectively. The size of linkage groups ranged from 3.6cM to 98.5cM with an average of 41.0cM. The number of markers per group varied from 2 to 44 with an average of 14.6. The female parent map contained 42 linkage groups, to which 285 markers (167microsatellites and 118AFLPs) were assigned. The male parent map contained 26 linkage groups, to which 299 markers (175microsatellites and 124AFLPs) were assigned. The length of female parent map was 1404.4cM, which covered 75.8% of silver carp genome, while the length of the male parent map was 859.1cM, covering 83.0% of the silver carp genome. Across two parental maps, 131 microsatellites were assigned and distributed on 31 female and 21 male parent linkage groups. These loci bounded a set of common intervals which were 755.4cM and 345.5cM in female and male parents respectively. Comparison of the syntenic loci of two parental maps identified a recombination suppressed region on LG12, which may be associated with sex determination.3. Effect of sperm competition on the recombination difference between sexesThe balance between shuffling (through recombination) and preserving (through sperm competition) might determine the differential recombination between sexes. To test this assumption, the parental linkage maps of Cross A (♀bighead carp×♂silver carp) made with induced spawning and mixed milt fertilization were compared with those of Cross B (♀silver carp×♂silver carp) made with induced spawning and natural external fertilization. The length of the two parental maps of Cross A was similar (965.8cM vs.904.7cM), whereas those of Cross B was obviously different (1404.4cM vs.859.1cM). In Cross A, the total lengths of the common intervals bounded on both sides by crossly assigned microsatellites were 333.7cM and 328.7cM respectively (P=0.7436), whereas that of Cross B were 755.3cM and 343.8cM (P=0.0001). In comparison with Cross B where competition among sperms within an individual ejaculate in nature external fertilization caused the differential recombination between sexes, Cross A has not proportionally reduced the length of parental maps, which demonstrated the avoidance of sperm competition by the mixed milt fertilization may caused the loss of differential recombination between the sexes and the depression of culturing performance. Although the results of only this study cannot be a mature theory, the attempt by the new perspective with sperm competition and map construction provides new evidences for resolving those issues.
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