Construction Of Genetic Linkage Map For Hypophthalmichthys Molitrix And Megalobrama Amblycephala Using Outbreeding Mapping Strategy And RAPD Markers

1. Producing Mapping Population by Outbreeding and Using RAPD Fragments as Genetic Markers A set of 100 random 10-base oligonucleotide primers was amplified for random amplified polymorphic DNA (RAPD) fragments within outbreed heterozygous parents Hypophthalmichthys molitrix, Megalobrama amblycephala and their progeny. In these 100 primers, a total of 58 yield visible sequence polymorphisms in the particular cross. The polymorphismic ratio between parents was 59.1%. Markers presented in the progeny were all inherited from their parents. 54 polymorphic locus fit to expected Mendelian ratio and the frequency of segregation distortion was low (3.6%) These outbred progeny are available in research on constructing fish genetic map. 2. Constructing Fish Genetic Maps Using a Outbreeding Mapping Strategy in Combination With the Random Amplified Polymorphic DNA (RAPD) Markers We have used an outbreeding mapping strategy in combination with the random amplified polymorphic DNA (R.APD) assay to construct two genetic linkage maps for Hypophthalmichthys molitrix and Megalobrama amblycephala. In the cross between two heterozygous individuals many R.APD markers will be 3 heterozygous in one parent, null in the other and therefore segregation 1:1 in their Fl progeny following a testcross configuration. Meiosis and segregation in each individual can be directly and efficiently analyzed using RAPD markers. We screened 100 primers of arbitrary sequence and selcct~.28. tQ ~rnplify a total of SO markers. These markers were grouped at LOD~3.O, 0 ~O.38, resulting in thednaternal Hypophthalmichthys mojifrix map having a total of 18 makers into S linkage groups (352.9cM) and the paternal Megalobrama amblycephala (282.5cM) map with 14 markers in 4 groups. Framework maps ordered with a likelihood support ~ 1000:1 were assembled covering 54.1% and 45.8% of the estimated Hypophthalmichthys molitrix and Megaiobrama wnblycephala genomes, respectively. We propose the combined use of RAPD markers and the outbreeding configuration as a general strategy for the construction of single individual genetic linkage maps in outbreed fish as well as in any highly heterozygous sexually reproducing living organism. It can be immediately applied to map any species without any prior genetic information. The only requirement is sexual reproduction between two individuals that result in the generation for a progeny large enough to allow the estimation of recombination frequencies between segregating markers. The method of analysis employed here should be applicable to many other outbrcd heterozygous species for which defined inbred lines are not available or need long time. 3. Optimizing Rtacting Condition of RAPD in Fishes RAPD markers do not require prior sequence information and can be viewed directly by agarose gel electropboresis without the need of specific probe libraries and radioisotope detection. The shortcomings of RAPD markers are their reliability and the transferability between laboratories. So it is very important to optimize the reacting condition before any formal amplification. We studied some essential factors affecting RAPD patM u~hI~kuldke optimal condition of ~mp1ification for RA.PD assay on fish in our laboratory. In 25 ul reaction solution, there were 10mM Tris-HCI (pH8.0), 10mM KC1, 8mM (NH4)2S04, 2mM MgC12, 0.05% NP-40, 0.2mM dNTP, 2Ong random...