Construction Of Genetic Linkage Map For Hunchuntao×Zhengmanzaosheng Using AFLP Markers

Hunchuntao X Zhengmanzaosheng genetic linkage map has been constructed with the 32 F| population by optimized AFLP marker technique. The result indicated that:Constructed peach AFLP technique system. Complete digestion system: in 25μl total reaction volume, including 500ng DNA, 5U EcoR I and 5U Mse I restriction enzymes, 2.5μl 10. One-Plus All Buffer, 37癈 digested 3~12h; Linkage adapter system: in 50μl total reaction volume, including 25 full digestion results, 2.5μl 10 X Buffer 30ng EcoR I and 50ng Mse I adapter, 1 ~3U T4 DNA ligase, 37癈 linked 3h; Preamplificated system: in 50μl total reaction volume, including diluted 10. linkage adapter results, 5μ1 10XPCR buffer, 1.75mM MgCl2, 0.25mM dNTP, 30ng E+l and 30ng M+l Primer, 1U Taq DNA polymerase, initial denaturation at 94℃ 5min, 30 cycles of PCR reaction at 94℃ 30s, 56℃ Imin, 72℃ lmin; Selective amplification system: in 20μl total reaction volume, including 5μl diluted 10X preamplification results, 2μ1 10 X PCR buffer, 1.5mMMgCl2, 0.2mM dNTP, 15ng E+3 and 30ng M+3 Primer, 0.4U Taq DNA polymerase, initial denaturation at 94 ℃ 5min, 13 cycles of PCR reaction at 94℃ 30s, 65℃ Imin, 72℃ Imin, followed by 33 cycles of 94℃ 30s, 56℃ 30s, 72℃ Imin.In this study 16 primer combinations which could have clear amplified bands type and high polymorphism were screened, amplifying 750 bands between two parents and FI population, each primer generate 46.88 bands on average-, the whole polymorphic bands were 172, the average was 10.75, the rate of polymorphism testing was 22.94 percent on average.AFLP markers' segregation patterns in Hunchuntao X Zhengmanzaosheng F] were divided into three types: Mendelian separation, distorted separation, abnormal separation. It accounted for 11.07, 11.87 and 3.86 percent on total marker sites, 48.26,51.74 percent on segregation sites respectively. So, it was feasible to construct Hunchuntao saturated linkage map with this population.There were 12 linkage groups in this map, including 28 distorted markers and 38 Mendelian markers, the length was 358.17cM, the average distance was 5.43cM among markers. The longest one had 13 distorted marker and 20 Mendelian markers, the length was 86.48cM, the average distance was 2.62cM; the shortest one had only 2 Mendelian markers, the length was 6.26cM, the average distance was 3.13cM. The largest genetic distance among linkage groups was 28.26cM, the smallest was O.OlcM.