| Magnolia officinalis belongs to Magnolia genus Magnoliaceae family, one of China’s three major woody herbs, which widely distributed in subtropical and north tropical regions alongwith the Yangtze River. In the 1980 s, M.officinalis had been excessive excavated because of its important medicinal value. The number of wild plants reduced drastically, and their habitat presents fragmentation. Recent studies show that gene flow among natural populations of M.officinalis is blocked. Facing the anxious survival situation,it is imminent to carry genetic diversity conservation and restoration work for M.officinalis.We conducted pollination tests between different groups of M.officinalis based on the genetic diversity of Magnolia officinalis. This paper studied the genetic diversity and relationships of M. officinalis Hybrid parenst and offspring with SSR molecular marker, and revealed genetic diversity differences between hybrid parents and the offspring at the molecular level. The main results are as follows:(1) Primers Screening and reaction system optimization of M.officinalisFactors affecting PCR reaction(DNA templates, primers, Mg2+, d NTPs and Taq polymerase) were analyzed using an orthogonal experimental design based on SSR primers screening. 13 pairs of SSR primers revealing clear bands and rich polymorphism were successfully selected. The impact of factors on PCR reaction system was found as follow: primer > Taq enzyme > Mg2+ > d NTPs > template DNA. The optimal PCR system for SSR analysis was 2 ng?μL-1 template DNA, 0.6 μmol?L-1 primer, 2.0 mmol?L-1 Mg2+,0.5 mmol?L-1 d NTPs and 0.03 U?μL-1 Taq polymerase in 25 μL reaction solution, and the augmentation procedure was predenaturation at 94 ℃ for 4 min, denaturation at 94 ℃ for 30 s, annealing at 48~59 ℃ for 30 s, extension at 72 ℃ for 1 min, reaction with 35 cycles, and extension at 72 ℃ for 10 min.(2) Genetic diversity analysed of M. officinalis hybrid parents, hybrids progeny and free pollinated progeny.To investigate the gene divisity in hybrid progeny between different populations, hybrid parents and free pollination progeny of M.officinalis,13 microsatellite primers were screened out for our study. The results showed that:the 13 microsatellite primers can stably amplify in all individuals with a total of 73 bands amplified. The order of gene divisity was hybrid progeny(I=1.4544,Nei=0.7193,He=0.7287) > hybrid parents(I=1.3958,He=0.6955,Nei=0.6899)> free pollination progeny(I=1.2118,Nei=0.6423,He=0.5910). Only two locus( M6D10 and Stm0246) had a negative Fis values among the the totle 13 locus in free-pollinated progeny, and the remaining were positive.It displayed homozygotes excess while heterozygotes obviously insufficient that there may be a higher degree of inbreeding in free-pollinated progeny. It showed a positive correlation between hybrid parents’ genetic distance and hybrid progeny in genetic diversity, but the correlation has not reached significant level(r=0.3924). There was a more closer relationships between hybrid progeny and male parent than female parent in UPGMA clustering. The higher genetic diversity in hybrid progeny than free pollination progeny showed a feasibility of the protection measure by artificial control pollination to accelerate gene flow between populations.It provided some references for parent selection when we take hybridization as a endangered species protection measure. The research results laied the theoretical basis for conservation biology research of M. officinalis and other recession species gene diversity recovery and population reconstruction. |
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