Construction Of RAOD-SCAR System For Anoectochilus Roxburghii (Wall.) Lindl And Their Utilization To Assess Genetic Diversity Of Germplasm

Anoectochilus roxburghii (Wall.) Lindlwas a rare medicinal plant in our country, which widely varying effects on inhancing immunity,anti-aging and decreasing blood sugar. The resources of Anoectochilus roxburghii (Wall.) Lindl were on the brink of extinctionalong with the excavation of a large number of wild resources in recent years. At same time, there were fuzzy genetic relationship among resources from differentecological regions, whichbrought many difficulties for the preservation and identification of Anoectochilus roxburghii (Wall.) Lindl resources. In this study,a set of 20 Anoectochilus roxburghii (Wall.) Lindlresources werecollected and their preservation technologies were in detail studied. Based on that, the RAPD-SCAR marker systems were developed to identify genetic relathionship of these resources collected in this study. The study results were as follow.1、The optimization of Anoectochilus roxburghii (Wall.) Lindltissue culture.Explant’sdisinfection was important step of tissue culture. Disinfection reagents and corresponding prossesing time had a significant influence on the vigour of explants. In this study, the sterilization methods of Anoectochilus roxburghii (Wall.) Lindl explants were optimized based on two factors (2%Sodium Hypochlorite and 0.1% mercuric chloride) and 5 levels (5min-25min with 5 min intervel) experiments. Each level set 20 bottles biological repeat, in which there were five stems in each bottle.After 40 days, the best treatment was captured through observing rate of the infection and germination for different treatments. As a result, the 0.1% mercuric chloride treatment for 10 minutes is the most optimized condition, in which the germination rate with this method was70%.The orthogonal experiment with 3 factors (MS,6-BA and NAA) and 3 levels were designed. That resulted in 9 experimental groups with 3 repeats under same culture condition. At the same time,the germination rate and average plant height were considered as indexesto evaluategrowth potential of Anoectochilus roxburghii (Wall.) Lindl stems derived from different groups. These two indexes were observed at 40 days,80 days and 120 days, respectively. The results showed that the growth potential of Anoectochilus roxburghii (Wall.) Lindl stems on 1 time MS with 2 mg/L NAA and 0.5 mg/L 6-BA were significantly higher than the other group in 40 days,80 days, 120 days.2、The optimization of Anoectochilus roxburghii (Wall.) LindlDNA extraction methods. As Anoectochilus roxburghii (Wall.) Lindl contain rich polysaccharide andpolyphenol, which seriously interferenced the quality of Anoectochilus roxburghii (Wall.) Lindl. To ensure quality of DNA in subsequent marker and clone experiment, the extraction method wasoptimized by adjusting extraction steps and modifying extraction reagents on basis of traditional CTAB extraction method. The result showed that it could significantly reduce the polysaccharide andpolyphenol remains. When the NaCl and PVP contention in the extraction regents were changed into 2.0 mol/L and 4% level respectively.Simultaneously, the step reprecipitating DNA fortwo times by high salt TE(100 mmol/L Tris-HC1,pH 8.0; 20 mmol/LEDTA, pH 8.0; 1.4 mol/L NaCl) could bring a beneficial improvment for DNA quality. In addition, the remain of polyphenol were obviously reduced when regents with phenol:chloroform:isoamyl alcohol (25:24:1)were replaced by chloroform:isoamyl alcohol (24:1) in extraction step. The high quality DNA obtained by CTAB optimizational method provided an important basis for DNA molecular marker developing.3、Construction of RAPD-SCAR system for Anoectochilus roxburghii (Wall.) Lindland their utilization to assess genetic diversity of germplasm. RAPD-SCAR marker had characteristics of efficient,high repetition rate and stability.In this study the polymorphic RAPD (Random Amplified Polymorphic DNA) and specific SCAR markers were developed based on 20 different germplasms from various places.28 of 100 RAPD primers had significant polymorphism and generated 135 polymorphic bands among 20 germplasms. On the basis of RAPD results,20 germplasms were clustered into 6 groups on genetic distance of 0.748. Clustering analysis showed that there were significant genetic differences among germplasms derived from different regions. A total of 5 specific bands from RAPD results were transferred into SCAR (Sequence Characterized Amplified Region) markers. Amplified results of SCAR markers among different germplasm showed that SCAR markers are significant specific to different germplsm. This study laid a solid foundation for accelerating breeding of Anoectochilus roxburghii (Wall.) Lindl.