Molecular Marker Identification And Superior Plant Screening In Anoectochilus Roxburghii.

Anoectochilus roxburghii is a rare and endangered medicinal plant of the genus Anoectochilus in the family Orchidaceae,with the effects of clearing heat,cooling the blood,strengthening the body and pacifying the liver,etc.It is known as the"King of Medicine"in folklore.Fujian Province is one of the major production areas of A.roxburghii in China,and it is also widely cultivated in the southwest of China.However,in the production process,the quality of A.roxburghii varies because of the complicated strains,irregular cultivation techniques and difficulties in identification.Therefore,it is important to investigate the genetic relationship between different A.roxburghii germplasm resources and to innovate the screening techniques for the identification of superior A.roxburghii plants for the establishment of the A.roxburghii variety selection system.This study collects Fujian Province native A.roxburghii varieties and their closely related genus materials to lay the foundation for A.roxburghii variety selection through germplasm resource identification and evaluation,identification and screening techniques for hybrid progeny,polyploid superior plants and in vitro flowering techniques to provide technical support for A.roxburghii variety selection and breeding.The research of this project will help the breeding of goldenrod and promote the development of A.roxburghii industry.The specific research contents and results are as follows:1.Ten germplasm resources of A.roxburghii were collected and analyzed for morphological index determination and for medicinal content determination.The 10 germplasm resources could be classified into two categories based on the color of the leaf reticulation veins,and differentiated according to the stem and leaf traits.The raw medicinal indexes of different strains of A.roxburghii differed significantly,and the best overall quality was concluded from the scoring of the main component quality traits for the native variety’Wuyi No.1’from Wuyishan,Fujian.Exploring the effect of light quality on the growth and quality of A.roxburghii,the results showed that red light（R2:B1）had a general promoting effect on the morphological indices of A.roxburghii such as plant height,leaf length and leaf width,while blue light（R1:B2）had a general inhibiting effect.The effect of light quality on medicinal content varied considerably depending on the strain.2.Establish the A.roxburghii ISSR-PCR and CDDP-PCR reaction system:25μL,12.5μL 2×Taq Master Mix,1μL 10μmol/L primer,1μL DNA（50 ng/μL）,and add sterilized dd H₂O to make up.The reaction procedure was 95℃,5 min;95℃,30 s;45-60℃,30 s;72℃,1.5 min;35cycles;and the final extension at 72℃ for 10 min.Using the four selected ISSR primers and nine CDDP primers to analyze the germplasm resources of A.roxburghii,the clustering results of ISSR and CDDP association analysis showed that the genetic distance of the 10 germplasm resources ranged from 0.38 to 0.85,the genetic distance of ISSR clustering results ranged from 0.33 to 0.96,and the genetic distance of CDDP clustering results ranged from 0.38 to 0.81.3.We used ISSR-PCR and CDDP-PCR molecular markers to identify the parents and hybrid offspring of’Wuyi No.1’with and without leaf veins,and screened four ISSR primers and six CDDP primers to effectively identify the differences between hybrid parents,and two of them,UBC866 and WRKY-R3B,were able to effectively identify the hybrid offspring.From the population of hybrid offspring（F1 generation）,nine good monocultures were screened,and the morphological and medicinal contents of the nine monocultures were compared,and two optimal hybrid monocultures were identified:U11-1 and U12-3.4.Eight polyploid monocultures were identified by microscopic observation of chromosome entries in the pre-induced polyploid plants;the main morphological indices such as plant height,stem thickness,leaf length and leaf width of these monocultures were measured,combined with the analysis of three main medicinal contents of goldenrod,namely flavonoids,total polysaccharides and goldenrod glycosides,and finally the 7D-1 polyploid superior plant was selected.5.We conducted a study on the flowering technology of’Wuyi No.1’,’Gongxiang No.2’,’Gongxiang No.5’and’Gongxiang No.6’in vitro.The flowering rate was calculated from the flowering seedlings and the cross fertility rate was calculated from the flowering seedlings of’Hongxia’.The results showed that the flower bud differentiation rates of ’Hongxia’,’Gongxiang No.6’and’Wuyi No.1’were 78.6%,57.1%and55.6%,respectively,and the highest fruiting rates of the hybrid combination Hongxia×Wuyi No.1and the hybrid combination Hongxia×Gongxiang No.5 were 25%,Meanwhile,the seed germination rate of Hongxia×Wuyi No.1 was 66.7%.