| This paper applies uniform design and orthogonal design methods to set up the target region amplification polymorphism-PCR (TRAP-PCR) system that suitable for Magnolia officinalis subsp.biloba. Then using the TRAP optimization system to study16different provenances of M.officinalis subsp.biloba in the nationwide main producing areas of M.officinalis subsp.biloba. Through that the TRAP fingerprints of various sources of M.officinalis subsp.biloba are constructed. And further the genetic diversity and genetic relationship of various sources of M.officinalis subsp.biloba are analyzed. On the other hand, the main seedling characters of12provenances of M.officinalis subsp.biloba are analyzed and screening out the fine provenance according to the seedling monitoring value. The study mainly gets the following conclusions:一、TRAP analysis of16provenances of M.officinalis subsp.biloba1、The25μL TRAP-PCR reaction system that suitable for M.officinalis subsp.biloba contains40ng template DNA,1.5mmol·L-1of Mg2+,0.28mmol·L-1of dNTPs,0.3μmol·L-1of fixed primer and arbitrary primer and0.75U Taq DNA polymerase. PCR program:predenaturing5min at94℃; the first five cycles run at94℃,lmin,40℃,lmin, and72℃,lmin, for denaturing, annealing and extension, respectively; Then the annealing temperature is raised to50℃for another38cycles; Run10min at72℃for extension2、The TRAP amplifications for16provenances of M.officinalis subsp.biloba by16pairs of primers screened from72pairs of TRAP primers detect180loci which are clear, stable and reproducible in total, among which146loci are polymorphic. The average polymorphism rate is81.1%. Fragment numbers of each primer pair can detect are between7-15, with an average of11.25. Amplification results show that the polymorphism between provenances of M.officinalis subsp.biloba is rich.3、The numbers of polymorphic bands that each pair of primer from the screened16pairs TRAP primers can amplify out are between4-13, each pair can amplify out9.13on average; Amplification band types of each primer pair are7～16, with an average of13. The technology of TRAP has strong ability of germplasm identification. The digital1D codes of16provenances of M.officinalis subsp.biloba are established based on9TRAP primer pairs Leafy/Eml、GAI1/Em8、psbA-trnH/Em6、Leafy/Em4、 Leafy/Em6、Leafy/Em9、psbA-trnH/Em5、rbcL/Em4and rbcL/Em8.4、Results of TRAP analyses of genetic diversity and genetic relationship for16provenances of M.officinalis subsp.biloba by Popgene32:The number of alleles is1.8111, the efficient alleles is1.4950, the index of genetic diversity is0.2873, the Shannon’s information index is0.4290and the total genetic diversity is0.2873. The genetic similarity is between0.5611to0.8389, with an average of0.6935; The genetic distance is between0.1757to0.5778, with an average of0.3702. There have abundant genetic diversity and large genetic differences among the provenances of M.officinalis subsp.biloba. The genetic relationship between provenances WFJya and WFJpn is the most recent and the genetic relationship between provenances WFJmx and WAHhf is the farthest.5、Clustering with UPGMA based on the TRAP genetic distance among provenances. The clustering result:The16tested provenances of M.officinalis subsp.biloba can be divided into three categories at the genetic distance of0.3714. The first category:WSCpzh、WAHhf、WHByc、WJSnj、WJSlyg、WHNxX、 WHNdx and WSXxa; The second category:WFJsc; The third category:WFJya, WFJpn、WFJmx、WJXda、 WJXls、WHBns and WSCmy. The results will be not very reliable if analysis of genetic differences based solely on geographic origin.二、 Seeding test of12provenances of M. officinalis subsp.biloba1、The difference levels of M.officinalis subsp.biloba seedling height among provenances and months are all significant. The6provenances WJSlyg、WHByc、WAHhf、WHNxx、WHBns and WSXxa have better growth potential of seedling height. WJSlyg is the best, followed by WHByc; WJXda is the worst, followed by WJXls. The growth of seedling height is relatively slow between June and July. The growth more concentrated in the7-9months, and then slows down after September. Differences of seedling height among provenances are mainly caused by the genetic factors. It is reliable for seedling height as the evaluation index of seedling selection.2、The difference levels of M.officinalis subsp.biloba ground diameter among provenances and months are all significant. The4provenances WHByc、WHBns、WJSlyg and WSCmy have better growth potential of ground diameter. WHByc is the best, followed by WHBns; WJXda is the worst, followed by WJXls. The growth of ground diameter is relatively slow between June and July and then to speed up from July. The growth period of ground diameter of M.officinalis subsp.biloba is longer than seedling height. Differences of ground diameter among provenances are mainly caused by the genetic factors. It is reliable for ground diameter as the evaluation index of seedling selection. Compared ground diameter with seedling height, seedling height as the evaluation index of seedling selection is more reliable.3、The correlations between seedling height、ground diameter and geographical and climatic factors of M.officinalis subsp.biloba:The seedling height has positive correlation with latitude、longitude and average annual sun exposure and negative correlation with average annual temperature and frost-free days. And the correlations are not remarkable overall. Among them north-south distribution has larger effect on seedling height of M.officinalis subsp.biloba. The ground diameter has negative correlation with latitude、 average annual temperature、average annual rainfall、average annual sun exposure and frost-free days and positive correlation with longitude. And the correlations are not remarkable overall. The west-east variation of ground diameter is more obvious than seedling height.4、The correlations between seedling height、ground diameter and biomass of M.officinalis subsp.biloba:The seedling height has significant positive correlation with shoot fresh weight、root fresh weight、total fresh weight、shoot dry weight、root dry weight and total dry weight, weakly positive correlation with root/shoot ratio and weakly negative correlation with seedling moisture content. The ground diameter has significant positive correlation with shoot fresh weight、root fresh weight、total fresh weight、shoot dry weight、root dry weight and total dry weight, weakly positive correlation with root/shoot ratio and significant negative correlation with seedling moisture content.5、The seedling biomass of M.officinalis subsp.biloba that the values of biomass indexes are significantly different among provenances. The correlations between seedling biomass and geographical and climatic factors of M.officinalis subsp.biloba are not significant. Its geographic variation pattern has great similarity with that in seedling height、ground diameter.6、The relative content of chlorophyll in leaves at seedling stage of M.officinalis subsp.biloba: Differences of relative content of chlorophyll in leaves are mainly caused by the genetic factors and the differences are significant. The correlations between relative content of chlorophyll in leaves and biomass indexes are not remarkable overall. The relative content of chlorophyll in leaves has no significant effect on biomass.7、The clustering results based on seedling height、ground diameter that the12tested provenances of M.officinalis subsp.biloba can be divided into four categories. The first category:WJXda、WJXls and WFJsc; The second category:WJSnj、WAHhf、WHNxx、WSXxa and WSCpzh; The third category: WSCmy; The fourth category:WHByc WJSlyg and WHBns. Preliminary screening out three fine provenances of M.officinalis subsp.biloba:WHBy、WJSlyg and WHBns. And three poor provenances of M.officinalis subsp.biloba:WJXda、WJXls and WFJsc. |
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