Amplified Fragment Length Polymorphism (AFLP) Analysis Of Genetic Variation And Cloning Of Allelopathy-Related Gene From Eupatorium Adenophorum

Eupatorium adenophorum Sprengel [Synonym: Ageratina adenophora (Sprengel)](Asteraceae), originally found in Mexico and Costarica, has rapidly spread and invaded about30 countries and regions at tropical and subtropical zones. In these regions, it has causedserious economic loss and threatened native biodiversity, ecosystem integrity and even thehealth of human and livestock. It was introduced into China from Burma in 1940s for the firsttime. Now it has been widespread to most of south-western China and formed steadygeographic patterns of variations to certain extent. It has most representative characters ofinvasive alien plants in China. In the present study, the AFLP technique was used tocharacterize genetic variability and partition of E. adenophorum populations throughoutsouth-western China. Furthermore, geographic patterns of variations were also determined.The results obtained may provide useful information on the life history and invasiveness ofthe weed. Besides, the F3'H gene of E. adenophorum was cloned and sequenced and therelationship between F3'H and allelopathy ofE. adenophorum was discussed. The results maybe helpful to interpret the invasive mechanism of E. adenophorum, and thus can be aguideline in sustainable controlling its further spread and expansion. The main results wereshown as follows.1. The AFLP markers proved an efficient method for detecting genetic variation amongdifferent geographic populations of E. adenophorum. The 3 primer pairs selected from the 64primers kit produced a total of 490 bands in the 62 E. adenophorum populations in China, ofwhich 328 bands were polymorphic (PPB=66.9%). Diversity levels within populations wererelatively high (mean expected heterozygosity H=0.15, mean Shannon index I=0.236).These indices at the species level were H=0.171 and I=0.273. The estimation of geneticdiversity showed that E. adenophorum populations at Yunnan province were more diverse,and those at Chongqing were less diverse.2.. Genetic variation (Gst=0.287) at nucleus DNA level were detected among the 62 E.adenophorum populations, indicating the genetic diversification among E. adenophorumpopulations at various habitats. The AMOVA analysis showed that 70.25% genotypic variation existed within populations, 8.04% variation was due to regional differences, and theremaining 21.71% was due to population differences among populations within the provincialregions.3. Cluster analysis based on the UPGMA method and principal coordinate analysis (PCO)grouped majority of the weed populations into four main clusters that corresponded with thegeographic regions. The regression analysis indicated a significant positive relationshipbetween Shannon genetic diversity and elevation (R²=0.256, P＜0.005), where geneticdiversity increased with increasing elevation. However, there was a negative correlationbetween Shannon genetic diversity and latitude, as well as between genetic diversity andlongitude of its habitat. Mantel test indicated that there was a positive correlation betweengeographic and genetic distances among populations (r=0.37, p=0.0004). It is concludedthat genetic diversity level of E. adenophorum in newly invaded area was lower than that informer invaded areas.4. Based on the genetic diversity of different populations and the results of cluster analysis,we speculated that the seeds of E. adenophorum were dispersed mainly by wind and water.The relatively high genetic differences of E. adenophorum within populations may largely bea result of multiple introductions from different sources. Since it was introduced into theCangyuan county of Yunnan from Burma firstly, E. adenophorum have spread towardnorthern and eastern Yunnan gradually. Some populations spread northward were dispersed toPanzhihua, then to Liangshan of Sichuan province; the others dispersed to Yibin of Sichuan,then to Chongqing province through the Yangtze River by waterflow. Most populations spreadeastward were dispersed from Simao and Wenshan of Yunnan to Baise of Guangxi, then to thesouth of Guangxi and the center of Guizhou.5,The flavonoid 3'-hydroxylase gene(F3'H) was cloned with RACE kit. The completenucleotide sequence of F3'H gene of E. adenophorum contains 1722 base pairs, deducing 530amino acids(NCBI Genebank accession No. EF137714). It was comprised of 5'-noncodingregion(37bp), coding region(1533bp) and 3'-noncoding region (252bp). The molecular massof the deduced protein is 56.8kDa and the isoelectric point is 6.44. The homology rate ofamino acid sequences of F3'H gene between E. adenophorum and Callistephus chinenis was 64.4%. Then the F3'H gene was classified to the CYP75B subfamily of cytochrome P₄₅₀.6. Southern blot analysis showed that there was one copy of F3'H in E. adenophorum.Northern blot analysis showed that the highest expression level of F3'H was detected in theleaf, the medium level in the stem, and the lowest in the root. The expression of F3'H can beinduced by 2mmol 9-Oxo-agerphorona, impling that F3'H gene was related to allelopathy ofthis weed. SDS-PAGE analysis of IPTG induced recombinant E. coli showed that a predicted56.8 kDa fusion protein was expressed in the culture and the expression level increased withthe extention of treated time.The results provide proofs to reveal the invasion history as well as expansion pathwaysof E. adenophorum, thus may be used as groundwork in sustainable control of its furtherspread and expansion.