249 AGRIVITA VOLUME 36 No. 3 OCTOBER - 2014 ISSN : 0126-0537 CLASSIFICATION OF POPULATION STRUCTURE FOR ALLELOPATHIC PROPERTIES IN ITCHGRASS (Rottboellia cochinchinensis) Apirat Bundit1), Anyamanee Auvuchanon2) and Tosapon Pornprom1*) 1) Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand 2) Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand *) Corresponding author E-mail: [email protected] Received: September 23, 2014/ Accepted: October 27, 2014 ABSTRACT INTRODUCTION Biodiversity of itchgrass from different areas were A synergistic relationship between the studied by morphological traits and AFLP allelopathic potential and genetic, chemicals, and analysis for classify of an allelopathic ability. The ecological factors, not only affects both the stable correlation of the similarity/distance between and unstable allelopathic potentials, but also AFLP markers (Jaccard coefficient) and morpho- relatively strong or weak allelopathic abilities (Zuo logical traits (Euclidean distance) was significant et al., 2007). Allelopathic activity was identified in with r = − 0.84**. Itchgrass were divided into two many plant species. Such as Avena sativa L. groups from both UPGMA and STRUCTURE (Kato-Noguchi et al., 1994), Helianthus annuus L. analyses: the group A consisted of itchgrass from (Ohno et al., 2001), and Rottboellia exaltata L. f. Chaehom-Lampang, Si Thep-Phetchabun, Phrom (Kobayashi et al., 2008, Meksawat and Phiram-Phitsanulok, Amphur Muang-Nakhon Pornprom, 2010), have been found to have Sawan, Kamalasai-Kalasin, Amphur Muang- allelopathic properties. However, the age and Chachoengsao and Bang Yai-Nonthaburi, genotype, location or environment, and cropping whereas itchgrass from Amphur Muang-Chiang system, all of which influence the production of Mai, Pak Chong-Nakhon Ratchasima and allelochemicals that impact on plant growth, in Kamphaeng Saen-Nakhon Pathom constituted the field, laboratory, or greenhouse (Weston et the group B. Allelopathic properties of itchgrass al., 2013). Therefore, a better understanding of as representative from different group were the allelopathic potential of physiological, determined in bioassay test, the aqueous extract biochemical, and genetic levels will advance our of itchgrass from Chaehom-Lampang area has a knowledge on the role of allelopathic properties. strong allelopathic ability on growth of Echino- Itchgrass [Rottboellia cochinchinensis chloa crus-galli, Bidens pilosa and Lactuca sativa (Lour.) W.D. Clayton] is an annual upland weed, than the other group. Molecular analysis was self-pollinated, and widely distributed in the strongly supported in morphological clustering tropical and subtropical regions (Alloub et al., with bioassay test, specific morphological traits 2005, Alves et al., 2003, Millhollon and Burner, were soft trichomes, dark purple stems and roots 1993). It is a dominant species in some regions which can be used for the preliminary including Thailand where has a serious weed classification of allelopathic ability. This suggests problem from itchgrass in several crops. that itchgrass classification by morphological Conversely, farmers in Chaehom-Lampang area in traits is related to the analysis of the genetic northern Thailand cultivated itchgrass as a relationship of itchgrass with AFLP analysis, that mulching material for weed control in vegetable allowing the assessment of bio-diversity of fields (Kobayashi et al., 2008, Meksawat and itchgrass and their allelopathic potentials. Pornprom, 2010). Itchgrass is a wide diversity in the morphological, biological, and physiological Keywords: AFLP analysis, allelopathy, genetic features between itchgrass populations in diversity, itchgrass, morphological Thailand. The climatic, edaphic, and biotic factors traits can affect the morphological and physiological Accredited SK No.: 81/DIKTI/Kep/2011 http://dx.doi.org/10.17503/Agrivita-2014-36-3-249-259 250 Apirat Bundit et al.: Classification of Population Structure for Allelopathic Properties……………………………………… characteristics of weeds and cause the evolution classification of the population structure. Each of new weed biotypes (Zimdahl, 1993). Thus, the location were represented a distinct geographical appearance and dispersal of itchgrass in different region in each part of the different areas. For areas needs to be considered to classify itch- example, Chaehom-Lampang (CH-LP) and grass accessions into certain phenoltypic groups Amphur Muang-Chiang Mai (AM-CM) were relevant to itchgrass management. Millhollon and located in northern region. The sampling areas in Burner (1993) analyzed itchgrass collected from central region were Phrom Phiram-Phitsanulok 34 countries and identified five biotypes based on (PR-PL), Si Thep-Phetchabun (ST-PB), Amphur general morphologies and growth patterns. Alves Muang-Nakhon Sawan (AM-NS), Bang Yai- et al. (2003) classified itchgrass collected from six Nonthaburi (BY-NB), and Kamphaeng Saen- Brazilian regions using both molecular marker Nakhon Pathom (KPS-NP). Pak Chong-Nakhon and morphological traits. Furthermore, three Ratchasima (PC-NR) and Kamalasai-Kalasin (KL- major groups were reported in Peninsular KS) itchgrass were located in north-east region Malaysia based on the variations in growth whereas Amphur Muang-Chachoengsao (AM- behavior of itchgrass (Alloub et al., 2005). CS) was situated in east region (Figure 1). The Techniques based on DNA sequencing seeds were stored at 7°C until the initiation of the have great accuracy in taxonomic studies, and experiment. amplified fragment length polymorphism (AFLP) The itchgrass-powder was prepared from markers are used to determine genetic variation mature itchgrass plants, which were grown under across different populations (Powell et al., 1996). uniform environmental and agronomical condi- AFLP analysis was successfully utilized to reveal tions. They were harvested at maturity and then genetic diversity among Echinochloa species separated into shoot and root. These plant (Tabacchi et al., 2006). To assess genetic diver- portions were individually cut and air-dried at sity, AFLPs were applied on Glycine max L. Merr. 40°C for one week, and ground into powder with (Tara Satyavathi et al., 2006) and Triticum an electrical grinder to pass a 0.5 mm screen aestivum L. (Eivazi et al., 2007). Molecular mark- mesh and stored in plastic bottles at −20°C. ers can establish associations between genotype Three test plant species such as Bidens and morphological traits. Zhao et al. (2007) pilosa L. var. radiata Sch. Biq., Echinochloa crus- reported the genetic variation of Brassica rapa galli L. P. Beauv., and Lactuca sativa L. var. OP with AFLP and the morphological data. Kantartzi were used in bioassay test. Test plant seed were and Stewart (2008) identified 58 cotton germ- germinated (radicle~0.02 m) before used in the plasms to evaluate fiber trait and their diversity by experiment. means of DNA markers. Skot et al. (2005) studied the heading date of natural populations of Morphological Traits perennial ryegrass and identify genetic diversity A single itchgrass seedling in each location with AFLP markers. Thus, a formal compre- was transplanted to a pot and placed in a hension of the genetic diversity and morpho- greenhouse under controlling condition. The data logical traits is called for the purpose of the were collected at different growth stages of classification of the allelopathic potential of itch- itchgrass that included the 2−3 leaf stage, grass. The limited information is available on the tillering, flowering, and maturation with measure- morphological and genetic diversity of itchgrass in ments of inflorescence length, leaf length, plant Thailand is not fully understood, in each morpho- height, number of seeds: inflorescence, 1000 type may have different allelopathic abilities. seeds weight, seed size, trichomes, leaf blade Therefore, the objectives of this study were taken color, leaf sheath color, stem color and root color. in order to determine genetic diversity, population The morphological characteristic data were structure, and a potent allelopathic of itchgrass standardized and the Euclidean distance co- using morphological and AFLP markers. efficients were calculated for all pairs of the samples using the similarity of interval data MATERIALS AND METHODS (SimInt) module of the software package. The cluster analysis was performed according to the Plant Materials unweighted pair group method with arithmetic Itchgrass seed from ten areas within the averages (UPGMA method) based on: trichomes, main crop-growing regions were investigated for the color (ligule, leaf sheath, and stem), as well 251 Apirat Bundit et al.: Classification of Population Structure for Allelopathic Properties……………………………………… as seed size (width, length, and 1,000 seed frozen leaf was used for DNA extraction with the weight) with the SAHN algorithm. The goodness modified method as described by Doyle and of fit of the clustering compared with the basic Doyle (1987), and Cullings (1992). The DNA data matrix was tested by computation of the analysis using AFLP markers was conducted as cophenetic correlation coefficient using the described by Vos et al. (1995). The isolated normalized Mantel statistics Z test through the genomic DNA was digested with EcoRI and Coph and MxComp algorithm. All commands MseI restriction enzymes, a total of five AFLP