April - June 2007 - Kasetsart University

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216 min at 56°C, and 24 cycles of 1 min at 72°C, followed by an extension of 5 min at 72°C. Amplification products were diluted in 100 µl deionized H 2O and 2 µl were used for selective amplification in a total volume of 10 µl containing 1 µmol/L of 10X PCR Buffer, 5 µmol/L of Especific primer extended by 3 selective neucleotides (Table 2), 5 µmol/L of M-specific primer extended by 3 selective nucleotides (Table 2), 1 U of Taq DNA polymerase (Fermentas, Lithuania) and 1 µmol/L of each dNTPs. PCR was performed using a touchdown protocol with initial denaturation of a cycle of 30 s at 94°C, 30 s at 65° C (decreasing the temperature by 1°C after each cycle until 57°C) and 1 min at 72°C, followed by 30 cycles of 30 s at 94°C, 30 s at 56°C and 1 min at 72°C with a final extension of 4 min at 72°C. Following amplification, 10 µl of formamide loading dye was added to the PCR products. The products were electrophoresed on 8% nondenaturing polyacrylamide gel. The bands were visualized using silver stain. Data analysis Each accession was scored (1) for presence and (0) for absence of each polymorphic band. AFLP bands within accessions were scored as missing if they were poorly resolved on the gel or if the template DNA did not amplify well. Similarity coefficient was calculated on the basis of Dice similarity coefficients (Dice, 1945) and is <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) written as Cjk = 2a/(2a+b+c) In which Cjk is similarity coefficient, a is number of AFLP markers present in both j and k accessions, b is number of AFLP markers present only in j accessions and c is number of AFLP markers present only in k accessions. The similarity matrix was subjected to cluster analysis by the unweighted pair-group method with arithmetic mean (UPGMA) and a dendrogram was created using the NTSYS-pc version 2.01d program (Rohlf, 1997). RESULTS Five informative AFLP primer combinations generated a total of 364 reproducible amplification fragments across all species of Amomum, among which 122 bands were polymorphic (Table 2). The number of amplified AFLP bands per primer pair varied from 66 to 81 with an average of 72.8 bands. The average number of polymorphic bands detected was 24.4 per primer combination. The fragment sizes were determined by comparing each one with the standard DNA ladder, ranging from about 140 to 726 base pairs (bp). Two primer combinations (E- AGG, M-CAA (Figure 2) and E-ACC, M-CAA) produced 30 polymorphic bands, a relatively higher numbers of polymorphisms compared to the other primers used in this study. Table 2 AFLP primer pairs and their number of amplified and polymorphic bands for phylogenetic study of Thai Amomum. Primer combinations Amplified bands No. of polymorphic bands (EcoRI+3/MseI+3) E-AGG, M-CAA 81 30 E-ACC, M-CTA 73 17 E-ACC, M-CAA 66 30 E-AGC, M-CTC 74 25 E-AGG, M-CTC 70 20 Total 364 122 Mean 72.8 24.4
Cluster analysis Cluster analysis using UPGMA (unweighted pair group method with arithmetic mean) was performed to examine genetic relationships among Thai Amomum species. A dendrogram was produced from the UPGMA cluster analysis of genetic similarity (GS) matrix for 45 accessions, 40 accessions of Amomum species and 5 accessions of out taxa, based on AFLP markers varied from 43% to 88% with a total average genetic similarity of 74.5% (Table 3). Two main clusters (A and B) were separated at 57% GS. The A cluster was separated into 2 groups: C and D, at 58% genetic similarity. The D group is subdivided into 2 subgroups (I and II) at 59% GS while the B Cluster generated 2 groups (E and F) at 59% GS (Figure 3). The A cluster is characterized by spiny fruit (rarely smooth fruit). The C group contains Amomum koenigii 1, A. koenigii 2, A.uliginosum 1, Amomum sp.9, Etlingera littoralis, A. aculeatum, Amomum sp.12 and Geostachys sp. while D group is divided into two subgroups (I and II). Subgroup (I) consists of A. testaceum 1, A. testaceum 2, A. testaceum 3 and Amomum cf. testaceum. Subgroup (II) consists of A. testaceum 4, A. cf. villosum2, Amomum sp.5, Amomum sp.7, A. uliginosum2, A. cf. villosum1, A. rivale1, A. micranthum, A. rivale2, A. cf.rivale, Amomum sp. 8, Amomum sp.4, A. biflorum and Amomum sp.13. The B cluster is characterized by smooth, ridged or wing fruit (rarely spiny fruit). This cluster contains E and F groups. The E group consists Amomum sp.16, Amomum sp.3, Amomum sp.2, Amomum sp.17a, A. siamense, Amomum sp.6b, Amomum sp.6a, Elettaria cardamomum, Amomum sp.17b, A.dealbatum, Amomum sp.15, Amomum sp.10 and Alpinia nigra. The F group contains Amomum sp.1, Amomum sp.14, A. repoense, and Etlingera pavieana. Regarding the out group taxa; Alpinia nigra, Elettaria cardamomum and Etlingera pavieana were inserted in B group while Etlingera littoralis and Geostachys sp. were placed in A group. <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) 217 DISCUSSION In this study, 40 accessions of Thai Amomum species were fingerprinted including 5 outgroup taxa. One hundred twenty two polymorphic AFLP markers were produced from five primer combinations. UPGMA cluster analysis (Rohlf, 1997) with genetic similarity of 57% separated Amomum into 2 main clusters: A consists of C and D groups and B consists of E and F groups (Figure 1). Regarding the C group, A. koenigii 1 and A.koenigii 2 were collected from Kanchanaburi and Nakhon Nayok provinces, respectively. It is clear that both collections are closely related (74%), even though the peduncular lengths vary greatly. The plants from Nakhon Nayok have a much shorter peduncle than those found in Kanchanaburi. The variation in phenotype could be caused by differences in their respective habitats. The placement of this species is similar to morphological analysis that placed it in spiny fruit group. This result is confirmation of the paraphyletic relationship between A.koenigii and the spiny fruit species (A. uliginosum and A. aculeatum). A.aculeatum and Amomum sp.12 are placed together at 90% GS. These closely related species are similar in leafy stem but differ in peduncular length, colour and size of labellum. From the results, the species Amomum sp.12 should be established as a new variety. However, this is difficult to decide from only a single plant. More collections are needed to solve this problem. D group is divided into two subgroups (I and II). Subgroup D (I) consists of A. testaceum 1, A. testaceum 2, A. testaceum 3 and Amomum cf. testaceum. Regarding A. testaceum species complex, the dendrogram suggests that this species can be separated into at least three varieties; especially A. testaceum 4 which was isolated from the group. The placement of A. testaceum is rather close to the spiny fruit species (Amomum sp.1 and Amomum sp.14). This result does agree with Xia et al. (2004) whose work was based on ITS and
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Page 5 and 6: Kasetsart J. (Nat. Sci.) 41 : 205 -
Page 7 and 8: harvesting time which started from
Page 9 and 10: y Chen and Paull (2000). Figure 1 a
Page 11 and 12: pineapple fruit allowed the accumul
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Page 21 and 22: Table 3 (Continued) 1 2 3 4 5 6 7 8
Page 25 and 26: CONCLUSION AFLP markers classified
Page 29 and 30: difference of soil texture used in
Page 31 and 32: under wet soil condition planting.
Page 33 and 34: tropics. Following the expansion, w
Page 35 and 36: sealed in a plastic box with 1 cm w
Page 37 and 38: moisture content increment after so
Page 39 and 40: Frequency Frequency 30 20 10 0 20.0
Page 41 and 42: School, Kasetsart University. The a
Page 43 and 44: for combining ability of lines (Lam
Page 45 and 46: selective mass sibbing within indiv
Page 47 and 48: Although most of S 2-interfamily hy
Page 49 and 50: composite sets as used in this stud
Page 53 and 54: days. The numbers of calli producin
Page 55 and 56: the report of Ching (1982) showing
Page 57 and 58: clearly amplified bands generated b
Page 59 and 60: caused by either the recombination
Page 61 and 62: Kuhlmann, V. and B. Foroughi-Wehr.
Page 63 and 64: Xanthomonas fuscans subsp. aurantif
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was collected and washed with 70% e
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expected size was amplified with 35
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eaction technique has been used for
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Schaad, N.W., D. Opgenorth and P. G
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MATERIALS AND METHODS Model descrip
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Kasetsart J. (Nat. Sci.) 41(2) 277
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calculated TF value calculated TF v
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sincere gratitude and deep apprecia
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1989). In monogastrics, the inclusi
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of all LLS samples in this study we
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Melbourne, Parkville, Victoria. Goe
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considered responsible for low prod
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mineral supplementation suggested b
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Body weight chane (kg/head) 32 30 2
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CONCLUSION AND RECOMMENDATION With
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SAS. (Statistical Analysis System).
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genes covering a variety of desirab
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mg/l NAA and 0.5 mg/l zeatin) incub
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5. Purification by various sucrose
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as the culture period increased. So
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culture, pp. 1-20. In L.C. Fowke an
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Kasetsart J. (Nat. Sci.) 41 : 311 -
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GC. Analytical methods Total carboh
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ash. The crude hot water polysaccha
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spirulan (H-SP), and a desulfated c
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cells often displayed an increased
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LITERATURE CITED Bell, T.A. and D.V
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for alternative sources of supply.
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BR2.1.2 formed the same clade with
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supplement with glutamate (Iida et
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optimal for S. limacinum BR2.1.2 fo
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fluorescent lamp at 1.5 kLux develo
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Wisconsin, USA). Total extracted RN
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RESULTS Cloning and sequencing of m
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Expression of rmIL-2 and purificati
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other strains of mice have differen
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dose interleukin-2 therapy promotes
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The chitosano-oligosaccharides are
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enzyme and cells were maximized by
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of crude chitosanases. Fortunately,
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Relative activity (%) 100 80 60 40
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Uchida, K. Tateishi, O. Shida and K
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MATERIALS AND METHODS 1. Materials
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without coating and coated with pec
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in Table 5-7. It was found that the
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the cultures at 10,200 × g for 15
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incubation and reached the final pH
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y lysine- and tyrosine-decarboxylas
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during the stages of ripening as su
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As the 25 companies were divided in
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All the data comes up with informat
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as piece “A” or straight as pie
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et al. (1998); Zeng (2000); and Tal
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p 1, i * Kasetsart J. (Nat. Sci.) 4
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c ≤ c2 jq2 j n q p k p * 2, j , ,
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algorithm would consider RM 5, prio
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the maximum deviation of about 10%.
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Stock Quantities Using Heuristic Op
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which dynamically and automatically
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2. Framework architecture and compo
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Risk (VaR) calculation which was im
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Throughput (job/sec) 7 6 5 4 3 2 1
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Speed up 40 35 30 25 20 15 10 5 0 F
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and discovery, resource selection a
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April - June 2007 - Kasetsart University

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