To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA （RAPD） was performed using 120 random primers. Of the suc...To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA （RAPD） was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced （Genl3ank access number KU553472）. Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples.展开更多
The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) techni...The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.展开更多
The effects of development states on the artemisinin content of clone S1 of Artemisia anuua L. grown in a greenhouse were investigated in the present study. The artemisinin content increased gradually during the phase...The effects of development states on the artemisinin content of clone S1 of Artemisia anuua L. grown in a greenhouse were investigated in the present study. The artemisinin content increased gradually during the phase of vegetative growth and reached its highest level at 8-9 mg/g dry weight （DW） when the S1 was 6 months old on a long day （LD） photoperiod. Treatment with 9-18 d of short day （SD） photoperiod resulted in the artemisinin content reaching and being maintained at a higher level （2.059-2.289 mg/g DW）, twofold that of control plants and plants of S1 presented at the pro-flower budding and flower-budding stages. The artemisinin content varied in different parts of the plant. The artemisinin content of leaves was higher than that of florets and branches. The artemisinin content in middle leaves was higher than that of bottom leaves, and then top leaves. Different densities of capitate glands （the storage organ of artemisinin） located on the surface of leaves, florets, and branches explained the variations in artemisinin content in these parts of the plant. The correlation coefficient between artemisinin content and density of capitate glands on the surface of different organs was 0.987. The genetic marker for artemisinin content was screened using random amplified polymorphic DNA （RAPD） and sequence characterized amplified region （SCAR） techniques. The random primer OPAl5 （5＇-TTCCGAACCC-3＇） could amplify a specific band of approximately 1 000 bp that was present in all high-artemisinin yielding strains, but absent in all low-yielding strains in three independent replications. This specific band was cloned and its sequence was analyzed. This RAPD marker was converted into a SCAR marker to obtain a more stable marker.展开更多
DNA polymorphism in the cultivar species;Cucumis sativus L., C. melo L. and Benincasa hispida Cogn. of subtribe Cucumerinae (Cucurbitaceae) in the four northeastern provinces of Thailand was examined by using RAPD tec...DNA polymorphism in the cultivar species;Cucumis sativus L., C. melo L. and Benincasa hispida Cogn. of subtribe Cucumerinae (Cucurbitaceae) in the four northeastern provinces of Thailand was examined by using RAPD technique. Twenty 10-mer primers were produced 212 RAPD fragments, ranging from approximately 120 to 2531 bp. The genetic similarities were estimated from banding profiles using a NTSYS* version 2.1 as a basis for dendrogram construction via the UPGMA method. Cluster analysis divided the taxa under study into 2 clades. Moreover, a RAPD marker: Cm (OPJ11700) was specified to C. melo, and this marker was converted into sequence characterized amplified region (SCAR) marker: Cm (SCJ11516). A pair of sequence-specific primer of clones Cm (OPJ11700) amplified a distinct single band of the same size as the RAPD clones. The SCAR marker was developed successfully to identify C. melo genotype.展开更多
OPM<sub>05</sub>-M<sub>2100</sub>,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEM<...OPM<sub>05</sub>-M<sub>2100</sub>,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEM<sup>R</sup> -T Vector that was transformed into Escherichia.coli and cloned and sequenced.Based on the sequence of RAPD marker,the sequences characterized amplified region（SCAR） primers were designed by the aid of the software Oligo5.0.The forward primer is M<sub>05</sub>F<sub>2</sub> （5’-CGGGT CATGG CTGGA GGTAT CGT-3’）,and the backward primer is M<sub>05</sub>R<sub>1</sub>(5’-TGGCT CAATG GCAAA TCCTT CGTA-3’.The specific fragment (OPM<sub>05</sub>-M<sub>2100</sub>) was successfully converted to SCAR marker(SCAR-M<sub>05</sub>-X<sub>600</sub>) by using M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>,which was the specific markers of B.xylophilus.Then, the DNA of 92 isolates of Bursaphelenchus,B. mucronatus,B.hofmanni,Aphelenchoides macronucleatus and Seinura sp.which were isolated from dead pines,were marked,and the DNA of a single nematode extracted with a simple method was detected using this set of specific primers.The results indicated that the PCR product of all 81 isolates of B.xylophilus was a clear and bright fragment about 600 bp with M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>.But eight isolates of B. mucronatus,one B.hofmanni,one A.macronucleatus and one Seinura sp.had no any fragments.Assay M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub> also successfully detected single pinewood nematode.Therefore,the specific pairwises would be used for constructing identification kits of B. xylophilus,implementing the aim of quick detection, and achieving the purpose of identify juvenile successfully.展开更多
Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (R...Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.展开更多
基金Project(2014ZX09304307-002)supported by the Major Program of Science and Technology Foundation of ChinaProject supported by Technology Platform for Quality/Safety Inspection and Risk Management of Traditional Chinese Medicine,China+1 种基金Project(2014SK2001)supported by the Key Program Foundation of Hunan Provincial Science&Technology Department,ChinaProject(XSYK-R201502)supported by the Hunan Provincial Food and Drug Administration under Key Project of Science and Technology for Food and Drug Safety,China
文摘To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA （RAPD） was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced （Genl3ank access number KU553472）. Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples.
基金Supported by the"Twelfth Five-Year-Plan"of National Science and Technology for the Rural Development in China(No.2012AA10A411)the Public Welfare Project of the Ministry of Agriculture of China(No.200903030)
文摘The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.
文摘The effects of development states on the artemisinin content of clone S1 of Artemisia anuua L. grown in a greenhouse were investigated in the present study. The artemisinin content increased gradually during the phase of vegetative growth and reached its highest level at 8-9 mg/g dry weight （DW） when the S1 was 6 months old on a long day （LD） photoperiod. Treatment with 9-18 d of short day （SD） photoperiod resulted in the artemisinin content reaching and being maintained at a higher level （2.059-2.289 mg/g DW）, twofold that of control plants and plants of S1 presented at the pro-flower budding and flower-budding stages. The artemisinin content varied in different parts of the plant. The artemisinin content of leaves was higher than that of florets and branches. The artemisinin content in middle leaves was higher than that of bottom leaves, and then top leaves. Different densities of capitate glands （the storage organ of artemisinin） located on the surface of leaves, florets, and branches explained the variations in artemisinin content in these parts of the plant. The correlation coefficient between artemisinin content and density of capitate glands on the surface of different organs was 0.987. The genetic marker for artemisinin content was screened using random amplified polymorphic DNA （RAPD） and sequence characterized amplified region （SCAR） techniques. The random primer OPAl5 （5＇-TTCCGAACCC-3＇） could amplify a specific band of approximately 1 000 bp that was present in all high-artemisinin yielding strains, but absent in all low-yielding strains in three independent replications. This specific band was cloned and its sequence was analyzed. This RAPD marker was converted into a SCAR marker to obtain a more stable marker.
文摘DNA polymorphism in the cultivar species;Cucumis sativus L., C. melo L. and Benincasa hispida Cogn. of subtribe Cucumerinae (Cucurbitaceae) in the four northeastern provinces of Thailand was examined by using RAPD technique. Twenty 10-mer primers were produced 212 RAPD fragments, ranging from approximately 120 to 2531 bp. The genetic similarities were estimated from banding profiles using a NTSYS* version 2.1 as a basis for dendrogram construction via the UPGMA method. Cluster analysis divided the taxa under study into 2 clades. Moreover, a RAPD marker: Cm (OPJ11700) was specified to C. melo, and this marker was converted into sequence characterized amplified region (SCAR) marker: Cm (SCJ11516). A pair of sequence-specific primer of clones Cm (OPJ11700) amplified a distinct single band of the same size as the RAPD clones. The SCAR marker was developed successfully to identify C. melo genotype.
文摘OPM<sub>05</sub>-M<sub>2100</sub>,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEM<sup>R</sup> -T Vector that was transformed into Escherichia.coli and cloned and sequenced.Based on the sequence of RAPD marker,the sequences characterized amplified region（SCAR） primers were designed by the aid of the software Oligo5.0.The forward primer is M<sub>05</sub>F<sub>2</sub> （5’-CGGGT CATGG CTGGA GGTAT CGT-3’）,and the backward primer is M<sub>05</sub>R<sub>1</sub>(5’-TGGCT CAATG GCAAA TCCTT CGTA-3’.The specific fragment (OPM<sub>05</sub>-M<sub>2100</sub>) was successfully converted to SCAR marker(SCAR-M<sub>05</sub>-X<sub>600</sub>) by using M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>,which was the specific markers of B.xylophilus.Then, the DNA of 92 isolates of Bursaphelenchus,B. mucronatus,B.hofmanni,Aphelenchoides macronucleatus and Seinura sp.which were isolated from dead pines,were marked,and the DNA of a single nematode extracted with a simple method was detected using this set of specific primers.The results indicated that the PCR product of all 81 isolates of B.xylophilus was a clear and bright fragment about 600 bp with M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub>.But eight isolates of B. mucronatus,one B.hofmanni,one A.macronucleatus and one Seinura sp.had no any fragments.Assay M<sub>05</sub> F<sub>2</sub>/R<sub>1</sub> also successfully detected single pinewood nematode.Therefore,the specific pairwises would be used for constructing identification kits of B. xylophilus,implementing the aim of quick detection, and achieving the purpose of identify juvenile successfully.
文摘Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.
