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1.
Li HB Wu XQ Peng HZ Fu LZ Wei HL Wu QQ Jin QY Li N 《Applied microbiology and biotechnology》2008,81(2):303-309
At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains
of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique.
Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs
of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three
different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in
which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish
this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development
of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China. 相似文献
2.
In this study, three molecular marker systems including sequence related amplified polymorphism (SRAP), random amplified polymorphic
DNA (RAPD), and inter-simple sequence repeats (ISSR) were screened to select polymorphisms of 24 main commercial strains of
Lentinula edodes cultivated widely in China. Twenty-nine sequence characterized amplified region (SCAR) markers were developed to set up a
dendrogram using UPMGA based on nucleotide sequences of some SRAP, RAPD, and ISSR polymorphic fragments. The grouping showed
that the 24 strains were apparently clustered into five groups at a level of 0.68 similarity coefficient, and those that have
similar breeding background clustered preferentially into the same subgroup. Results also revealed that the 24 strains had
a low level of genetic diversity, and the breeding source of L. edodes should be broadened by exploiting wild types and introducing exotic strains. In addition, the tested strains of L. edodes could be clearly distinguished and identified from others by using different combinations of SCAR primers. Thus, results
of this work demonstrated that SCAR was an excellent genetic marker system to characterize and investigate genetic diversity
of L. edodes. Furthermore, this provided an alternative method to identify the genetic relationship of different strains of other fungi. 相似文献
3.
Loncaric I Donat C Antlinger B Oberlerchner JT Heissenberger B Moosbeckhofer R 《Journal of applied microbiology》2008,104(5):1433-1441
Aim: The aim of this study was to develop a specific and sensitive identification method for two Aureobasidium pullulans biocontrol strains, CF10 and CF40, based on a sequence-characterized amplified region (SCAR) derived from RAPD – and multiplex-RAPD PCR analysis. Methods and Results: The random amplified polymorphic DNA (RAPD) and multiplex RAPD-PCR techniques were used for a preliminary screening of A. pullulans genetic variability among 200 isolates. This approach allowed the selection of ten fragments present solely in strains CF10 and CF40. The RAPD fragments were cloned, sequenced and used to design two SCAR primers. Two primer pairs obtained from SCH3RAPD fragment of CF 40 and 6RAPD of CF10 were highly specific and sensitive. Conclusions: In this study, we developed strain-specific multiplex-PCR based on sequence-characterized amplified region (SCAR) markers to simultaneously detect both strains in a single PCR. Significance and Impact of the Study: This new multiplex-PCR provides a valuable tool for specific and sensitive identification of CF10 and CF40, and could be used in studies on the efficacy and persistence of introduced strains of A. pullulans for fire blight control. 相似文献
4.
Li-Zhong Fu Hong-Yu Zhang Xue-Qian Wu Hai-Bo Li Hai-Long Wei Qing-Qi Wu Li-An Wang 《World journal of microbiology & biotechnology》2010,26(4):709-716
Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genetic diversity among 23 elite Lentinula edodes strains in China. A total of 138, 77 and 144 bands were detected by 16 RAPD primers, 5 ISSR primers and 23 SRAP primer combinations, among which 58.8%, 73.5% and 56.3% was polymorphic, respectively. By UPGMA clustering, a dendrogram was constructed based on each analysis. The three dendrograms showed that 23 L. edodes strains were clustered into three or four groups. The grouping exhibited similar structure and was generally consistent with their pedigrees. Twenty-three L. edodes strains shared great similarity indicated that the low level of genetic diversity of L. edodes strains and their relationship between each other. The important source of breeding material, such as wild and exotic types, must be introduced in order to broaden genetic base and decreases genetic vulnerability of L. edodes. 相似文献
5.
Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce 总被引:77,自引:3,他引:74
I. Paran R. W. Michelmore 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,85(8):985-993
Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites. 相似文献
6.
Xiao Yang Dai Yaohong Lu Yingying Liu Wei Wang Zhuoren Bian Yinbing 《World journal of microbiology & biotechnology》2011,27(7):1731-1734
Rapid and correct authentication of commercial strains is still important in today’s mushroom industry. Here for the first
time we reported the using of sequence characterized amplified region (SCAR) marker developed from inter-retrotransposon amplified
polymorphism (IRAP) marker to identify Lentinula edodes strain. Genomic polymorphism among 44 shiitake cultivars in China were surveyed by 24 IRAP primer combinations, from which
primer combination LTR1L/MarY1R could generate a unique and reproducible 1712 bp fragment to distinguish strain No. 4 from
other 43 strains. Based on this strain-specific fragment, a SCAR primer pair was designed and its validity was verified by
correctly amplifying a single strain-specific fragment from DNA samples of 100 L. edodes strains. Our study lays the foundation for developing strain-specific SCAR marker by retrotransposon-based marker technique
in fungi. 相似文献
7.
Hongyan Su Lei Wang Yihe Ge Ermei Feng Jie Sun Linde Liu 《World journal of microbiology & biotechnology》2008,24(7):1223-1226
In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified
by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated
as GL612F and GL612R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer
pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum. 相似文献
8.
Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes. 相似文献
9.
Sequence characterized amplified region markers tightly linked to the mating factors of Lentinula edodes. 总被引:1,自引:0,他引:1
Detecting the mating types in shiitake, Lentinula edodes (Berk.) Pegler, is important for making progress in the breeding of this mushroom and determining the compatibility of the pair to cross. Shiitake is a tetrapolar fungus with two unlinking mating factors, A factor and B factor. We screened molecular markers linked to the mating factors using the randomly amplified polymorphic DNA (RAPD) method to develop the mating type identification procedure. Using 147 oligonucleotide primers, a total of 6 linkage markers for the shiitake mating factors, 4 markers for the A factor and 2 markers for the B factor, were discovered with a logarithm of the odds threshold of 3.0 for linkage. Two RAPDs that perfectly segregated with each mating factor among 72 basidiospore strains were detected. Both of these RAPDs were cloned and sequenced to convert them to the sequence characterized amplified region (SCAR) markers. Four primers, two sets of primers, were designed according to the internal sequences of two RAPDs tightly linking to the A factor or B factor. Consequently, we determined the polymerase chain reaction condition for multiplex analyses of these SCAR markers. 相似文献
10.
Combined randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) were used to assess the genetic diversity of Pleurotus ostreatus strains cultivated in China. For the RAPD and SRAP analyses, 479 and 282 polymorphic bands were obtained from 20 P. ostreatus strains using 20 and 13 selected primers or primer pairs, respectively. A combined RAPD/SRAP dendrogram grouped the 20 strains into five clades with a coefficient of 0.690. The comparison of RAPD and SRAP was evaluated in the present study. The combined RAPD/SRAP markers provided reliable information regarding the relationships among the P. ostreatus strains. 相似文献
11.
In this paper, we report for the first time on authentication of Flammulina velutipes cultivars by using strain-specific sequence-characterized amplified region (SCAR) markers developed from inter-simple sequence repeat (ISSR) markers. The genomic DNA polymorphism was analyzed by the ISSR technique in 7 strains of F. velutipes presently cultivated in China on a commercial scale. Eight primers selected from 20 ISSR primers amplified 104 clear and stable bands, of which 81 bands were polymorphic. Among the selected primers, primer ISSR9 can distinguish strain No. 12 from the other 6 strains by amplifying a unique and reproducible band of approximately 750 bp. According to the sequence of the strain-specific fragment, a pair of SCAR primers was designed to diagnose strain No. 12 on the molecular level. The validity of the SCAR marker was confirmed by using DNA samples from another 12 strains of F. velutipes obtained from different parts of China. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for F. Velutipes. 相似文献
12.
利用SRAP和ISSR建立快速鉴定灵芝属菌株的SCAR标记 总被引:5,自引:0,他引:5
根据ERIC聚类分析的结果,把152株灵芝属菌株(包括128株来自中国的栽培菌株及24株国外菌株)建成48个DNA池。用SRAP和ISSR引物对48个DNA池进行扩增,筛选获得4个特异性标记,回收特异性条带,经克隆测序后设计了4对SCAR引物,并通过SCAR-PCR扩增验证,从而将SRAP标记和ISSR标记均成功地转化为特异性和稳定性更好的SCAR标记;将得到的4个SCAR标记在构成DNA池的152个菌株上验证,并建立多重PCR体系,最终证实了SCAR特异标记在菌株快速检测鉴定中的可行性和可靠性。 相似文献
13.
Fuli Liu Jianting Yao Xiuliang Wang Zimin Hu Delin Duan 《Journal of applied phycology》2011,23(4):709-713
To construct a molecular-marker-assisted selection (MAS) system, research was done on identifying molecular markers linking
to longer frond length, a crucial selection index in the breeding of the commercially important seaweed Saccharina japonica. An F2-segregant population of 92 individuals was obtained by crossing two prominent S. japonica strains. Genomic DNA from ten individuals with the longest frond and ten individuals with the shortest frond in the F2-segregant population were mixed to create two DNA pools for screening polymorphic markers. In bulked-segregant analysis (BSA),
out of 100 random amplified polymorphic DNA (RAPD) primers only two produced three polymorphic RAPD markers between the two
DNA pools. In conversion of the three RAPD markers into sequence-characterized amplified region (SCAR) markers, only one was
successfully converted into a SCAR marker FL-569 linking to the trait of longer frond. Test of the marker FL-569 showed that
80% of the individuals with longest fronds in a wild population and 87.5% of individuals with the longest fronds in an inbred
line “Zhongke No. 2” could be detected by FL-569. Additionally, genetic linkage analysis showed that the SCAR marker could
be integrated into the reported genetic map and QTL mapping showed that FL-569 linking to qL1-1. The obtained marker FL-569
will be beneficial to MAS in S. japonica breeding. 相似文献
14.
Field studies on the efficacy and persistence of an introduced strain of Beauveria bassiana for insect control require detection assays to differentiate the non-native strain from indigenous populations. In this study we developed strain-specific molecular markers based on polymerase chain reaction amplification of sequence-characterized amplified regions (SCAR) in combination with dilution plating on semi-selective medium to detect and estimate density of propagules of a commercial strain of B. bassiana (strain GHA) in field samples. Using random amplified polymorphic DNA (RAPD) analysis, unique fragments that distinguished GHA from other strains of B. bassiana were obtained. Three amplicons, OPA-14(0.44), OPA-15(0.44), and OPB-9(0.67), generated with RAPD primers were cloned and sequenced and used as bases for designing SCAR primers OPA14 F/R(445), OPA15 F/R(441), and OPB9 F/R(677), respectively. All three SCAR primers were highly sensitive, capable of detecting 100pg B. bassiana GHA genomic DNA, and thus could be used to detect varying levels of the fungus in the field. 相似文献
15.
Onivaldo Randig Michel Bongiovanni Regina M D G Carneiro Philippe Castagnone-Sereno 《Génome》2002,45(5):862-870
RAPD markers were used to characterize the genetic diversity and relationships of root-knot nematodes (RKN) (Meloidogyne spp.) in Brazil. A high level of infraspecific polymorphism was detected in Meloidogyne arenaria, Meloidogyne exigua, and Meloidogyne hapla compared with the other species tested. Phylogenetic analyses showed that M. hapla and M. exigua are more closely related to one another than they are to the other species, and illustrated the early divergence of these meiotically reproducing species from the mitotic ones. To develop a PCR-based assay to specifically identify RKN associated with coffee, three RAPD markers were further transformed into sequence-characterized amplified region (SCAR) markers specific for M. exigua, Meloigogyne incognita and Meloidogyne paranaensis, respectively. After PCR using the SCAR primers, the initial polymorphism was retained as the presence or absence of amplification. Moreover, multiplex PCR using the three pairs of SCAR primers in a single reaction enabled the unambiguous identification of each species, even in mixtures. Therefore, it is concluded that the method developed here has potential for application in routine diagnostic procedures. 相似文献
16.
Development of SCAR markers for the DNA-based detection of the Asian long-horned beetle,Anoplophora glabripennis (Motschulsky) 总被引:4,自引:0,他引:4
Kethidi DR Roden DB Ladd TR Krell PJ Retnakaran A Feng Q 《Archives of insect biochemistry and physiology》2003,52(4):193-204
DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence characterized amplified regions (SCARs) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after screening 230 random primers in a PCR-based assay system. Three pairs of nested 22-mer oligonucleotide primers were designed on the basis of the sequence of this fragment and were used to perform diagnostic PCR. The first pair of primers (SCAR1) amplified a single 745-bp fragment of ALB DNA, but this did not differentiate ALB from other species. The other two pairs of SCAR primers (SCAR2 and SCAR3) amplified bands of 1,237- and 2,720-bp, respectively, that were capable of differentiating ALB from other closely related non-native and native Cerambycids, such as A. chinensis (Forster), A. malasiaca (Thomson), A. nobilis (Ganglbauer), Monochamus scutellatus (Say), Plectrodera scalator (Fab), Saperda tridentata (Olivier), and Graphisurus fasciatus (Degeer). The latter two SCAR markers could be amplified using DNA extracted from body parts of ALB such as the wing, the leg, and the antennae as well as tissues from all the developmental stages including the egg, larva, pupa, and adult. These markers were also capable of identifying ALB using the DNA extracted from frass. Our results demonstrate that the SCAR markers we have identified can be used for unambiguously identifying ALB from other closely related Cerambycids using a simple PCR procedure. 相似文献
17.
The authors' studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed. 相似文献
18.
QS Zhang BL Xu LD Liu QQ Yuan HX Dong XH Cheng DL Lin 《World journal of microbiology & biotechnology》2012,28(5):2237-2248
To evaluate the genetic diversity of Pleurotus citrinopileatus Singer cultivars in China, 20 P. citrinopileatus strains were analyzed using morphological traits, inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism
(SRAP) molecular markers. Eleven ISSR primers amplified a total of 116 DNA fragments of which 96 (82.91%) were polymorphic,
whereas 8 SRAP primer pairs amplified 69 fragments of which 65 (93.47%) were polymorphic. Phylogenetic trees constructed on
the basis of ISSR, SRAP, and combined ISSR/SRAP analyses using the Unweighted Pair-group Method with Arithmetic Averages method
distributed the 20 strains into three or six major groups. The grouping exhibited great similarity and was generally consistent
with their morphological characters and antagonism test, which indicated a high level of genetic diversity among P. citrinopileatus Singer and relationship between each other. Based on the genetic analysis, the primary mini-core strains were constructed
with progressive sampling method of the smallest genetic distance. The mini-core germplasm collection included 4 strains (strain
2, 5, 7 and 11). Our findings will provide a scientific fundament for facilitating parent selection for broadening genetic
base, accelerating the genetic breeding, identification of cultivated strains and the development of bioactive products from
this commercially important medicinal mushroom. 相似文献
19.
The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields. 相似文献
20.
Development of new strains and related SCAR markers for an edible mushroom, Hypsizygus marmoreus 总被引:1,自引:0,他引:1
New fast-growing and less bitter varieties of Hypsizygus marmoreus were developed by crossing monokaryotic mycelia from a commercial strain (Hm1-1) and a wild strain (Hm3-10). Six of the better tasting new strains with a shorter cultivation period were selected from 400 crosses in a large-scale cultivation experiment. We attempted to develop sequence characterized amplified region (SCAR) markers to identify the new strain from other commercial strains. For the SCAR markers, we conducted molecular genetic analysis on a wild strain and the eight most cultivated H. marmoreus strains collected from various areas in East Asia by randomly amplified polymorphic DNA. Ten unique DNA bands for a commercial Hm1-1 strain and the Hm3-10 strain were extracted and their sequences were determined. Primer sets were designed based on the determined sequences. PCR reactions with the primer sets revealed that four primer sets successfully discriminated the new strains from other commercial strains and are thus suitable for commercial purposes. 相似文献