[Cloning and analyzing of the female-specific marker in the dioecious species Asparagus officinalis L].
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Abstract:
Sex-linked molecular markers are being obtained, which would be essential to be used in the screening of different sex of dioecious plants at the seedling stage. Furthermore, it is important in cloning the gene related to the sex. In this study the random amplified polymorphic DNA (RAPD) technique was employed with the objective to find markers linked to sex determination in Asparagus. A total of 100 primers were tested with the same PCR cycling procedure. A female-associated fragment with a length of about 867bp was generated with S12 primer. The fragment was cloned and sequenced, showing it is abundant in AT and contains 2 shorter open reading frames. In order to convert the RAPD marker into SCAR (sequence characterized amplified regions) marker, 24bp specific primers were constructed and used for PCR amplifying. The female-linked dominant SCAR marker was obtained, which would be efficient to identify the different sex of Asparagus officinalis L.Keywords:
Asparagus
Primer (cosmetics)
Cloning (programming)
Molecular marker
Marker gene
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To establish sequence characterized amplified region markers of Cornus officinalis and provide a scientific basis for molecular identification of C. officinalis.The random primer was screened through RAPD to obtain specific RAPD marker bands. The RAPD marker bands were separated, extracted, cloned and sequenced. Both ends of the sequence of RAPD marker bands were determined. A pair of specific primers was designed for conventional PCR reaction, and SCAR marker was acquired.Four pairs of primers were designed based on the sequence of RAPD marker bands. The DNA of the seven varieties of C. officinalis was amplified by using YST38 and YST43 primer. The results showed that seven varieties of C. officinalis were able to produce a single PCR product. It was an effective way to identify C. officinalis. The varieties with cylindrical and long-pear shape fruits amplified by YST38 showed a specific band, which could be used as the evidence of variety identification. Seven varieties of C. oficinalis were amplified by using primer YST39. But the size of band of the variety with spindly shape fruit (35,0400 bp) was about 300 bp, which was shorter than those of the variety with the other shape fruits of C. officinalis (650-700 bp). The variety with the spindly shape fruit could be identified through this difference. The primer YST92 could produce a fragment from 600-700 bp in the varieties with cylindrical and long-pear shape fruits, a fragment from 200-300 bp in the varieties with oval and short-cylindrical shape fruits and had no fragment in the varieties with long cylindrical, elliptic and short-pear shape fruits, which could be used to select the different shapes of C. officinalis.SCAR mark is established and can be used as the basis for breeding and distinguishing the verieties of C. officinalis.
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Molecular marker
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Asparagus
Liliaceae
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The random amplified polymorphic DNA (RAPD) technique was used to determine the male trait of Carica papaya L.. 214 10-mer primers were tested. A male-associated fragment was generated with Z18 primer. The marker fragment, named Z18-1000 existed in all male plants but not in the female and hermaphrodite plants so far analyzed. The fragment was cloned and sequenced. Four primers were designed based on the sequence to transform RAPD marker into SCAR marker. The RAPD marker was successfully converted into a SCAR marker, which was designated SD1000.
Carica
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Molecular marker
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Asparagus
Southern blot
Molecular marker
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Objective:To analyze genome difference between Asparagus male and female plants for screening male-or female-specific molecular markers.Method:Using amplified fragment length polymorphism(AFLP)technique,genomes of asparagus male and female plants were amplified respectively,by designing multiple primer combinations.Result:A total of 72selective primer pairs were screened,and only one primer pair(E-AAG/M-CAT)amplified a 555bp band(MLDA555)in male plants.The marker was AT rich in sequence(59%)and there was no sequence showed significant similarity to this marker by Blast test.This male-specific AFLP marker MLDA555 was converted into sequence-characterized amplified region(SCAR)marker using primers designed according its sequence.As expected,the SCAR primers were able to amplify a single DNA band of 523bp and the SCAR marker was efficient in the identification of male individuals from different asparagus populations.Conclusion:Male-specific AFLP and SCAR markers were identified from A.officinalis by AFLP technique,which could provide theoretical information and technical support for the understanding of sex determination mechanism and molecular marker assistant breeding of A.officinalis.
Asparagus
Primer (cosmetics)
Molecular marker
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While random amplification of polymorphic DNA (RAPD) markers linked to disease resistance genes have been widely used in plant breeding programs, they generally lack reproducibility. To overcome this major disadvantage and other drawbacks, RAPD markers can be converted into sequence characterized amplified region (SCAR) markers, which are genetically defined loci amplified by polymerase chain reaction (PCR) using specific primers. Thus, SCAR markers are typically more reproducible than RAPD markers, due to specific amplification of genomic regions. In this study, a previously identified RAPD marker AT9/917 that is linked to the Puccinia psidii Winter (rust) resistance gene 1 (Ppr1) in Eucalyptus grandis was successfully converted into a specific SCAR marker. Seven specific SCAR primers were designed based on cloning and sequencing of the RAPD marker AT9/917. Different pairs of SCAR primers were tested in an E. grandis family from a crossing between a resistant and a susceptible E. grandis. Prime pair SCAR AT99151L and AT9915914R produced amplicons of expected size. Restriction enzyme digestion of the amplicon revealed polymorphisms between the resistant and susceptible parents. Association analysis between phenotype (rust resistance) and SCAR genotypes in the E. grandis family suggests that this specific SCAR is useful for marker-assisted selection of E. grandis trees resistance to P. psidii Winter. Key words: Plant breeding, molecular markers, random amplified polymorphic DNA (RAPD), Mark-assisted selection, sequence characterized amplified region (SCAR).
Amplicon
Marker-Assisted Selection
Molecular marker
Rust (programming language)
Sequence-tagged site
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In this study the random amplified polymorphic DNA (RAPD) technique was employed with the objective of finding markers linked to sex determination in hemp. Ten male individual DNA samples or ten female individual DNA samples were prepared and contributed to two DNA pools: one was male DNA pool and another female DNA pool, with the intention of providing a common genetic background for each pool and leaving the sex linked materials as the primary difference between the two pools. A total of 30 10 mer primers (Table 1) was tested with at least three different PCR cycling procedures. A male associated fragment with a length of about 2.5 kb (Figs.1 and 2) was generated with S401 primer. The fragment was cloned and sequenced (Fig.3). In order to convert the RAPD marker into SCAR (sequence characterized amplified regions) marker, one 20 mer and another 22 mer specific primers were constructed and used for PCR amplifying. The male linked dominant SCAR marker was obtained (Fig.4), which would favor the screening of all gynoecious hemp lines.
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Sequence-tagged site
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Pistacia chinensis Bunge is a dioecious plant that originated in China, and its sex cannot be identified at the early stage of cultivation by only its appearance.Recent studies show that the seed of P. chinensis is an ideal feedstock for biofuel production.To guide the cultivation of this energy plant scientifically, a new method is urgently needed to identify the sex of P. chinensis seedlings.In this paper, from 21 random-amplified polymorphic DNA primers and 20 inter-simple sequence repeat primers, 2 sex-specific primers (S1 and S281) were identified that can amplify female-specific fragments of 473 and 1242 bp, respectively.However, only 1 fragment (FS281) was converted successfully into a sequence-characterized amplified region marker using S281-1 and S281-2 primers.When the annealing temperature was 64°C, a 636-bp specific sequence appeared in all female specimens but was absent in all the male samples tested.This study will offer some clues to sex selection in P. chinensis plantations.
Anacardiaceae
Pistacia
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Primer (cosmetics)
genomic DNA
Molecular marker
Bulked segregant analysis
Phoenix dactylifera
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