Advanced
Development of a SCAR Marker for Sex Identification in Asparagus
Development of a SCAR Marker for Sex Identification in Asparagus
Korean Journal of Plant Resources. 2014. May, 27(3): 236-241
Copyright © 2014, The Plant Resources Society of Korea
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : February 02, 2014
  • Accepted : May 05, 2014
  • Published : May 28, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Seong-Cheol Kim
Namhae Sub-Station, National Institute of Horticultural and Herbal Science, RDA, Namhae 668-812, Korea
kimsec@korea.kr
Yong-Hwan Jung
Jeju Biodiversity Research Institute, Jeju Technopark, Seogwipo 699-943, Korea
Ki-Cheol Seong
National Agricultural Research Center for Climate Change, NIHHS, RDA, Jeju 690-150, Korea
Seung-Jong Chun
National Agricultural Research Center for Climate Change, NIHHS, RDA, Jeju 690-150, Korea
Chun Hwan Kim
National Agricultural Research Center for Climate Change, NIHHS, RDA, Jeju 690-150, Korea
Chan Kyu Lim
National Agricultural Research Center for Climate Change, NIHHS, RDA, Jeju 690-150, Korea
Jae-Ho Joa
National Agricultural Research Center for Climate Change, NIHHS, RDA, Jeju 690-150, Korea
Dong-Sun Lee
Faculty of Biotechnology, Jeju National University, Jeju 690-756, Korea
Abstract
A sex-linked random amplified polymorphic DNA (RAPD) marker was identified from Asparagus officinalis L. and was converted into a sequence-characterized amplified regions (SCAR) marker for the large-scale screening of male and female plants. A total of 100 arbitrary decamer oligonucleotide primers were used for the RAPD analysis. Among them, the primer UBC347 amplified one female-specific 400 base pair DNA. Subsequently, the amplified RAPD fragment was cloned and sequenced. The fragment was abundant in AT and shared sequence homology with retrotransposon elements. On the basis of the sequence obtained, a pair of SCAR primer was designed. The amplification product, named F400, was the same size as the respective RAPD fragment from which it was derived. The F400 SCAR marker resulted to be female-specific in the three asparagus varieties tested in this study. This SCAR marker can be used for an early and rapid identification of female and male plants during breeding programs of asparagus.
Keywords
Introduction
Asparagus ( Asparagus officinalis L.) is an important perennial species with several functional substances and is native to Europe and Eastern Asia. Asparagus rarely produces hermaphroditic flowers and has homomorphic sex chromosomes (Telgmann-Rauber et al ., 2007; Jamilena et al ., 2008). The cultivated asparagus is dioecious with 1:1 sex ratio plant (male: M/m, female: m/m). Male plants are desired for yield, uniformity, and crop longevity; besides they do not produce seeds which give ‘asparagus weed’ in the production fields (Yeager and Scott, 1938; Ellison, 1986). On the other hand, it takes 2~3 years to distinguish male and female plants until asparagus plants flower. Therefore, early selection of a male asparagus among young seedlings is very important. Rapid identification of male and female asparagus varieties become also one of the main goals for breeding of this vegetable crop. If it is a purpose of super-male plants in breeding programs, the time generally takes about 5~6 years containing test cross processes (Reuther, 1984). Sexual dimorphism is known apparently controlled by gene(s) located on a pair of homomorphic L5 chromosomes in asparagus (Löptien, 1979). Telgmann-Rauber et al . (2007) determined and characterized genetic and physical maps around the sex-determining M-locus of the dioecious plant asparagus. Molecular markers have several advantages over traditional phenotypic markers. They can be detected in all phases of plant growth, whereas the latter can be identified only at a specific stage. The use of molecular markers can also improve the efficiency of crossbreeding because they are not modified by the environment. Their development has been greatly improved through the use of random amplified polymorphic DNA (RAPD) (Welsh and McClelland, 1990; Del Rio and Bamberg, 2000; Kim et al ., 2004), restriction fragment length polymorphism (RFLP) (Lízal and Relichová, 2004; Zhong et al ., 2003), amplified fragment length polymorphism (AFLP) (Lin and Kuo, 1995; Negi et al ., 2000), and simple sequence repeats (SSR) (Powell et al ., 1996). The RAPD technique, combined with BSA (bulked segregant analysis), can identify markers that are closely linked to economically important traits (Michelmore et al ., 1991; Paran and Michelmore, 1993). To facilitate MAS (marker-assisted selection), RAPD markers have been converted into valuable SCAR (sequence-characterized amplified region) markers (Barzen et al ., 1997; Gill et al ., 1998; Kim et al ., 2000; Vidal et al ., 2000). In case of asparagus, sex identification through the induction of early flowering was previously performed (Abe and Kameya, 1986; Yanosaka et al ., 1989). Since molecular marker techniques were developed, male- or female-specific molecular markers also have been developed for asparagus breeding program (Jamsari et al ., 2004; Lu et al . 2006; Gao et al ., 2007; Gebler et al ., 2007).
In this study, we report a SCAR marker to identify female and male plants in the dioecious asparagus.
Materials and Methods
Seeds of asparagus cultivars ‘Welcome’, ‘Greentower’ and ‘Shower’ were supplied by Sakata Seed Co. in Yokohama, Japan. Plants were cultivated from 2001 to 2004 at National Research Center for Climate Change, National Institute of Horticultural and Herbal Science, Rural Development Administration (NIHHS, RDA), Korea. Asparagus is a dioecious plant species containing separate staminate flowers and pistillate flowers on different individuals. Staminate flower looks superficially hermaphroditic, but its pistil size is much smaller than female flower due to a developmental arrest. In contrast, pistillate flowers develop only pistil without stamen ( Fig. 1 ). We collected three male and female plants from three varieties after flowering and immediately frozen in liquid nitrogen and stored at -70℃. Total DNA was purified from approximately 0.5 g of fresh leaves using the protein precipitation method of Dellaporta et al . (1983).
PPT Slide
Lager Image
Male (A) and female flowers (B) of asparagus. In case of female flower, petal was completely removed to show ovule and stigma. Scale bar = 5 mm.
RAPD analysis was performed with random decamer primers (Biotechnology Laboratory, University of British Columbia, Canada; UBC 301 to 400). Twenty nanograms of DNA was used as template in a total reaction volume of 25 ml that contained 2.5 ml of 10 × reaction buffer, 2.5 mM MgCl 2 , 400 mM of each dNTP, 4 pM of each primer, and 2.0 units Taq DNA polymerase (MBI Fermentas, St. Leon-Roth, Germany). Amplification was performed in a Gene Amp PCR System 9600 Thermal Cycler (Perkin Elmer Cetus, USA), with initial denaturation for 5 min at 94℃, then 30 cycles of 1 min at 94℃, 1 min at 38℃, and 1.5 min at 72℃; followed by 5 min of final extension at 72℃. The PCR products were separated on 1.2% agarose gels at 100 V for 40 min using 1 × Tris-borate-EDTA (TBE) buffer, and finally stained with ethidium bromide. After amplification, each RAPD marker fragment was purified with 1 × TBE (Tris-borate/EDTA) buffer on 2% low-melting-point agarose (Promega, USA) gel. After staining with ethidium bromide, the agarose block containing the DNA was excised from the gel under low-wavelength UV light. DNA was recovered using a Wizard® PCR Preps DNA purification system (Promega, USA), according to the manufacturer’s protocol (Soltis and Soltis, 1997).
DNA fragments were cloned into the pT-Adv vector, with the aid of an AdvanTAge TM PCR Cloning Kit (Clontech, CA), according to the manufacturer’s instructions. Nucleotide sequences of the cloned fragment were determined using Cy5-labeled vector primers -- M13 -40 and M13 reverse -- and a Cy 5TM AutoCycle TM Sequencing Kit (Pharmacia, USA) on an ALFexpress II DNA Sequencer. The samples were separated on 6% acrylamide-7 M urea gels in 0.5 × TBE buffer at 1500 V for 700 min. Sequences were aligned with CLUSTAL W (Thompson et al ., 1994) and then adjusted manually to align several conserved regions. Sites with missing data or gaps were excluded from all analyses. Four specific primers were designed from the sequences of the marker fragments. Each primer comprised a 10 base pair (bp) of original RAPD primer sequence and an additional 12 to 14 bp of internal sequence. The PCR reaction mixture (25 ml) contained 2.5 ml of 10 × reaction buffer, 2.5 mM MgCl 2 , 400 mM of each dNTP, 4 pM of each primer, and 2.0 units Taq DNA polymerase. Amplification was performed in a GeneAmp PCR System 9600 Thermal Cycler, with initial denaturation for 5 min at 94℃; followed by 1 min at 94℃, 1 min at 57℃, and 1.5 min at 72℃ (total of 30 cycles); with 7 min of final extension at 72℃. The PCR products were separated on 1.2% agarose gels at 100 V for 40 min using 1 × TBE buffer before being stained with ethidium bromide.
Results and Discussion
We utilized a RAPD approach to find sex-linked molecular markers from asparagus. Out of 100 primers screened (UBC300 to UBC400), only two primers UBC319 and UBC347 generated polymorphism PCR bands between male and female plants. The UBC347 primer produced a 400 bp fragment UBC400 347 linked to female plants ( Fig. 2 ). However, UBC319 primer showed a problem in reproducibility. Therefore, we performed further study only for PCR product amplified with UBC347 primer.
PPT Slide
Lager Image
RAPD marker UBC347 linked to sex. Arrow indicates a specific RAPD band for female plants.
Since RAPD markers show poor reproducibility, they have to be converted into SCAR markers. For this purpose, we cloned and determined its nucleotide sequence and compared sequences of UBC400 347 fragments from female plants ( Fig. 3A ). A 24-mer SCAR primer was designed on the basis of the UBC319 RAPD primer which amplified specific fragments only in male plants and a 22-mer SCAR primer was designed on the basis of the UBC347 RAPD primer which amplified specific bands only in female plants. The primer named F400 ( Table 1 ).
PPT Slide
Lager Image
Nucleotide sequences of a female specific fragment amplified with UBC347 primer and multiple sequence alignment. (A) Nucleotide sequences of a female specific fragment amplified with UBC347 primer. Underlines indicate primer sequences for SCAR primers. (B) Multiple sequence alignment of UBC400347 fragment and plant retrotransposon proteins belonging to UBN2 superfamily. The sequence alignment and conserved residues were displayed using the CLUSTALW and the BOXSHADE programs. Identical amino acid sequences are highlighted in black, and similar sequences in grey. The retrotransposon protein sequences used in this alignment are as follows: UBC347, Asparagus officinalis L. (in this study, GenBank Acc. No. JX508289); Oryza sativa Japonica Group (GenBank Acc. Nos. AAP53866 and AAO13467, respectively); Solanum lycopersicum (GenBank Acc. No. ABO36622); Nicotiana tabacum (GenBank Acc. No. P10978); Solanum chilense (GenBank Acc. No. AAK29467).
A SCAR primer designed from sequences of RAPD-PCR fragmant produced by UBC347 primer
PPT Slide
Lager Image
A SCAR primer designed from sequences of RAPD-PCR fragmant produced by UBC347 primer
The fragment amplified with UBC347 primer was abundant in AT with a 38% of low GC content. BlastX search against NCBI nr database search using the nucleotide sequences from UBC400 347 showed that it shared a 45% sequence identity (76% sequence similarity and 3e-12 of E-value) with a putative retrotransposon protein of rice, belonging to Ty1-copia subclass ( Fig. 3B ). Various types of retroelements have been found in heteromorphic or homomorphic sex chromosomes of dioecious plants (Fraser and Heitman 2005; Telgmann- Rauber et al ., 2007; Urasaki et al ., 2012). In this context, UBC400 347 fragment is likely a part of female-specific retroelements.
We tested the SCAR primer in eighteen male/female plants of the three cultivars (‘Welcome’, ‘Greentower’ and ‘Shower’). PCR amplification with the SCAR primer F400 revealed a 400 bp fragment with the same size as the cloned RAPD marker, which was female-specific in all the plants belonging to the three cultivars ( Fig. 4 ).
PPT Slide
Lager Image
Identification of sex using SCAR primer F400 for male and female plants of asparagus. Lane 1 and Lane 20, GeneRulerTM 100-bp DNA Ladder Plus; Lane 2 to 4, female of cv. ‘Welcome’; Lane 5 to 7, male of cv. ‘Welcome’; Lane 8 to 10, female of cv. ‘Greentower’; Lane 11 to 13, male of cv. ‘Greentower’; Lane 14 to 16, female of cv. ‘Shower’; Lane 17 to 19, male of cv. ‘Shower’. Arrow indicates a specific female fragment amplified with a SCAR primer F400.
There is no way to identify morphological differences between male and female at seedling stages (Kubituki and Rudall, 1998). And then, sex-linked molecular markers in asparagus are not only important for breeding but also for understanding the genetic mechanism of sex determination. SCAR markers are also advantageous over RAPD markers because they detect only a single locus and their amplification is less sensitive to reaction conditions (Weeden, 1994). Several sex-linked molecular markers have been identified from asparagus (Jiang and Sink, 1997; Lu et al ., 2006; Gao et al ., 2007; Gebler et al ., 2007).
To obtain sex-linked diagnostic markers and to better understand molecular and evolutionary mechanism of sex determination in asparagus, a BAC library was constructed and several BAC clones containing sex-linked markers were identified (Jamsari et al ., 2004). Telgmann-Rauber et al ., (2007) determined genetic and physical maps around the sex-determining M-locus using 4 closely linked BAC clones and also found an accumulation of repetitive sequences and a low gene density in the sex-determining region UBC400 347 fragment has no sequence homology with any other nucleotide sequences derived from asparagus on NCBI nr database and previously published literatures, indicating that the fragment is a new female-linked molecular marker located on a novel locus. Jiang and Sink (1997) reported that SCAR markers linked to the sex expression locus M from asparagus could be used for only one parents and their progeny. But F400 SCAR marker was found to be applicable to three asparagus germplasm. We suggest that the SCAR marker will be a useful marker for cultivation and breeding of a new variety in three asparagus varieties (‘Welcome’, ‘Greentower’ and ‘Shower’).
Acknowledgements
This study was carried out with the support of “Research Program for Agricultural Science & Technology Development (Project No. PJ007461)”, RDA, Republic of Korea. Authors also thank to Dr. Ho Bang Kim (BioMedic, Co. Ltd) for critical reading of the manuscript.
References
Abe T , Kameya T 1986 Promotion of flower formation by atrazine and diuron in seedling of Asparagus Planta 169 289 - 291
Barzen E , Stahl R , Fuchs E , Borchardt D.C , Salamin F 1997 Development of coupling repulsion-phase SCAR markers diagnostic for the sugar beet Rr1 allele conferring resistance to rhizomania Mol. Breed. 3 231 - 238
Dellaporta S.L , Wood J , Hick J.B 1983 A plant DNA minipreparation: version II Plant Mol. Rep. 1 19 - 21
Del Rio A.F , Bamberg J.B 2000 RAPD markers efficiently distinguish heterogenous populations of wild potato (Solanum) Gen. Res. Crop Evol. 47 115 - 121
Ellison J.H , Bassett M.J 1986 Breeding Vegetable Crops Avi Publishing Co Westport, USA Asparagus breeding 523 - 569
Fraser J.A , Heitman J 2005 Chromosomal sex-determining region in animals, plants and fungi Current. Opi. Genet. Dev. 15 645 - 651
Gao W.J , Li R.L , Li S.F , Deng C.L , Li S.P 2007 Identification of two markers linked to the sex locus in dioecious Asparagus officinalis plants Russian J. Plant Physiol. 54 816 - 821
Gebler P. , Wolko L. , Knaflewski. M. 2007 Identification of molecular markers for selection of supermale (YY) asparagus plants J. Appl. Genet. 48 129 - 131
Gill G.P , Harvey C.F , Gardner R.C , Fraser L.G 1998 Development of sex-linked PCR markers for gender identification in Actinidia Theor. Appl. Genet. 97 439 - 445
Jamsari A , Nitz I , Reamon-Büttner S.M , Jung C 2004 BAC-derived diagnostic markers for sex determination in asparagus Theor. Appl. Genet. 108 1140 - 1146
Jamilena M , Mariotti B , Manzano S 2008 Plant sex chromosomes: molecular structure and function Cytogenet. Genome Res. 120 255 - 264
Jiang C , Sink K.C 1997 RAPD and SCAR markers linked to the sex expression locus M in asparagus Euphytica 94 329 - 333
Kim C.S , Lee G.P , Han D.H , Ryu K.H , Lee C.H 2000 SCARs markers derived from RAPD for cultivar identification on Pyrus pyrifolia J. Kor. Soc. Hort. Sci. 41 125 - 128
Kim S.C , Jung Y.H , Kim M , Koh S.C , Song K.J , Kim H.B 2004 Characterization of a RAPD fragment unique to species with hairy fruit skin in the genus Actinidia J. Plant Biol. 47 210 - 215
Kubituki K , P.J Rudall , Kubituki K. 1998 Families and Genera of Vascular Plants Springer-Verlag Berlin, Heiderberg, Germany Asparagaceare 125 - 128
Lin J.J , Kuo J 1995 A novel PCR-based assay for plant and bacterial DNA fingerprinting Focus 17 70 - 71
Lízal P , Relichova J 2004 Localization of seven new late-flowering mutations on the genetic map of Arabidopsis thaliana using a newly generated CAPS marker Plant Sci. 167 143 - 149
Löptein H 1979 Identification of sex chromosome pair in asparagus (Asparags officinalis L.) Z. Pflanzenzuecht 82 162 - 173
Lu L.D , Li R.L , Gao W.J , Deng C.L , Wang L.J 2006 Cloning and analyzing of the female-specific marker in the dioecious species Asparagus officinalis L Fen Zi Xi Bao Sheng Wu Xue Bao 39 281 - 284
Michelmore R.W , Paran I , Kesseli R.V 1991 Identification of markers linked to disease resistant genes by bulked segregant analysis: A rapid method to detect markers in specific genome regions using segregating populations Proc. Natl. Acad. Sci. USA 88 9828 - 9832
Negi M.S , Devic M , Delseny M , Lakshmikumaran M 2000 Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to SCAR marker for rapid selection Theor. Appl. Genet. 101 146 - 152
Paran I , Michelmore R.W 1993 Development of reliable PCR-based markers linked to downy mildew resistance in lettuce Theor. Appl. Genet. 85 985 - 993
Powell W , Machray G.C , Provan J 1996 Polymorphism revealed by simple sequence repeats Trends Plant. Sci. 1 215 - 222
Reuther C , Sharp R.S , Evans D.V , Ammirato P.V , Yamada Y 1984 Handbook of Plant Cell. MacMillan Publishing Co New York, USA Asparagus 211 - 239
Reamon-Büttner S.M 2000 AFLP-derived STS marker for the identification of sex in Asparags officinalis L Theor. Appl. Genet. 100 432 - 438
Soltis D.E , Soltis P.S 1997 Phylogenetic relationships in Saxifragaceae sensu lato: A comparison of topologegies based on 18S rDNA and rbcL sequences Amer. J. Bot. 84 504 - 522
Telgmann-Rauber A , Jamsari A , Kinney M.S , Pires J.C , Jung C 2007 Genetic and physical maps around the sex-determining M-locus of the dioecious plant asparagus Mol. Genet. Genomics 278 221 - 234
Thompson J.D , Higgins D.G , Gibson T.J 1994 CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting position specific gap penalties and weight matrix choice Nucl. Acids Res. 22 4673 - 4680
Urasaki N , Tarora K , Shudo A , Ueno H , Tamaki M , Miyagi N , Adaniya S , Matsumura H 2012 Digital transcriptome analysis of putative sex-determination genes in papaya (Carica papaya) Plos One 7 e40904 -
Vidal J.R , Delavault P , Coarer M , Defontaine A 2000 Design of grapevine (Vitis vinifera L) cultivar-specific SCAR primers for PCR fingerprinting Theor. Appl. Genet. 101 1194 - 1201
Welsh J , McClelland M 1990 Fingerprinting genomes using PCR with arbitrary primers Nucleic Acids Res. 18 7213 - 7218
Yanosaka K , Iwamura H , Fujita T 1989 Flower induction in seedling of Asparagus officinalis L by N-phenylcarbamates Z. Naturforch. 44c 226 - 232
Yeager A.F , Scott H 1938 Studies of mature asparagus plantings with special reference to sex survival and rooting habits Proc. Amer. Soc. Hort. Sci. 36 513 - 514
Zhong S , Effertz R.J , Jin Y , Franckowiak H.D , Steffenson B.J 2003 Molecular mapping of the leaf rust resistance gene Rph6 in barley and its linkage relationships with Rph5 and Rph7 Phytopathology 93 604 - 609