Garlic (A. sativum L.) alliinase gene family polymorphism reflects bolting types and cysteine sulphoxides content

BMC Genetics

Ladislav Kučera¹  Katarína Mitrová¹  Jaroslava Ovesná¹ 

[1] Crop Research Institute, Drnovská 507/73, Prague-Ruzyně, 161 06, Czech Republic

关键词: Bolting type;    Molecular marker;    Polymorphism;    Alliinase;    Garlic;    Allium sativum L.;   

Others  :  1216023
DOI  :  10.1186/s12863-015-0214-z

 received in 2015-01-13, accepted in 2015-05-11,  发布年份 2015

【 摘 要 】

Background

Alliinase is an important enzyme occurring in Allium species that converts precursors of sulfuric compounds, cysteine sulfoxides into a biologically active substance termed allicin. Allicin facilitates garlic defense against pests and produces health-promoting compounds. Alliinase is encoded by members of a multigene family that has not yet been sufficiently characterized, namely with regard to the copy numbers occurring within the genome and the polymorphisms among the family members.

Results

We cloned 45 full-length alliinase amplicons of cultivar (cv.) Jovan. Sequence analyses revealed nine different sequence variants (SVs), confirming the multilocus nature of this gene family. Several mutations in exons, mainly occurring in the first exon coding for vacuolar signal peptide, were found. These results enabled us to identify sequences with putatively modified vacuole-targeting abilities. We found additional sequence variants using partial amplicons. We estimated that the minimum number of gene copies in the diploid genome of the investigated cultivar was fourteen. We obtained similar results for another three cultivars, which differed in bolting type and place of origin. The further identification of high degree of polymorphisms in the intron regions allowed us to develop a specific polymerase chain reaction assay capable to capture intron length polymorphism (ILP). This assay was used to screen 131 additional accessions. Polymorphic data were used for cluster analysis, which separated the bolting and non-bolting garlic types and those with high cysteine-sulfoxide contents in a similar way as AFLP analysis in previous study. These newly developed markers can be further applied for the selection of desirable garlic genotypes.

Conclusions

Detailed analysis of sequences confirmed multigenic nature of garlic alliinase. Intron and exon polymorphism analysis generated similar results as whole genome variability assessed previously by AFLP. Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes. ILP markers capable to detect intron polymorphisms were newly developed. Developed markers could be applied in garlic breeding. Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.

【 授权许可】

   
2015 Ovesná et al.; licensee BioMed Central.

【 图 表 】

Fig. 1.

Figure 1.

【 参考文献 】

• [1]Ipek M, Ipek A, Almquist SG, Simon PW. Demonstration of linkage and development of the first low-density genetic map of garlic, based on AFLP markers. Theor Appl Genet. 2005; 110:228-36.
• [2]Park HJ, Kim IS. Antioxidant activities and anticancer effects of red yeast rice grown in the medium containing garlic. Food Sci Biotechnol. 2011; 20(2):297-302.
• [3]Lancaster JE, Dommisse EM, Shaw ML. Production of flavour precursors (S-alk(en)yl-L-cysteine sulphoxides) in photo callus of garlic. Phytochemistry. 1988; 27(7):2123-4.
• [4]Lanzotti V. Bioactive polar natural compounds from garlic and onions. Phytochem Rev. 2012; 11:179-96.
• [5]Kim YS, Baek HH, Chung IM, Kwon B, Ji GE. Garlic Fermentation by Lactic Acid Bacteria. Food Sci Biotechnol. 2009; 18(5):1279-83.
• [6]Kuettner EB, Hilgenfeld R, Weiss MS. Purification, characterization, and crystallization of alliinase from garlic. Arch Biochem Biophys. 2002; 402:192-200.
• [7]Shimon LJW, Rabinkov A, Shine I, Miron T, Mirelman D, Wilchek M et al.. Two structures of alliinase from Alliium sativum L.: Apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the PLP cofactor. J Mol Biol. 2007; 366:611-25.
• [8]Weiner L, Shin I, Shimon LJW, Miron T, Wilchek M, Mirelman D et al.. Thiol-disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum). Protein Sci. 2009; 18(1):196-205.
• [9]Wang J, Cao Y, Sun B, Wang C, Mo Y. Effect of ultrasound on the activity of alliinase from fresh garlic. Ultrason Sonochem. 2011; 18(2):534-40.
• [10]Van Damme EJ, Smeets K, Torrekens S, Van Leuven F, Peumans WJ. Isolation and characterization of alliinase cDNA clones from garlic (Allium sativum L.) and related species. Eur J Biochem. 1992; 209:751-7.
• [11]Rabinkov A, Zhu XZ, Grafi G, Galili G, Mirelman D. Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning. Appl Biochem Biotech. 1994; 48(3):149-71.
• [12]Do GS, Suzuki G, Mukai Y. Genomic organization of a novel root alliinase gene, ALL1, in onion. Gene. 2004; 325:17-24.
• [13]Drugă B, Şuteu D, Rosca-Casian O, Pârvu M, Sragos N. Two Novel Alliin Lyase (Alliinase) Genes from Twisted-Leaf Garlic (Allium obliquum) and Mountain Garlic (Allium senescens var. montanum). Not Bot Hort Agrobot Cluj. 2011; 39(2):293-8.
• [14]Ovesná J, Kučera L, Horníčková J, Svobodová L, Stavělíková H, Velíšek J et al.. Diversity of S-alk(en)yl cysteine sulphoxide content within a collection of garlic (Allium sativum L.) and its association with the morphological and genetic background assessed by AFLP. Sci Hortic. 2011; 129:541-7.
• [15]Nwachukwu ID, Slusarenko AJ, Gruhlke MCH. Sulfur and sulfur compounds in plant defence. Nat Prod Commun. 2012; 7(3):395-400.
• [16]Viswanathan V, Phadatare AG, Mukne A. Antimycobacterial and antibacterial activity of Allium sativum bulbs. Indian J Pharm Sci. 2014; 76(3):256-60.
• [17]Rana SV, Pal R, Vaiphei K, Sharma SK, Ola RP. Garlic in health and disease. Nutr Res Rev. 2011; 24(1):60-71.
• [18]Trio PZ, You S, He X, He J, Sakao K, Hou DX. Chemopreventive functions and molecular mechanisms of garlic organosulfur compounds. Food Funct. 2014; 5(5):833-44.
• [19]King JJ, Bradeen JM, Bark O, McCallum JA, Havey MJ. A low-density genetic map of onion reveals a role for tandem duplication in the evolution of an extremely large diploid genome. Theor Appl Genet. 1998; 96(1):52-62.
• [20]Ohri D, Pistrick K. Phenology and genome size variation in Allium L. - a tight correlation? Plant Biology. 2001; 3(6):654-60.
• [21]Kondrashov FA. Gene duplication as a mechanism of genomic adaptation to a changing environment. Proc Biol Sci. 2012; 279:5048-57.
• [22]Etoh T, Simon PW. Diversity, fertility and seed production of garlic. In: Allium Crop Sciences: Recent Advances. Rabinowitch HD, Currah L, editors. CAB International, Wallingford, UK; 2002: p.101-17.
• [23]Fischer I, Dainat J, Ranwez V, Glémin S, Dufayard J-F, Chantret N. Impact of recurrent gene duplication on adaptation of plant genomes. BMC Plant Biol. 2014; 14:151. BioMed Central Full Text
• [24]Endo A, Imai Y, Nakamura M, Yanagisawa E, Taguchi T, Torii K et al.. Distinct intraspecific variations of garlic (Allium sativum L.) revealed by the exon-intron sequences of the alliinase gene. J Nat Med. 2014; 68(2):442-7.
• [25]Kim DW, Jung TS, Nam SH, Kwon HR, Kim A, Chae SH et al.. GarlicESTdb: an online database and mining tool for garlic EST sequences. BMC Plant Biol. 2009; 9:61. BioMed Central Full Text
• [26]Paris N, Neuhaus JM. BP-80 as a vacuolar sorting receptor. Plant Mol Biol. 2002; 50(6):903-14.
• [27]Pompa A, De Marchis F, Vitale A, Arcioni S, Bellucci M. An engineered C-terminal disulfide bond can partially replace the phaseolin vacuolar sorting signal. Plant J. 2010; 61(5):782-91.
• [28]Pereira C, Pereira S, Pissarra J. Delivering of Proteins to the Plant Vacuole-An Update. Int J Mol Sci. 2014; 15(5):7611-23.
• [29]Son JH, Park KC, Lee SI, Jeon EJ, Kim HH, Kim NS. Sequence Variation and Comparison of the 5S rRNA Sequences in Allium Species and their Chromosomal Distribution in Four Allium Species. J Plant Biol. 2012; 55(1):15-25.
• [30]Cavagnaro P, Masuelli R, Simon PW. Molecular data suggest multiple members comprising the alliinase gene family in garlic. Hort Science. 2003; 38:804-5.
• [31]Cantsilieris S, Baird PN, White SJ. Molecular methods for genotyping complex copy number polymorphisms. Genomics. 2013; 101(2):86-93.
• [32]Pohl G, Shih IM. Principle and applications of digital PCR. Expert Rev Mol Diagn. 2004; 4(1):41-7.
• [33]Ma KH, Kwag JG, Zhao W, Dixit A, Lee GA, Kim HH et al.. Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic (Allium sativum). Sci Hortic. 2009; 122(3):355-61.
• [34]Chen S, Chen W, Shen X, Yang Y, Qi F, Liu Y et al.. Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits. Biochem Syst Ecol. 2014; 55:260-7.
• [35]Chinnappareddy LRD, Khandagale K, Chennareddy A, Ramappa VG. Molecular markers in the improvement of Allium crops. Czech J Genet Plant Breed. 2013; 49:131-9.
• [36]Ipek M, Ipek A, Senalik D, Simon PW. Characterization of an unusual cytoplasmic chimera detected in bolting garlic clones. J Amer Soc Hort Sci. 2007; 132(5):664-9.
• [37]Ovesná J, Leišová-Svobodová L, Kučera L. Microsatellite analysis indicates the specific genetic basis of Czech bolting garlic. Czech J Genet Plant Breed. 2014; 50(3):226-34.
• [38]Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000; 132:365-86.
• [39]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-10.
• [40]Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H et al.. ClustalW and ClustalX version 2.0. Bioinformatics. 2007; 23(21):2947-8.
• [41]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28:2731-9.
• [42]Kapustin Y, Souvorov A, Tatusova T, Lipman D. Splign: algorithms for computing spliced alignments with identification of paralogs. Biol Direct. 2008; 3:20. BioMed Central Full Text
• [43]Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003; 31(13):3784-8.
• [44]Sigrist CJ, de Castro E, Cerutti L, Cuche BA, Hulo N, Bridge A et al.. New and continuing developments at PROSITE. Nucleic Acids Res. 2013; 41(D1):D344-7.
• [45]Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR et al.. Pfam: the ptotein families database. Nucleic Acids Res. 2014; 42:222-30.
• [46]Emanuelsson O, Brunak S, von Heijne G, Nielsen H. Locating proteins in the cell using TargetP, SignalP, and related tools. Nat Protoc. 2007; 2(4):953-71.
• [47]Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 1997; 10(1):1-6.
• [48]Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011; 8:785-6.
• [49]Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis Version 6.0. Mol Biol Evol. 2013; 30(12):2725-9.
• [50]Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4:406-25.
• [51]Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985; 39:783-91.
• [52]Zuckerkandl E, Pauling L. Evolutionary divergence and convergence of proteins. In: Evolving Gene and Proteins. Bryson V, Vogel HJ, editors. Academic, New York; 1965: p.97.
• [53]Letunic I, Bork P. Interactive tree of life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acid Res. 2011; 39:475-8.