Cover Image

DNA Barcoding in Selected Species and Subspecies of Rye (Secale) Using Three Chloroplast Loci (matK, rbcL, trnH-psbA)

Lidia SKUZA, Izabela SZUĆKO, Ewa FILIP, Anastazja ADAMCZYK


DNA barcoding is a relatively new method of identifying plant species using short sequences of chloroplast DNA. Although there is a large number of studies using barcoding on various plant species, there are no such studies in the genus Secale. In this study the plant material consisted of 10 cultivated and non-cultivated species and subspecies of rye genus. Three chloroplast DNA regions (rbcL, matK, trnH-psbA) were tested for their suitability as DNA barcoding regions. Universal primers were used, and sequenced products were analyzed using Neighbor Joining and the Maximum Likelihood in the MEGA 7.1 program. We did not observe high variability in nucleotide sequences within the matK and rbcL regions. Only 2.2% of the sequences showed polymorphism in the rbcL region, while 6.5% in the matK region. The most variable trnH-psbA (15.6%) intergenic region was the most useful for rye barcoding. Individual application of the studied regions did not provide the expected results. None of the regions used in the study allowed the division of rye species and subspecies according to the adopted classification of the genus Secale. The results confirm that the use of matK and rbcL is insufficient for DNA barcoding in rye species, and better discrimination within the genus Secale can be obtained only in combination with the non-coding trnH-psbA sequence. Our results also indicate the necessity of using a different region. All of the new sequences have been deposited in Genbank.



In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 1, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI number will become active after the article will be included in the complete issue.


barcoding DNA; rye; cpDNA; intergenic spacer

Full Text:



Ajmal AM, Gyulai G, Hidwegi N, Kerti B, Al Hemaid F, Pandey AK, Lee J (2014). The changing epitome of species identification – DNA barcoding. Saudi Journal of Biological Sciences 21:204-231.

Bartoš J, Paux E, Kofler R, Havránková M, Kopecký D, Suchánková P, … Doležel J (2008). A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R. BMC Plant Biology 8:1-12.

Bauer E, Schmutzer T, Bauer E, Schmutzer T, Barilar I, Mascher M, … Scholz U (2017). Towards a whole-genome sequence for rye (Secale cereale L.). The Plant Journal 89(5):853-869.

Bellstedt DU, Linder HP, Harley EH (2001). Phylogenetic relationships in Disa based on noncoding trnL-trnF chloroplast sequences: evidence of numerous repeat regions. American Journal of Botany 88:2088-2100.

Bieniek W, Mizianty M, Szklarczyk M (2015). Sequence variation at the three chloroplast loci (matK, rbcL, trnH-psbA) in the Triticeae tribe (Poaceae): comments on the relationships and utility in DNA barcoding of selected species. Plant Systematics and Evolution 301:1275-1286.

Bolibok-Bragoszewska H, Targonska M, Bolibok L, Kilian A, Rakoczy-Trojanowska M (2014). Genome-wide characterization of genetic diversity and population structure in Secale. BMC Plant Biology 14:184.

Bolson M, de Camargo SE, Brotto MA, Silva-Pereira V (2015). ITS and trnH-psbA as Efficient DNA Barcodes to Identify Threatened Commercial Woody Angiosperms from Southern Brazilian Atlantic Rainforests. PLoS One 10(12):e0143049.

CBOL Plant Working Group (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States of America 106:12794-12797.

Chen S, Yao H, Han J, Liu C, Song J, Shi L, … Leon C (2010). Validation of the ITS2 region as a novel DNA barcode for identifying medicinal. PLoS One 5(1):e8613.

Cowan RS, Chase MW, Kress WJ, Savolainen V (2006). 300,000 species to identify: problems, progress and prospects in DNA barcoding of land plants. Taxon 55:611-616.

Cuadrado A, Jouve N (2002). Evolutionary trends of different repetitive DNA sequences during speciation in the genus Secale. Journal of Heredity 93(5):339-345.

DeBustos A, Jouve N (2002). Phylogenetic relationships of the genus Secale based on the characterization of rDNA ITS sequences. Plant Systematics and Evolution 235:147-154.

Dedio W, Kaltsikes PJ, Larter EN (1969). Numerical chemotaxonomy in the genus Secale. Canadian Journal of Botany 47:1175-1180.

Del Pozo JC, Figueiras AM, Benito C, De La Pena A (1995). PCR derived molecular markers and phylogenetic relationships in the Secale genus. Biologia Plantarum 37:481-489.

Delipavlov D (1962). Secale rhodopaeum Delipavlov - A new species of rye from the Rhodope Mountains. Doklady Bolgarskoi Akademii Nauk 15:407-411.

DeMattia F, Bruni I, Galimberti A, Cattaneo F, Casiraghi M, Labra M (2011). A comparative study of different DNA barcoding markers for the identification of some members of Lamiaceae. Food Research International 44:693-702.

Du ZY, Qimike A, Yang CF, Chen JM, Wang QF (2011). Testing four barcoding markers for species identification of Potamogetonaceae. Journal of Systematic and Evolution 49:246-251.

Fazekas AJ, Burgess KS, Kesanakurit PR, Graham SW, Newmaster SG, Husband BC, … Barrett SCH (2008). Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well. PLoS One 3(7):e2802.

Frederiksen S, Petersen G (1998). A taxonomy revision of Secale (Triticeae, Poceae). Nordic Journal of Botany 18:399-420.

Gamache J, Sun G (2015). Phylogenetic analysis of the genus Pseudoroegneria and the Triticeae tribe using the rbcL gene. Biochemical Systematics and Ecology 62:73-81.

Ge SA, Li B, Lu R, Zhang SZ, Hong DY (2002). A phylogeny of the rice tribe Oryzeae (Poaceae) based on matK sequence data. American Journal of Botany 89:1967-1972.

Hamilton MB, Braverman JM, Soria-Hernanz DF (2003). Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in New World species of the Lecythidaceae. Molecular Biology and Evolution 20:1710-1721.

Hartmann S, Nason JD, Bhattacharya D (2002). Phylogenetic origins of Lophocereus (Cactaceae) and the senita cactus-senita moth pollination mutualism. American Journal of Botany 89:1085-1092.

Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003). Biological identification through DNA barcodes. Proceedings of the Royal Society of London 270:313-322.

Hollingsworth ML, Clark A, Forrest LL, Richardson J, Pennington RT, Long DG, … Hollingsworth PM (2009). Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants. Molecular Ecology Resources 9:439-457.

Hollingsworth PM, Graham SW, Little DP (2011). Choosing and using a plant DNA barcode. PLoS One 6(5):e19254.

Hunt HV, Badakshi F, Romanova O, Howe CJ, Jones MK, Heslop-Harrison JSP (2014). Reticulate evolution in Panicum (Poaceae): the origin of tetraploid broomcorn millet, P. miliaceum. Journal of Experimental Biology 65(12):3165-3175.

Kelly LJ, Ameka GK, Chase MW (2010). DNA Barcoding of African Podostemaceae (river-weeds): A test of proposed barcode regions. Taxon 59:251-260.

Khush GS (1962). Cytogenetic and evolutionary studies in Secale. II. Interrelationships of the wild species. Evolution 16:484-496.

Klak C, Hedderson TA, Linder P (2003). A molecular systematic study of the Lampranthus group (Aizoaceae) based on the chloroplast trnT-trnF and nuclear ITS and 5S NTS sequence data. Systematic Botany 28:70-85.

Kress WJ, Erickson DL (2007). A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region. PLOS ONE 2(6):e508.

Kress WJ, Erickson DL, Andrew JF, Swenson NG, Perez R, Sanjur O, Bermingham E (2009). Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proceedings of the National Academy of Sciences of the United States of America 106:18621-18626.

Kubicka H, Puchalski J, Niedzielski M, Luczak W, Martyniszyn A (2006). Gromadzenie i ocena zasobów genowych ?yta [Collection and evaluation of rye gene resources]. Biulletin of Plant Breeding Acclimatization Instituite IHAR 40(241):141-149.

Muellner AN, Samuel R, Johnson SA, Cheek M, Pennington TD, Chase MW (2003). Molecular phylogenetics of Meliaceae (Sapindales) based on nuclear and plastid DNA sequences. American Jouranl of Botany 90:471-480.

Murai K, Naiyu X, Tsunewaki K (1989). Studies on the origin of crop species by restriction endonuclease analysis of organellar DNA. III. Chloroplast DNA variation and interspecific relationships in the genus Secale. The Japanese Journal of Genetics 64:36-47.

Newmaster SG, Ragupathy S (2009). Testing plant barcoding in a sister species complex of pantropical Acacia (Mimosoideae, Fabaceae). Molecular Ecology Resources 9:172-180.

Parmentier I, Duminil J, Kuzmina M, Philippe M, Thomas DW, … Hardy OJ (2013). How effective are DNA barcodes in the identification of African rainforest trees?. PLoS One 8(4): e54921.

Petersen G, Doebley F (1993). Chloroplast DNA variation in the genus Secale (Poaceae). Plant Systematics and Evolution 187:115-125.

Rabinovich SV (1998). Importance of wheat-rye translocations for breeding modern cultivar of Triticum aestivum L. Euphtytica 100:323-340.

Reddy P, Appels R, Baum BR (1990). Ribosomal DNA spacer-length variation in Secale spp. (Poaceae). Plant Systematics and Evolution 171:203-220.

Ren TH, Chen F, Zou YT, Jia YH, Zhang HQ, Yan BJ, Ben ZL (2011). Evolutionary trends of microsatellites during the speciation process and phylogenetic relationships within the genus Secale. Genome 54(4):316-326.

Roy S, Tyagi A, Shukla V, Kumar A, Singh UM, Chaudhary LB, … Tuli R (2010). Universal plant DNA barcode loci may not work in complex groups: A case study with Indian Berberis species. PLoS One 5:e13674.

Rzepka-Plevnes D (1990). Wlasciwosci miedzygatunkowych mieszancow zyta (Secale sp.) [Properties of cross-species hybrid rye (Secale sp.)]. Bulletin of Plant Breeding Acclimatization Institute IHAR 32/3(4):27-36.

Rzepka-Plevnes D (1993). Badania nad mieszancami S. cereale × S. vavilovii Gross. w aspekcie ich przydatnosci w hodowli odmian zyta odpornych na porastanie. Czesc I [Utility properties of hybrids S. cereale × S. vavilovii Gross. in terms of their suitability in growing rye varieties resistant to sprouting. Part I]. Bulletin of Plant Breeding Acclimatization Institute IHAR 37/5(6):69-79.

Rzepka-Plevnes D, Tomczak P (1993). Badania nad mieszancami S. cereale × S. vavilovii Gross. w aspekcie ich przydatnosci w hodowli odmian zyta odpornych na porastanie. Czesc II [Utility properties of hybrids S. cereale × S. vavilovii Gross. in terms of their suitability in growing rye varieties resistant to sprouting. Part II]. Bulletin of Plant Breeding Acclimatization Institute IHAR 37/5(6):81-91.

Sakai MA, Kanazawa A, Fujii F, Thseng S, Abe J, Shimamoto Y (2003). Phylogenetic relationships of the chloroplast genomes in the genus Glycine inferred from four intergenic spacer sequences. Plant Systematics and Evolution 239:29-54.

Samuel RT, Stuessy F, Tremetsberger K, Baeza CM, Siljak-Yakovlev S (2003). Phylogenetic relationships among species of Hypochaeris (Asteraceae, Cichorieae) based on ITS, plastid trnL intron, trnL-F spacer, and matK sequences. American Journal of Botany 90:496-507.

Sang TD, Crawford J, Stuessy TF (1997). Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). American Journal of Botany 84:1120-1136.

Sass C, Little DP, Stevenson DW, Specht CD (2007). DNA Barcoding in the Cycadales: Testing the potential of proposed barcoding markers for species identification of cycads. PLoS One 2:e1154.

Schittenhelm S, Kraft M, Wittich KP (2014). Performance of winter cereals grown on field-stored soil moisture only. European Journal of Agronomy 52(B):247-258.

Schlegel R (2006). Rye (Secale cereale L.): A younger crop plant with a bright future. Singh RJ, Jauhar PP (Eds). In: Genetic Resources, Chromosome Engineering, and Crop Improvement: Cereals. Vol 2. CRC Press, US pp 365-394.

Shang H-Y, Wei Y-M, Wang X-R, Zheng Y-L (2006). Genetic diversity and phylogenetic relationships in the rye genus Secale L. (rye) based on Secale cereale microsatellite markers. Genetics and Molecular Biology 29:685-691.

Singh R, Robbelen G (1975). Comparison of somatic Giemza banding pattern in several species of rye. IG Pfalnzenzuchtg 75:270-285.

Skuza L, Filip E, Szucko I (2015) Intergenic spacer length variability in cultivated, weedy and wild rye species. Open Life Science 10(1):175-181.

Skuza L, Rogalska SM, Bocianowski J (2007). RFLP analysis of mitochondrial DNA in the genus Secale. Acta Biologica Cracoviensia Series Botanica 49(1):77-87.

Spencer HA, Hawkes JG (1980). On the origin of cultivated rye. Biological Journal of the Linnean Society 13:299-313.

Stoeckle MY, Thalerd S (2014). DNA barcoding works in practice but not in (neutral) theory. PLoS One 9:e100755.

Stracke S, Schilling AG, Forster J, Weiss C, Glass C, Miedaner T, Geiger HH (2003). Development of PCR-based markers linked to dominant genes for male-fertility restoration in Pampa CMS of rye (Secale cereale L.). Theoretical and Applied Genetics 106:1184-1190.

Su X, Liu YP, Chen Z, Chen KL (2016). Evaluation of candidate barcoding markers in Orinus (Poaceae). Genetic Molecular Research 15(2):gmr.15027714.

Theodoridis S, Stefanaki A, Tezcan M, Aki C, Kokkini S, Vlachonasios KE (2012). DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çesme-Karaburun Peninsula (Turkey). Molecular Ecology Resources 12:620-633.

Tripathi AM, Tyagi A, Kumar A, Singh A, Singh S, Chaudhary LB, Roy S (2013). The Internal Transcribed Spacer (ITS) region and trnH-psbA are suitable candidate loci for DNA barcoding of tropical tree species of India. PLoS One 8:e57934.

Vences FJ, Vaquero F, de la Vega MP (1987a) Phylogenetic relationships in Secale: An isozymatic study. Plant Systematics and Evolution 157:33-47.

Vences FJ, Vaquero F, Garcia P, de la Vega MP (1987b). Further studies on phylogenetic relationships in Secale: On the origin of its species. Plant Breeding 98:281-291.

Xu DH, Sakai AJ, Kanazawa M, Shimamoto A, Shimamoto Y (2000). Sequence variation of non-coding regions of chloroplast DNA of soybean and related wild species and its implications for the evolution of different chloroplast haplotypes. Theoretical and Applied Genetics 101:724-732.

Yan HF, Hao G, Hu CM, Ge XJ (2011). DNA Barcoding in closely related species: A case study of Primula L. sect. Proliferae Pax (Primulaceae) in China. Journal of Systematics and Evolution 49:225-236.

Zhang B, Luo L, Zhang X, Li R, Song Y, Zhang D, … Wei Y (2011). Bioinformatics analysis on ribulose-1,5-bisphosphate carboxylase/ oxygenase large subunits in different plant. Molecular Plant Breeding 2(15):101-108.

Zimmermann T, Bocksberger G, Brüggemann G, Berberich W (2013). Phylogenetic relationship and molecular taxonomy of African grasses of the genus Panicum inferred from four chloroplast DNA-barcodes and nuclear gene sequences. Journal of Plant Research 126(3):363-371.


June 1, 2018: Notulae Botanicae Horti Agrobotanici Cluj-Napoca in Scopus – Elsevier CiteScore 2017=0.78, Horticulture; Agronomy and Crop Science; Plant Science