MOSCOW, RUSSIA

MOSCOW, RUSSIA
Timiryazev Institute of Plant Physiology
Moscow State University
Belozerskii Institute of Physicochemical Biology

Nucleotide sequence analysis of SCAR markers from Zea mays somaclones (Zea mays L.)

--Osipova, ES, Troitskij, AV, Dolgikh, YuI, Shamina, ZB, Gostimskij, SA

The deviations of the somaclones from the initial line A188 at the molecular level have been determined by RAPD- and ISSR- analysis (Osipova et al., MNL 74:52, 2000). Based on some specific RAPD- and ISSR-fragments, which were typical both for individual somaclones and for the groups of related ones, six SCAR-markers (Sequence Characterized Amplified Region) were produced (Osipova et al., MNL 77:53, 2003).

When we compared the nucleotide sequence of the SCAR-markers, using GenBank and PlantGDB, the most interesting results were obtained for the M-10 fragment. This marker contains the famous retrotransposon Opie-1 5′LTR (90% identity, error probability 3e^-54) (Fig. 1).

Figure 1. Finding the retrotransposon Opie-1 5′LTR in SCAR marker M-10.

AF050451 Zea mays retrotransposon Opie-1 5′ LTR, partial sequence

Length = 1231

Score = 220 bits (111), Expect = 3e-54
Identities = 171/190 (90%), Gaps = 2/190 (1%)
Strand = Plus / Minus

M-10: 46 acacaactcag--agttctctactcaaatggagctctagttgctatcacaaagaatcgaa 103
||||||||||| ||||| | ||| ||||||||||||| |||||||| ||||||||| ||
Opie-1: 776 acacaactcagcgagttcacaacttaaatggagctctaattgctatcgcaaagaatcaaa 717

M-10: 104 tgcgcggaaatgaggtcttggtgcttaggaatgctcaagggatgcttggtgtactcctcc 163
||||||||| || ||||||||||||||||||||| | | ||||||||||| |||||||
Opie-1: 716 tgcgcggaatcgaagtcttggtgcttaggaatgcttagagaatgcttggtgtgctcctcc 657

M-10: 164 atgcgcctaggggtcccttttatagctccaaggcagctaggagccgttgagagcattcca 223
|||||||||||||||| ||||||||| |||||||||||||||||||||||| ||||||||
Opie-1: 656 atgcgcctaggggtcctttttatagccccaaggcagctaggagccgttgagtgcattcca 597

M-10: 224 ggaaggcaat 233
||||||||||
Opie-1: 596 ggaaggcaat 587

The M-10 fragment was also homologous to part of the gene, which corresponded to the first exon of Zea mays ribosomal protein — L26 mRNA (Fig. 2). The deletion, consisting of 23 nucleotides and resulting in the shift of the reading frame, was found in the M-10 sequence, which corresponded to the L26 gene. A discrepancy of the sequences at the end of this gene encoding site and fragment M-10 was also noticed. Thus, we can assume that such destructive changes in this gene resulted from somaclonal variation within the process of cultivation.

Figure 2. Finding the part of the gene sequence encoding the first exon of Zea mays ribosomal protein L26 mRNA (marked in grey) in SCAR marker M-10.

M-10 CCGCTGCCATGAAGCGCAATCCCCGCATCACGAGCTCCCGCCGGAAGTGC 400
Zea_mays_ribosomal_protein_L26 CCGCCGCCATGAAGCGCAATCCCCGCGTCACGAGCTCCCGCCGGAAGTGC 96
**** ********************* ***********************

M-10 AGCAAGGCGCACTTCACGGCCCCGTCCTCCGTCCGCTGCGTTCTCATGTC 450
Zea_mays_ribosomal_protein_L26 CGCAAGGCGCACTTTACGGCCCCGTCCTCCGTCCGCCGCGTTCTCATGTC 146
************* ********************* *************

M-10 CGACGGGCTGTCGACGGAGCTCC-----------------------ATCC 477
Zea_mays_ribosomal_protein_L26 CGCGGGGCTGTCGACGGAGCTCCGCCACAAGTACAACGTGCGCTCCATCC 196
** ******************* ****

M-10 CGATCCACAAGGACGACGAGGTGCAGGTCGTGCGCGGCACCTACAAAGGC 527
Zea_mays_ribosomal_protein_L26 CGATCCGCAAGGACGACGAGGTGCAGGTCGTGCGCGGCACCTACAAGGGC 246
****** *************************************** ***

M-10 CGTGAGGGGAAGGTGGTGCAGGTGTACCACCGCCGCTGGGTCATCCACGT 577
Zea_mays_ribosomal_protein_L26 CGTGAGGGGAAGGTGGTGCAGGTGTACCGCCGCCGCTGGGTCATCCACGT 296
**************************** *********************

M-10 CGAGCGGATCACACGCGAGAAGGTGAACAGCTCTGTGCCTGGATGAGCGA 627
Zea_mays_ribosomal_protein_L26 CGAGCGGATCACCCGCGAGAAGGTGAACGGCTC----------------- 329
************ *************** ****

M-10 TCATCCAAGAGATAGACTAGGCTCTATGCCTGGATGAGCGATCCGCAAGG 677
Zea_mays_ribosomal_protein_L26 --------------------------------------------------

When analyzing the Leb marker, it was found that this marker could contain either two unannotated repeats or the site homologues of the unknown rice gene. There are hypothetical acceptor and donor sites in the Leb sequence at the bounds of the identity segment (Fig. 3). Perhaps this fragment has some homology with the analogous maize genes which haven’t been sequenced yet.

Figure 3. Finding the hypothetical acceptor and donor sites (marked in red) in the SCAR marker Leb located at the ends of the identified segment. The site homologues of the unknown rice gene is marked in grey.

TGTATAGACTCATCAAAAGCCTGGACCCATGGGGAGCCCATGATGATTTCCGGGGAAGACAGGCTTCGGCCCCGTAGACCAGGAAGAACGGGGTATCCCC
GGTGGCACGGCTGGGTGTTGTCCGGTTCCCCCATAGCACGGATGGGAGTTCACTGACCCAATTCGCACCATGCTTCTTCAAGTAATCGTAGGTGCGCGTC
TTGAGTCCCCTGAGGATCTCTGCATTTGCCTTTCCAACTTGGCCATTACTCCTGGGGTGGGCCACGGACGCGAAGCAGAGCTGGGTGTCGATGCCCTTGC
AATACTCCTGGAAGATCTGACTTGTAAACTGGGTTCCATTGTCCGTAATGATACGGTTTGGGACCCTAAATCTTCAGACGATGGACTTGAGGAAGGCGAC
AGCGGCACCCTAGGTGATGTTGACCACAGGGGTGGCCTTTGGCCACTTGGTGAACTTGTCGATGGTGACGAAGAGGTACCGGTACCCGCCAACGGCCCTG
GGAAATGGTCCCAGGATATCCACCCCCCATATGACGAATGGCCAGGAGGGTGGAATCATCTTCAGAATCTAAGCTGGTGTGTGTATCTGCTTTGCGTGGA
ATTAACACGCCTTGCAGGACTTTACCATCTCAGCTGCATCCTGGAGAGCGGTTGGCCAGTAGAAGCCATGCCAGAAGGCCTTACCGACCAGTGTGCAGGA
TGATGAATGACTTCCGCACTCTACTCCATGGATCTCCGTGAGCAACTCACGGCCCTCCTCCTGGGTAATGCACCGCATGTGAATGTCGTTGGCGCCGCGG
TGGTAGAGATCCCCTTCCACCACCGTGTAGCATTTAGCCAACCGCACTATGCGCTCTGCAGACACATGGTCTTCAGGAAGGATATTCTCCTTCAGGTAGT
CCCGGATCTCGGAGACCCATGCATCGGGACCACTCTG

Some repeating sequences were revealed in markers QR-A and QR-2. As a result of sequencing, it was found that the QR-2 fragment (1018 bp), which was characteristic of the somaclones, differed from the QR-A fragment (1428 bp), which was characteristic of the initial line A188 (Fig. 4). They differed in the deletion located from the 490 to 944 position, which was probably the result of somaclonal variation.

Figure 4. Comparison of nucleotide sequences of SCAR markers QR-2 and QR-A.

++++++++++++++++++++++++++++++++++++++ ++++ ++++++ ++++++++
QR-2 (420) AATCCCGGTGATCTACATCCCTGCCATCTTGGCTGGCAGAAACgaGAGTGCCGCTGAATT
QR-A (420) AATCCCGGTGATCTACATCCCTGCCATCTTGGCTGGCAAAAAC–-GAGTGCTGCTGAATT

+++++++++++++
QR-2 (480) CAAACACAGCTAA-----------------------------------------------
QR-A (478) CAAACACAGCTAAgggcctgtttggaagtagagttattccatagttttcatgcaatacca

QR-2 (493) ------------------------------------------------------------
QR-A (538) tagtatatagaaataccatggtatttcaaacccaaagctgtttggttggactttagaaaa

QR-2 (493) ------------------------------------------------------------
QR-A (598) cagagttttgaataccatagttttatcaataccatggtatttctgtggtattgaaaactg

QR-2 (493) ------------------------------------------------------------
QR-A (658) aagtccagaccaaagtttttcctttcttgcgcgtgttggactgcaccattatctttttct

QR-2 (493) ------------------------------------------------------------
QR-A (718) gcgctttactagttttgcttcatgtccgattggtccagatgttgacaatgtcttgtttac

QR-2 (493) ------------------------------------------------------------
QR-A (778) ggaaaaacctgtgacttatgtgacatggctccaaacatgtggtagtatctagggatacct

QR-2 (493) ------------------------------------------------------------
QR-A (838) tggttttaccttgggattagtaaaagggtagtattatgtcataacaattgttagactacg

+++++++++
QR-2 (493) --------------------------------------------------TACTTGGTT-
QR-A (898) gtatttctaaaccataggtttacaaaattgtgtttccaaacagggcctaaTACTTGGTTg

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
QR-2 (502) CCCTGTTCTCTTGCTCCAGTCAACTACTTCCATCCACCAGGATGTAGATCCTAATGCTGG
QR-A (958) CCCTGTTCTCTTGCTCCAGTCAACTACTTCCATCCACCAGGATGTAGATCCTAATGCTGG

The OPC-09 marker could contain either two unannotated repeats or the site homologues of the rice gene gag/pol, which is typical for encoding sites of retrotransposons (Table 1).

The results of our analysis demonstrated that these fragments mostly contained the repeats (Table 1). Such repeating sequences in specific RAPD- and ISSR-fragments were characteristic for somaclones. This corresponded to the available data of other researchers. In accordance to these, the somaclonal changes more often take place in the non-encoding sites of the genome.

Table 1. Analysis of nucleotide sequences of SCAR markers.

Name of the SCAR marker	Hits according to BLAST (NCBI)	Repeats according to Repeatmasker	Conclusion
M10 (806 bp)	There are substantial hits in Zea mays ribosomal protein L26 mRNA and Zea mays retrotransposon Opie-1 5′ LTR.	There is a masked repeat (2-236 bp) characteristic of cereals.	Known repeats and pseudogenes are possible. There is a frameshift in exon.
Leb (937 bp)	There are two hits in Z. mays, both in unannotated parts.	There is a masked repeat (63-761 bp) characteristic of cereals.	Two new repeats or genes are possible.
OPC-09 (640 bp)	There are two hits in Z. mays, both in unannotated parts.	No repeats were found.	Two new repeats or the destructive gene, gag/pol, characteristic of retrotransposons are possible.
QR-2 (1018 bp)	No hits were found.	No repeats were found.	New repeat is possible.
QR-A (1428 bp)	No hits were found.	No repeats were found.	New repeat is possible.
Q-20 (796 bp)	No hits were found.	No repeats were found.	No data.
NO-15 (204 bp)	No hits were found.	No repeats were found.	No data.

No homologues were revealed for the NO-15 and Q-20 markers. This allows us to assume these fragments were specific for the somaclones investigated.

To clarify the nature of the Q-20 marker and its distribution in the genome, the Southern-blot hybridization analysis was applied. Hybridization of the Q-20 probe revealed one major 550 bp fragment at the second group of somaclones. For the R105 regenerant, the same fragment was also found in a high-molecular zone that assumes the duplication of this fragment (Fig. 5).

Figure 5. Southern-blot analysis of EcoRI, EcoRV, XbaI-digested DNA hybridised to the alpha ³²P-dATP-labelled Q-20 SCAR probe. A188 — the initial maize inbred line; R11-R54 — the somaclones of the first group, obtained after two months subculturing; R105-R119 — the somaclones of the second group, obtained after eight months subculturing; M — the molecular-weight marker (1 kb DNA ladder “GIBCO BRL”).

Thus, by means of sequencing SCAR markers, which were created on the basis of some polymorphic RAPD- and ISSR-fragments, some homology with known sequences of Zea mays and some other cereals was found. The changes were revealed both among repeating and unique sequences. The finding of the retrotransposon in the M-10 fragment corresponds to the assumption about the possible activation of some mobile element in in vitro culture. This may be one of the reasons for the appearance of some genetic changes.