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Supplementary material 1
Intraspecific and intraorganismalcopy number dynamics of retrotransposons and tandem repeat
inAegilops speltoidesTausch (Poaceae, Triticeae)
“Protoplasma”
Imad Shams and Olga Raskina*
Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Aba-Hushi Avenue 199, Mount Carmel, Haifa 3498838, Israel
*Corresponding author:
Phone: (972) 4 8240785
Email:
Methods: Extraction of genomic DNA from the spike tissues
Genomic DNA was extracted from the pistils and anthers using the NucleoSpin Plant II kit (MACHEREY-NAGEL GmbH & Co. KG, Germany) with modifications. Specifically, the young spikes at the microsporogenesis stage were collected in fixative (3:1 absolute ethanol to glacial acetic acid) and stored at +4 °C until further analysis.The procedures ofgDNA extraction were performed in 1.5 ml microcentrifuge tubes; the solutions were carefully changed using a manual pipettorafter a short spin at a maximum speed of 2,000 rpm.Prior to gDNA extraction, similar to the FISH procedures, the fixed material was thoroughly washed (3 x 5 min) in distilled water and subsequently incubated in an enzyme buffer (10 mM citrate buffer at pH 4.6) and partially digested in 6% pectinase plus 0.5% cellulase (NBC Biomedicals, UK) and 5% cellulase “Onozuka” R-10 (Yakult Honsha Co., Ltd) at 370C (pistils for 30 min, pre-meiotic anthers for 40 min, anthers at the meiosis I stages for 50 min, and anthers at the meiosis II stages for 60 min) followed by washes with enzyme buffer (3 x 5 min) and distilled water (3 x 5 min). Subsequently, the plant material was transferred to lysis buffer PL1 based on the established CTAB procedures containing RNase A using a standard series of steps according to the manufacturer's instructions.Specifically, the procedures were as follows: cell lysis for 10 min at 650C; filtration/clarification of lysate in spin columns by centrifugation for 2 min at 11,000 g; binding of DNA to the silica membrane in PC buffer for 1 min at 11,000g; washing and drying of silica membrane in PW1 (1 x 1 min)and PW2 buffers(2 x 1 min) at 11,000g; DNA elution with nuclease-free double distilled water at 650C for1 min.In the cases of low amount of gDNA, concentration was increased using Vacuum Concentrator (Eppendorf) up to final concentration of 30 ng/µl in 20-25 µl of DDW. Minimum of 440 ng (anthers at the meiosis II stages, genotype C1) and maximum of 4,000 ng (anthers at the meiosis I stages, genotype F1_GK/A1) of gDNA were extracted. The minimal amount of gDNA extracted from the pistils was 550 ng for genotype F1_K5/A2.
Methods: In situ hybridization procedures
Cells were spread as described in Raskina et al. (2004a) and were treated with 1g/mL of DNase-free RNase A in 2SSC (0.3 M NaCl plus 30 mMtrisodium citrate) for 60 min at 37C followed by 3 3 min washes with 2SSC at 37C. The preparations were then dehydrated in an ethanol series (70, 90 and 100%, 3 min each) at room temperature, washed 2 2 min with 2SSC, and then allowed to air-dry. The hybridization mixture (20 L per slide under theglass coverslip 22 x 22 mm) contained 10% dextran sulfate, 2SSC, and 50 ng each of DNA probe. DNA probes and chromosome spreads were simultaneously denatured at 950C for 3 minand hybridized using ThermoBriteStatSpinSystem. Hybridization was carried out at 63C for 2 h. After removal of the coverslips, the slides were washed for 2 5 min in 2SSC at 63C, additionally once in 0.1SSC for 5 min at 63C to increase stringency, then cooled to 370C and washed for 2 x 5 min in 0.1xSSC, cooled to room temperature, washed in distilled water for 1 min, allowed to air-dry for 20 min, and mounted in antifade (Vector Laboratories).To re-probe after the microscopic analysis, the coverslips were removed in distilled water, the slides were dehydrated in an ethanol series (70, 90 and 100%, 3 min each) at room temperatureand used for FISH with a new set of DNA probes as it is described above.
Supplementary Table S1 Transposable elements, gene, and tandem repeat accessions and primer combinations for quantitative real-time PCR
Target / Accessions / Primers combination for qPCR / IdentityWilma,
Ty3-gypsy / TREP1438; AY494981.1 / W-For 5’-CTAGCCAAATTCTTTGCACCAA-3’
W-Rev 5’-TCTCATGGCAAAACTGGGTTT-3’ / internal domain
Angela,
Ty1-copia / FM242577.1 / A-For 5’-ACGAGGGTACGTGGACAACAC-3’
A-Rev 5’-AAAAATTCCTACGCACACGCA-3’ / LTR
Stasy,
LINE / TREP1820;
AY146587.2 / S-For 5’-GGGTCGTCGCATCCTTAGTG-3’
S-Rev 5’- CAAATGCGAAGACCCTTGCT-3’ / endonuclease domain
VRN-1,
gene / AY747606.1 / V-For 5'-TGGGAGAGGATCTTGAATCTTTG-3'
V-Rev5'-GATATGTTTCAGTGAGCTTTCCAGC-3' / Kraitshtein
et al. 2010
Spelt1, tandem repeat / Y09217.1 / Splt-For 5’-CTTGCTCCTCTATGGCACGG-3’
Splt-Rev 5’-AAATGTTGTGAACGGCGCTT-3’
Supplementary Table S2 Amplicon sequences for real-time quantitative PCR
Target / Amplicon / IdentityWilma,
Ty3-gypsy / 5’-CTAGCCAAATTCTTTGCACCAAGTAGAGATACT
ACATGTGCTTCTGAATATCCTTAAACCCAGTTTTGCCATGAGA-3’ / internal domain
Angela,
Ty1-copia / 5’-ACGAGGGTACGTGGACAACACTCTCCCCTCTCG
TTGCTATGCATCACCATGATCTTGCGTGTGCGTAGGAATTTTTT-3’ / LTR
Stasy,
LINE / 5’-GGGTCGTCGCATCCTTAGTGGCGCGCTACATCG
GCGCCCGGATGTAGTGGAAATTGAGCAAGGGTCTTCGCATTTG-3’ / endonuclease domain
Spelt1,
tandem repeat / 5’-CTTGCTCCTCTATGGCACGGCCAGAAGAACTTA
TTGCTATCCAAACCATCCCCGTCAAGCGCCGTTCACAACATTT-3’
VRN1,
gene / 5’-TGGGAGAGGATCTTGAATCTTTGAATCTCAAGGAGTTG CAGCAACTGGAGCAGCAGCTGGAAAGCTCACTGAAACATATC-3’ / Kraitshtein et al. 2010
Supplementary Table S3 Copy numbers in absolute quantification per haploid genome of Angela, Wilma, and Stasy retrotransposons and Spelt1 tandem repeat in leaves of Ae. speltoidesindividual genotypes
TEs, TR in leaves / Angela / Wilma / Stasy / Spelt1Genotypes / copies/1C / sample/
T.urartu / copies/1C / sample/
T. urartu / copies/1C / sample/
T. rartu / copies/1C
♀Giv’atKoach x ♂Ankara-1
♀GK / 6414 / 2.0 / 1452 / 1.2 / 42 / 1.5 / 75745
♂A1 / 7607 / 2.4 / 1682 / 1.4 / 43 / 1.6 / 287
Average ♀♂ / 7010 / 2.2 / 1567 / 1.3 / 42 / 1.6 / 38016
F1_GK/A1 / 5383 / 1.7 / 1524 / 1.2 / 46 / 1.7 / 43749
(% to ♀♂) / 77% / 97% / 110% / 115%
F2-1_GK/A1 / 7256 / 2.3 / 1720 / 1.4 / 59 / 2.2 / 40386
F2-4_GK/A1 / 5606 / 1.8 / 1380 / 1.1 / 42 / 1.5 / 48559
F2-5_GK/A1 / 5665 / 1.8 / 1458 / 1.2 / 47 / 1.7 / 47776
Average/set / 6593 / 2.1 / 2631 / 2.2 / 46 / 1.7 / 42750
♀Katzir-5 x ♂Ankara-2
♀K5 / 10385 / 3.3 / 2446 / 2.0 / 75 / 2.8 / 89095
♂A2 / 10695 / 3.4 / 2887 / 2.4 / 84 / 3.1 / 258
Average ♀♂ / 10540 / 3.3 / 2667 / 2.2 / 79 / 2.9 / 44676
F1_K5/A2 / 7843 / 2.5 / 1879 / 1.5 / 56 / 2.1 / 57948
(% to ♀♂) / 74% / 70% / 70% / 130%
F2-1_K5/A2 / 7520 / 2.4 / 1635 / 1.3 / 59 / 2.2 / 75785
F2-2_K5/A2 / 10197 / 3.2 / 1888 / 1.5 / 69 / 2.6 / 60329
F2-3_K5/A2 / 9673 / 3.0 / 2161 / 1.8 / 69 / 2.6 / 74443
Average/set / 9835 / 3.1 / 3681 / 3.0 / 68 / 2.5 / 74492
♀Cankiri-1 x ♂Katzir-17
♀C1 / 8122 / 2.5 / 1654 / 1.4 / 60 / 2.2 / 1564
♂K17 / 6825 / 2.1 / 1404 / 1.2 / 47 / 1.7 / 114164
Average ♀♂ / 7474 / 2.3 / 1529 / 1.3 / 53 / 2.0 / 57864
F1_C1/K17 / 7817 / 2.4 / 1117 / 0.9 / 53 / 2.0 / 82806
(% to ♀♂) / 105% / 73% / 100% / 143%
F2-2_C1/K17 / 6853 / 2.1 / 1582 / 1.3 / 44 / 1.6 / 51846
F2-3_C1/K17 / 5911 / 1.9 / 1353 / 1.1 / 49 / 1.8 / 49936
F2-4_C1/K17 / 5605 / 1.8 / 1209 / 1.0 / 35 / 1.3 / 51127
Average/set / 5531 / 1.7 / 2318 / 1.9 / 43 / 1.6 / 58574
T. urartu / 3192 / 1.0 / 713 / 0.6 / 27 / 1.0 / 1
Supplementary Table S4 Copy numbers in absolute quantification per haploid genome of Angela, Wilma, and Stasy retrotransposons and Spelt1 tandem repeat in spikes of Ae. speltoidesindividual genotypes
TEs, TR in spikes / Angela / Wilma / Stasy / Spelt1Genotypes / copies/1C / sample/
T.urartu / copies/1C / sample/
T. urartu / copies/1C / sample/
T.urartu / copies/1C
F1_GK/A1-leav* / 5383 / 1.7 / 1524 / 1.2 / 46 / 1.7 / 43749
F1_GK/A1-pst / 8224 / 2.6 / 1038 / 0.9 / 65 / 2.4 / 144002
F1_GK/A1-prm / 8812 / 2.8 / 1313 / 1.1 / 62 / 2.3 / 186266
F1_GK/A1-mei / 7733 / 2.4 / 935 / 0.8 / 58 / 2.2 / 74079
F1_GK/A1-pol / 10466 / 3.3 / 1316 / 1.1 / 66 / 2.4 / 116305
Average/genotype / 8124 / 1225 / 60 / 112880
pol/pst, ratio / 127% / 127% / 101% / 81%
prm/pst, ratio / 107% / 127% / 96% / 129%
F1_K5/A2-leav* / 7843 / 2.5 / 1879 / 1.5 / 56 / 2.1 / 57948
F1_K5/A2-pst / 8523 / 2.7 / 1023 / 0.8 / 61 / 2.3 / 192378
F1_K5/A2-prm / 11255 / 3.5 / 1410 / 1.2 / 63 / 2.3 / 204561
F1_K5/A2-mei / 11800 / 3.7 / 1620 / 1.3 / 64 / 2.4 / 107083
F1_K5/A2-pol / 12180 / 3.8 / 1712 / 1.4 / 62 / 2.3 / 134214
Average/genotype / 10320 / 1529 / 61 / 139237
pol/pst, ratio / 143% / 167% / 101% / 70%
prm/pst, ratio / 132% / 138% / 104% / 106%
F1_C1/K17-leav* / 7817 / 2.4 / 1117 / 0.9 / 53 / 2.0 / 82806
F1_C1/K17-pst / 9485 / 3.0 / 1365 / 1.1 / 64 / 2.4 / 191959
F1_C1/K17-prm / 9688 / 3.0 / 1536 / 1.3 / 58 / 2.2 / 216074
F1_C1/K17-mei / 19189 / 6.0 / 2943 / 2.4 / 90 / 3.3 / 283109
F1_C1/K17-pol / 16317 / 5.1 / 2232 / 1.8 / 82 / 3.0 / 120095
Average/genotype / 12499 / 1839 / 69 / 178808
pol/pst, ratio / 172% / 164% / 127% / 63%
prm/pst, ratio / 102% / 113% / 91% / 113%
F2-3_C1/K17-leav* / 5911 / 1.9 / 1353 / 1.1 / 49 / 1.8 / 49936
F2-3_C1/K17-pst / 6997 / 2.2 / 1392 / 1.1 / 37 / 1.4 / 168279
F2-3_C1/K17-mei / 9546 / 3.0 / 2333 / 1.9 / 55 / 2.0 / 115002
F2-3_C1/K17-pol / 9479 / 3.0 / 2189 / 1.8 / 55 / 2.0 / 169164
Average/genotype / 7983 / 1817 / 49 / 125595
pol/pst, ratio / 135% / 157% / 149% / 101%
prm/leaves, ratio / 118% / 103% / 75% / 337%
C1-leav* / 8122 / 2.5 / 1654 / 1.4 / 60 / 2.2 / 1564
C1-pst / 8633 / 2.7 / 2285 / 1.9 / 63 / 2.3 / 23777
C1-prm / 15942 / 5.0 / 3197 / 2.6 / 113 / 4.2 / 17065
C1-mei / 13229 / 4.1 / 2725 / 2.2 / 84 / 3.1 / 15176
C1-pol / 18279 / 5.7 / 3761 / 3.1 / 117 / 4.3 / 17436
Average/genotype / 12841 / 2725 / 87 / 15004
pol/pst, ratio / 212% / 165% / 186% / 73%
prm/pst, ratio / 185% / 140% / 179% / 72%
K17-leav* / 6825 / 1404 / 47 / 114164
K17-prm / 9696 / 1759 / 56 / 203375
K17-mei/pol / 17092 / 3319 / 70 / 241449
Average/genotype / 11205 / 2160 / 57 / 186329
prm/leaves, ratio / 142% / 125% / 119% / 178%
pol/prm, ratio / 176% / 189% / 125% / 119%
T. urartu / 3192 / 1.0 / 713 / 0.6 / 27 / 1.0 / 1
* - The copy numbers in the leaves are the same as indicated in Supplementary Table S3 and included for convenience to compare with spike tissues. Abbreviations: leav – leaves; pst – pistils; prm – pre-meiotic anthers; mei – anthers at the meiosis I stages; pol – anthers at the meiosis II/young free pollen grain stages.