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Genetic characterisation of centuries-old oak and linden trees using SSR markers

Svitlana Bilous
Svitlana Bilous
, 
Larysa Prysiazhniuk
Larysa Prysiazhniuk
, 
Snizhana Chernii
Snizhana Chernii
, 
Serghii Melnyk
Serghii Melnyk
, 
Yurii Marchuk
Yurii Marchuk
 oraz 
Artur Likhanov
Artur Likhanov
   | 11 mar 2022
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Article Category: Original Article
Data publikacji: 11 mar 2022
Zakres stron: 58 - 68
Otrzymano: 05 lip 2021
Przyjęty: 08 lut 2022
DOI: https://doi.org/10.2478/ffp-2022-0006
Słowa kluczowe
© 2022 Svitlana Bilous et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Results of PCR testing of samples of common oak with marker: A – ssrQrZAG 11: 1 – Yuzefin oak; 2 – Shevchenko oak; 3 – Rylskyi oak; 4 – centuries-old oak in the botanical garden of the NULES; 5 – Vytautas oak; 6 – centuries-old oak on the territory of NULES Ukraine; B – ssrQrZAG 65: 1 – Rylskyi oak; 2 – centuries-old oak in the NULES botanical garden; 3 – Vytautas oakNote: M – molecular weight marker 100-bp DNA ladder O’GeneRuler (Thermo Scientific), NULES – National University of Life and Environmental Sciences, PCR – polymerase chain reaction.
Results of PCR testing of samples of common oak with marker: A – ssrQrZAG 11: 1 – Yuzefin oak; 2 – Shevchenko oak; 3 – Rylskyi oak; 4 – centuries-old oak in the botanical garden of the NULES; 5 – Vytautas oak; 6 – centuries-old oak on the territory of NULES Ukraine; B – ssrQrZAG 65: 1 – Rylskyi oak; 2 – centuries-old oak in the NULES botanical garden; 3 – Vytautas oakNote: M – molecular weight marker 100-bp DNA ladder O’GeneRuler (Thermo Scientific), NULES – National University of Life and Environmental Sciences, PCR – polymerase chain reaction.

Figure 2

Cluster distribution of Quercus robur samples by SSR markersNote: Q1 – Yuzefin oak; Q2 – Shevchenko oak; Q3 – Rylskyi oak; Q4 – centuries-old oak in the NULES botanical garden; Q5 – Vytautas oak; Q 6 and Q7 - centuries-old oak near the territory of NULES Ukraine; NULES – National University of Life and Environmental Sciences, SSR – simple sequence repeat.
Cluster distribution of Quercus robur samples by SSR markersNote: Q1 – Yuzefin oak; Q2 – Shevchenko oak; Q3 – Rylskyi oak; Q4 – centuries-old oak in the NULES botanical garden; Q5 – Vytautas oak; Q 6 and Q7 - centuries-old oak near the territory of NULES Ukraine; NULES – National University of Life and Environmental Sciences, SSR – simple sequence repeat.

Figure 3

Cluster distribution of Tilia cordata samples by SSR markersNote: L.1 T.H. Shevchenko Tilia cordata, L.2 – P. Mohyla Tilia cordata, L.3 Tilia cordata of St. Theodosius of Pechersk, L.4 a centuries-old Tilia cordata of the Feofaniia Monument Park, L.5 a centuries-old Tilia cordata (the village of Irsha), L.6 a centuries-old Tilia cordata in the Holosiivskyi forest.
Cluster distribution of Tilia cordata samples by SSR markersNote: L.1 T.H. Shevchenko Tilia cordata, L.2 – P. Mohyla Tilia cordata, L.3 Tilia cordata of St. Theodosius of Pechersk, L.4 a centuries-old Tilia cordata of the Feofaniia Monument Park, L.5 a centuries-old Tilia cordata (the village of Irsha), L.6 a centuries-old Tilia cordata in the Holosiivskyi forest.

Figure 4

Relationship between genetic distances of Quercus robur samples according to geographical coordinates and SSR markers (SSR – simple sequence repeat)
Relationship between genetic distances of Quercus robur samples according to geographical coordinates and SSR markers (SSR – simple sequence repeat)

Figure 5

Relationship between genetic distances of Tilia cordata samples according to geographical coordinates and SSR markers (SSR – simple sequence repeat)
Relationship between genetic distances of Tilia cordata samples according to geographical coordinates and SSR markers (SSR – simple sequence repeat)

Material characteristics

Centuries-old trees nomenclature Location Age [years] Geographical coordinates
Quercus robur
Q1 Yuzefinskyy oak Hlynne, Rivne region nearly 1000 51°55′33″N 27°37′86″E
Q2 Oak T. Shevchenko Kyiv more than 600 50°49′59″N 30°45′16″E
Q3 M. Rylskyi oak Kyiv nearly 600 50°38′68″N 30°51′01″E
Q4 Centuries-old oak in the NULES botanical garden Kyiv more than 200 50°38′16″N 30°50′28″E
Q5 Oak Vitovta Kyiv more than 400 50°38′42″N 30°49′73″E
Q6 Centuries-old oak 1 on the territory of NULES Ukraine Kyiv more than 400 50°38′38″N 30°50′53″E
Q7 Centuries-old oak 2 on the territory of NULES Ukraine Kyiv more than 400 50°38′37″N 30°50′61″E
Tilia cordata
L1 Linden T. Shevchenko Sedniv, Chernihiv region more than 600 51°63′76″N 31°56′95″E
L2 Linden P. Mohyla Kyiv more than 600 50°45′75″N 30°51′71″E
L3 Centuries-old linden tree of the Feofaniia Monument Park Kyiv more than 400 50°43′25″N 50°43′25″E
L4 Linden tree of St. Feodosiya Pechers′koho Kyiv more than 700 50°34′00″N 30°48′’68″E
L5 Centuries-old linden tree in Holosiivskyi forest Kyiv more than 200 50°72′18″ N 29°44′92″E
L6 Centuries-old linden tree v. Irsha, Radomyshl district, Zhytomyr region more than 200 50°38′29″ N 30°50′51″E

Sizes of obtained alleles for Quercus robur with SSR markers

SSR Allele size, bp
Q1* Q2 Q3 Q4 Q5 Q6 Q7
ssrQrZAG 7 132 120, 140 132 152 152 140 146
ssrQrZAG 11 300 332 268, 300 290 290 268, 300 290
ssrQrZAG 25 116 206 206 206 194 158, 206 112, 158
ssrQrZAG 30 204, 226 212, 230 182 192 212 192, 212 204
ssrQrZAG 31 146, 182 146, 182 140, 158 174 182 158 146, 182
ssrQrZAG 44 116, 156 124, 180 144 144, 180 144 156, 216 156
ssrQrZAG 65 272, 320 264, 312 312 312, 340 296, 320 396 388

Characteristics of SSR markers for assessment of Quercus robur polymorphism

SSR Nucleotide sequences of primers, 5′→3′ Motive The expected size of the amplicons, bp
ssrQrZAG 7 F: CAACTTGGTGTTCGGATCAA (TC)17 150
R: GTGCATTTCTTTTATAGCATTCAC
ssrQrZAG 11 F: CCTTGAACTCGAAGGTGTCCTT (TC)22 273
R: GTAGGTCAAAACCATTGGTTGACT
ssrQrZAG 25 F: GATATGAAAGATTCTTATTCCATCC (GA)32 135
R: GTTAGAACCAATGTACCAAAGTCC
ssrQrZAG 30 F: TGCTCCGTCATAATCTTGCTCTGA (GA)26 211
R: GCAATCCTATCATGCACATGCACAT
ssrQrZAG 31 F: CTTAGTTTGGTTGGGAAGAT (GA)31 190
R: GCAACCAAACAAATGAAAT
ssrQrZAG 44 F: ACCCTTGTAGTCATGTTCGTTG (GA)29 (TG)31 145
R: GAAATCTCACCTGCTCCCTATC
ssrQrZAG 65 F: CAGTGGTGTCAACTCCTCCCAG (TC)21(TA)10 270
R: GTCAGGTGACCATTCAAACCTAGAA

Sizes of obtained alleles for Tilia cordata with SSR markers

SSR Allele size, bp
L1* L2 L3 L4 L5 L6
Tc927 152, 168 152 152 152, 168 152 152
Tc5 154 154, 174, 180 154 158 154 154, 180
Tc915 154 154, 168 154 154, 174 182 168
Tc920 218, 232 224, 240 218, 232 232 232, 252 252
Tc937 152 152 152 162 162 162
Tc963 246 246 238 238 246 246

PCR protocol for Quercus robur and Tilia cordata

Step name Q. robur T. cordata
parameters [°C/time] cycle number parameters cycle number
Initial denaturation 95/3 min 1 95/3 min 1
Denaturation 94 (89)*/30 s 33* 94/30 s 35
Annealing 50/30s 54**/1 min
Extension 72/30 s 72/30 s
Final extension 72/3 min 1 72/3 min 1

Characteristics of SSR markers for assessment of Tilia cordata polymorphism

SSR Nucleotide sequences of primers, 5′→3′ Motive The expected size of the amplicons, bp
Tc5 F: TTTTCATACATTTAGAGACTTTTAGCA (AG)12 150
R: TGCATGATTTGTATGTTTAGGG
Tc915 F: ACATCGATTGTATTTCCCTTTAAC (CT)16 165
R: GTTGTATTTTGCCCTTAACATTG
Tc920 F: AAATGTCTTCAGAGTGACTAGATGG (GA)2(GT)15 (AG)4 232
R: TGCCTCATTATTCTCCTAATTCTC
Tc927 F: AGTCCTCCTGTCAAATGCTG (AG)10 157
R: ATCACACTCGTTTATGACATCTTG
Tc937 F: AGCCAACCAACTTTTACAATACAG (AG)13 162
R: AGATAAAAGCACATAAATCGATGG
Tc963 F: CTAACCCCATTCTCTTTAATTCTG (CT)11 238
R: GCTTTCATTTCAGTTTTCCTCTAC

Composition of reaction mixture for PCR of Quercus robur and Tilia cordata

Components Final concentration
Q. robur T. cordata
PCR buffer* 1× 1×
MgCl2 1.5 μM 2 μM
dNTP (dATP, dTTP, dGTP, dCTP) 100 μM 200 μM
Polymerase Taq 1 U 0.5 U
Primer (F) 1 μM 0.2 μM
Primer (R) 1 μM 0.2 μM
DNA 50 ng 50 ng

Characteristics of the obtained alleles by SSR markers for the samples of Quercus robur and Tilia cordata

SSR Number of alleles Allele size range Alleles’ frequency PIC
Q. robur
ssrQrZAG 7 5 120–152 0.07–0.29 0.77
ssrQrZAG 11 4 268–332 0.14–0.43 0.69
ssrQrZAG 25 5 112–206 0.07–0.50 0.68
ssrQrZAG 30 6 182–230 0.07–0.29 0.80
ssrQrZAG 31 5 140–182 0.07–0.36 0.76
ssrQrZAG 44 6 116–216 0.07–0.36 0.76
ssrQrZAG 65 8 264–396 0.07–0.29 0.84
T. cordata
Tc5 4 154–180 0.08–0.58 0.60
Tc915 4 154–182 0.08–0.50 0.65
Tc920 5 218–252 0.08–0.25 0.72
Tc927 2 152–168 0.17–0.83 0.28
Tc937 2 152–162 0.5 0.50
Tc963 2 238–246 0.33–067 0.44
eISSN:
2199-5907
ISSN:
0071-6677
Język:
Angielski
Częstotliwość wydawania:
4 razy w roku
Dziedziny czasopisma:
Life Sciences, Plant Science, Medicine, Veterinary Medicine
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