Supporting information to the paper: Environmentally-related genotypic, phenotypic and metabolic diversity of oat landraces based on 67 Polish accessions.Genetic Resources and Crop Evolution
Maja Boczkowska1, 2, Jacek Zebrowski3, Jarosław Nowosielski2, Izabela Kordulasińska2, Dorota Nowosielska2, Wiesław Podyma 1, 2
1 Polish Academy of Sciences, Botanical Garden - Center for Biological Diversity Conservation in Powsin, Warsaw, Poland
2 Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute, Radzików, Poland Polish
3 Institute of Applied Biotechnology and Basic Plant Sciences, University of Rzeszow, Poland
corresponding author: Maja Boczkowska
e-mail:
Detailed information on methods of genetic analysis.
DNA isolation:
Bulk DNA of 25 plantlets was isolated from young healthy leaf tissue separately for each accession using CTAB procedure (Murray & Thompson 1980) for AFLPs and using the Genomic Mini AX Plant (A & A Biotechnology) for ISSRs. DNA quality was evaluated by electrophoresis on 1.5 % (w/v) agarose gel and its concentration was determined spectrometrically (NanoDrop Spectrophotometer ND-1000).
AFLP
Four the most informative primer pairs EcoRI-ACT/MseI-CAA, EcoRI-AAG/MseI-CAC, EcoRI-AAG/MseI-CAG, EcoRI-AAG/MseI-CAT out of 64 combinations tested were used for selective PCR. All amplification reactions were accomplished using the Biometra T1 Thermocycler at the speed of temperature change 1°C/s. Electrophoresis was conducted on 36 cm long plates in denaturing polyacrylamide gel (4.5%) for 4 h at 2,400 V. The analysis was performed using fluorescent labelling and detecting technology using ABI PRISM 377 XL device (PE Applied Biosystems).
The length of the analyzed fragments, ranging from 35 to 500 bp, was determined using GENESCAN software with accuracy of 1 bp, and the minimum accepted height of the peak was 100 points.
ISSR
PCR amplification was carried out in a 10 μl volume of a mixture containing 25 ng of genomic DNA, 1 μ SuperHotStartTaq polymerase (Bioron), 1x Taq buffer, 1.9 mM MgCl2, 0.2 mM dNTP’s mix and 1.5 μM primer. PCR amplification was performed under the following temperature profile: 5 min at 94 °C followed by 45 cycles, each one included 30 s at 94 °C, 45 s at 58 or 54 °C, 2 min at 72 °C and the final extension for 10 min at 72 °C in a thermocycler Verity 96 Thermal Cycler (Applied Biosystems).
Based on preliminary experiments a set of 8 ISSR di-nucleotide sequence primers anchored at 5′ or 3′ end (University of British Columbia: UBC807, UBC825, UBC834, UBC841, UBC856, UBC857, UBC884, UBC885) were found to be sufficiently polymorphic to be used for the assessment. Each of them was labelled at the 5′ end with one of four fluorochromes: 6-FAM, VIC, NED or PET (Applied Biosystem). The amplified products were separated and visualized using the capillary sequencer ABI 3130xl Genetic Analyzer. The 36 cm capillaries array filled with polymer POP-7 was used. The length of fragments was assessed against the standard size GeneScan1200 LIZ Size Standard (Applied Biosystems).
Table 1 The list of accessions used in the study with eco-geographic description of collection sites
Accession number / Botanical variety / Acquisition year / Collection site descriptionLatitude / Longitude / Region / Altitude [mamsl] / Annual average temperature [oC] / Annual precipitation [mm]
PL50079 / 7, 1 / 1982 / 49 24 N / 20 01 E / S / 718 / 6.7 / 864
PL50338 / 4 / 1976 / 49 31 N / 22 09 E / SE / 386 / 8 / 817
PL50345 / 6, 2 / 1978 / 50 18 N / 21 45 E / SE / 209 / 8.7 / 660
PL50381 / 4 / 1978 / 50 18 N / 21 45 E / SE / 209 / 8.7 / 660
PL50411 / 7, 1 / 1974 / 50 27 N / 23 17 E / SE / 308 / 7.9 / 685
PL50432 / 7, 1 / 1976 / 49 24 N / 20 18 E / S / 578 / 7.4 / 840
PL50438 / 2 / 1974 / 50 40 N / 23 10 E / E / 226 / 8.2 / 627
PL50503 / 2, 6 / 1973 / 50 44 N / 23 15 E / E / 210 / 8.2 / 596
PL50512 / 7 / 1978 / 49 30 N / 20 14 E / S / 783 / 6.3 / 908
PL50520 / 2, 4 / 1973 / 52 16 N / 22 19 E / E / 150 / 8.1 / 555
PL50521 / 2, 4 / 1978 / 49 33 N / 20 59 E / S / 461 / 7.8 / 882
PL50524 / 6, 2 / 1978 / 49 32 N / 22 06 E / SE / 342 / 8.2 / 802
PL50528 / 1, 7 / 1976 / 49 29 N / 20 04 E / SE / 440 / 7.8 / 624
PL50529 / 4 / 1979 / 53 38 N / 23 09 E / NE / 145 / 7.5 / 578
PL50530 / 7, 1 / 1973 / 50 44 N / 23 15 E / E / 210 / 8.2 / 596
PL50531 / 2, 6, 4 / 1979 / 53 01 N / 23 21 E / C / 155 / 7.6 / 575
PL50554 / 2, 6 / 1973 / 49 32 N / 21 02 E / S / 473 / 7.7 / 872
PL50556 / 7 / 1978 / 49 30 N / 20 14 E / S / 783 / 6.3 / 908
PL50561 / 2 / 1973 / 52 16 N / 22 19 E / E / 150 / 8.1 / 555
PL50587 / 2, 6 / 1973 / 51 15 N / 22 34 E / E / 185 / 8.3 / 581
PL50593 / 7, 1 / 1978 / 49 30 N / 20 14 E / S / 783 / 6.3 / 908
PL50613 / 5, 3 / 1977 / 50 39 N / 21 54 E / E / 192 / 8.8 / 580
PL50616 / 2 / 1976 / 49 35 N / 20 28 E / S / 516 / 7.6 / 819
PL50622 / 2 / 1978 / 49 36 N / 21 03 E / S / 409 / 8 / 841
PL50627 / 7, 5 / 1977 / 52 15 N / 21 53 E / E / 162 / 8.1 / 577
PL50673 / 2, 6 / 1974 / 50 33 N / 23 36 E / E / 222 / 8.2 / 609
PL50694 / 2, 4 / 1976 / 49 27 N / 22 20 E / SE / 394 / 8 / 818
PL50698 / 2, 6 / 1977 / 50 49 N / 22 19 E / SE / 269 / 8.1 / 662
PL50705 / 2, 6 / 1976 / 49 24 N / 20 18 E / S / 578 / 7.4 / 840
PL50706 / 2, 6 / 1976 / 49 24 N / 20 18 E / S / 578 / 7.4 / 840
PL50709 / 2 / 1978 / 49 48 N / 21 54 E / SE / 309 / 8.3 / 738
PL50712 / 2 / 1977 / 50 22 N / 21 50 E / SE / 192 / 8.7 / 642
PL50718 / 2, 6 / 1974 / 51 02 N / 23 00 E / E / 212 / 8.2 / 592
PL50725 / 2 / 1976 / 49 23 N / 20 08 E / S / 785 / 6.4 / 889
PL50754 / 2 / 1976 / 49 30 N / 20 30 E / S / 583 / 7.3 / 865
PL50758 / 4 / 1979 / 54 20 N / 22 56 E / NE / 183 / 7 / 638
PL50760 / 2 / 1977 / 50 39 N / 22 18 E / E / 183 / 8.6 / 658
PL50902 / 2, 6 / 1978 / 49 22 N / 20 11 E / S / 880 / 5.9 / 938
PL50904 / 2 / 1978 / 49 22 N / 20 11 E / S / 880 / 5.9 / 938
PL50925 / 4 / 1978 / 49 30 N / 20 24 E / S / 535 / 7.5 / 801
PL50945 / 2 / 1978 / 49 22 N / 20 11 E / S / 880 / 5.9 / 938
PL51519 / 2 / 1984 / 52 03 N / 22 18 E / E / 159 / 8.1 / 549
PL51521 / 4 / 1984 / 51 50 N / 22 29 E / E / 155 / 8.2 / 553
PL51522 / 4 / 1984 / 51 07 N / 23 20 E / E / 233 / 8 / 569
PL51599 / 2 / 1985 / 50 52 N / 23 04 E / E / 217 / 8.2 / 604
PL51600 / 1, 7 / 1986 / 50 10 N / 22 36 E / SE / 175 / 8.7 / 638
PL51603 / 7, 1, 2 / 1986 / 50 00 N / 22 40 E / SE / 224 / 8.5 / 670
PL51604 / 7, 1 / 1986 / 49 41 N / 22 29 E / SE / 335 / 8.1 / 795
PL51605 / 1, 7 / 1986 / 49 41 N / 22 31 E / SE / 393 / 7.8 / 816
PL51606 / 2, 6 / 1986 / 52 24 N / 19 47 E / C / 100 / 8.7 / 535
PL51607 / 7, 1 / 1986 / 50 00 N / 22 35 E / SE / 237 / 8.5 / 677
PL51610 / 2, 6 / 1986 / 50 03 N / 22 34 E / SE / 212 / 8.6 / 666
PL51611 / 2 / 1986 / 50 01 N / 22 38 E / SE / 217 / 8.6 / 666
PL51612 / 4, 2 / 1987 / 50 32 N / 23 36 E / E / 228 / 8.2 / 612
PL51614 / 2, 6 / 1985 / 50 37 N / 22 59 E / E / 245 / 8.2 / 708
PL51615 / 6, 2 / 1985 / 50 51 N / 23 26 E / E / 245 / 8 / 594
PL51616 / 7, 1, 2, 5 / 1987 / 49 21 N / 20 12 E / S / 913 / 5.7 / 1012
PL51617 / 2, 7 / 1987 / 49 22 N / 20 11 E / S / 880 / 5.9 / 938
PL51618 / 2, 7, 6 / 1987 / 49 22 N / 20 11 E / S / 880 / 5.9 / 938
PL51619 / 7, 2 / 1987 / 49 22 N / 20 11 E / S / 880 / 5.9 / 938
PL52191 / 7, 2 / 1997 / 49 21 N / 20 12 E / S / 913 / 5.7 / 1012
PL52265 / 2, 6 / 1997 / 49 45 N / 19 16 E / S / 576 / 7.4 / 1066
PL52306 / 2, 6 / 1998 / 50 48 N / 23 14 E / E / 231 / 8.1 / 600
PL52338 / 2, 6 / 1999 / 50 26 N / 20 26 E / S / 285 / 8.4 / 599
PL52343 / 7, 2 / 1999 / 52 54 N / 22 52 E / E / 134 / 7.9 / 568
PL52344 / 7 / 1999 / 49 30 N / 19 45 E / S / 729 / 6.6 / 855
PL52351 / 7, 1, 5, 3 / 1999 / 53 01 N / 22 50 E / NE / 129 / 7.8 / 563
[Botanical variety: 1, aristata; 2, aurea; 3, cinerea; 4, flava; 5, grisea; 6, krausei; 7, mutica; Region: C, Central; E, East; NE, North –East; S, South; SE, South-East]
Table 2 List of 25 morphological traits. The analysis was carried out on 20 randomly chosen plants for each accession. The morphological traits were set on the base of descriptors described by Slabonski (1949), IBPGR (1985) and UPOV (1994).
Morphological trait / Stages of traitsAngle of flag leaf to culm / All plants with acute leaves; 25% with obtuse leaves; 50%.....; 75%.....; all plants with obtuse leaves
Angle to culm of leaves / Acute; intermediate; obtuse
Awn type / Absent; weak; medium; strong;
Awnedness / 0-6% no awns; 7-15% weak awns; 16-100% strong awns
Axis nodes in panicle / Number
Color of leaves / Yellow-green; light green; green; dark green; other
Color of panicle / Yellow-green; light green; green; dark green; other
Erectness of spikelet / Erect; drooping erect
Growth habit / erect; semierect; intermediate; semiprostrate; prostrate
Hairiness of basal part of the primary grain / Absent; weak; medium; strong; very strong
Hairiness of leaf margin / Absent; weak; medium; strong; very strong
Hairiness of leaf sheath / Absent; weak; medium; strong; very strong
Hairiness of lemma / Absent; weak; medium; strong; very strong
Hairiness of rachilla / Absent; weak; medium; strong; very strong
Intensity of leaves spirality / Lack of rotation; weak rotation (¼ torsion); medium rotation (1/2 torsion); strong rotation (3/4 torsion); very strong rotation (1 torsion)
Lemma color / White; yellow; brown; grey; black
Length of rachilla / Very short; short; medium; long; very long
Nodes hairiness / Absent; weak; medium; strong; very strong
Number of grains in spikelet / [number]
Rigidity of leaves / Erect; bent; weak dropping, dropping; strongly dropping
Shape of basal part of the primary grain / Convex; intermediate; flax
Shape of panicle / Erect; semi-erect; horizontal, dropping; strongly dropping
Shape of primary grain / Convex; intermediate; slender; peaked
Spirality of leaves / Left; right
Type of panicle / Unilateral; sub-unilateral; equilateral
Table 3 Range and mean of twelve evaluation traits assessed in field experiments. The accession number is in parentheses.
minimum / mean / maximumdays to heading / days / 70 (PL51618) / 76 / 82 (PL51612)
lodging at mature stage / scale 1-9 / 0.5 (PL50530) / 4.7 / 6.7 (PL51618)
crop height / cm / 110 (PL50530) / 128 / 147 (PL51520)
panicle length / cm / 22 (PL51610) / 26 / 29 (PL51616)
powdery mildew / % / 0 (PL51604) / 1 / 6 (PL51610)
crown rust / % / 0.3 (PL50698) / 3.5 / 16.5 (PL50345)
stem rust / % / 0 (PL52306) / 0.5 / 2.9 (PL50712)
number of panicles / no / 21 (PL50758) / 34 / 53 (PL50945)
days to maturity / days / 114 (PL51618) / 116 / 118 (PL51604)
Yield / g / 832 (PL52351) / 1456 / 2058 (PL52344)
thousand seed weight / g / 23 (PL50381) / 28 / 35 (PL51615)
test weight / kg/hl / 29 (PL50524) / 32 / 35 (PL50554)
Table 4Pairwise Mantel test results for all methods and eco-geographic data
Genotype / ISSR / AFLP / Phenotype / Morphology / Evaluation / Metabolome / Proteins / Lipids / Geographic distance / Altitude / Annual precipitation / Annual average temperatureGenotype / 0.272 / 0.311 / ns / ns / 0.129 / ns / ns / 0.211 / 0.131 / 0.208
ISSR / ns / 0.261 / 0.307 / ns / ns / 0.153 / ns / ns / 0.240 / 0.152 / 0.258
AFLP / ns / ns / ns / ns / ns / ns / ns / ns / ns / ns
Phenotype / ns / 0.220 / ns / 0.122 / 0.163 / 0.113 / 0.180
Morphology / 0.169 / ns / 0.240 / ns / ns / 0.180 / 0.130 / 0.188
Evaluation / ns / ns / ns / ns / ns / ns / ns
Metabolome / ns / ns / ns / ns
Proteins / ns / ns / 0.188 / 0.109 / 0.200
Lipids / 0.145 / ns / ns / ns
[-, not calculated; ns, not significant p=0.05]
Table 5The Rc valuefor pairwise GPA of all methods
ISSR / AFLP / Morphology / Evaluation / Proteins / LipidsISSR / - / 0.593 / 0.659 / 0.598 / 0.626 / 0.609
AFLP / 0.593 / - / ns / ns / ns / ns
Morphology / 0.659 / ns / - / 0.668 / 0.639 / 0.608
Evaluation / 0.598 / ns / 0.668 / - / ns / ns
Proteins / 0.626 / ns / 0.639 / ns / - / 0.684
Lipids / 0.609 / ns / 0.608 / ns / 0.684 / -
[-, not calculated; ns, the Rc value of the original data was > 95% of Rc values from 10000 permutation.]
Figure 1The value of Nei’s coefficient (Lynch and Milligan 1994) of genetic diversity within oat landrace collection for all described morphological traits (standard error indicated).
Figure 2 (a) Typical ATR-FTIR spectrum of dehusked oat grains with denoted main bands within regions characteristic for lipids (3000-2800 cm-1), proteins (amide I (1652 cm-1) +amide II (1543 cm-1)) and carbohydrates (1200-900 cm-1). (b) Expanded spectral region of prominent absorbance assigned to the C-H asymmetric (2927 cm-1) and symmetric (2854 cm-1) stretching vibrations of methylene –CH2-group. Locations of very weak shoulders due to the C-H stretching vibrations of methyl (–CH3) group at 2959 cm-1 (asymmetric) and at 2873 cm-1 (symmetric) are also marked. (c) Carbohydrate IR spectral region of the oat grain (upper) and starch (lower). The arrows point out contribution of the starch bands to the spectral profile of the oat grain.
Figure 3 The value of Nei’s coefficient (Lynch and Milligan 1994) of genetic diversity within oat landrace collection for each of the methods used in the study (standard error indicated)
Figure 4 The value of genetic diversity within groups created based on passport and eco-geographic data (region of origin, altitude of origin place and annual average precipitation and temperature in collection sites.The Fisher's least significant differences (LSD)ploted as errors bars.