Theor Appl Genet (1996) 93:717 721
,g Springer-Verlag 1996
M . Baranyi 9 J. Greilhuber 9 W. K. gwi~cicki
Genome size in wild Pisumspecies
Received: 22 March 1996 ' Accepted: 12 April 1996
Abstract Genome size was measured in 75 samples of the wild pea species Pisum abyssinicum, P. elatius, P. fuh'um and P. humile by ethidium-bromide (EB) flow cytometry (internal standard: Triticum monococcum) and Feulgen densitometry (internal standard: Pisum sativum "Kleine Rheinl~inderin'). Total variation of EBDNA between samples covered 97.7% to 114.92/o of the P. satit, um value, and Feulgen DNA values were strongly correlated with EB-DNA values (r=0.9317, P < 0.001). Only P.fidt, um was homogeneous in genome size (108.9% ofP. sativum). Wide variation was observed between samples in P. abyssinicum (100.9-109.7%), P. elatius (97.7 114.9%) and P. humile (98.3 111.1% of P. sativum). In view of the world-wide genome size constancy in P. sativum, the present data are interpreted to show that the pea taxa with variable genome size are genetically inhomogeneous and that the current classification is not sufficient to describe the biological species groups adequately. Key words P i s u m 9 Wild peas 9 Genome-size variation 9 Flow cytometry 9 Feulgen densitometry
size variation (Schweizer and Davies 1972; Baranyi and Greilhuber 1995. 1996) is available. Using flow cytometry Baranyi and Greilhuber (1996) demonstrated genome-size constancy in P. sativum accessions of very different origins, including wild samples, landraces and high-bred cultivars, and confirmed the earlier results of Greilhuber and Ebert (1994), which were obtained with Feulgen densitometry. However, Baranyi and Greilhuber (1995, 1996) found larger genomes in some accessions of P. darius (up to 119.5% of the P. sativum value) and P. humile (108.9%), while other accessions of these taxa were not different from P. sativum. P. abyssinicum and P. ofldvUm had about 107% of P. sativum in these studies. In view of the general usefulness of genome-size data in biosystematic work (Greilhuber and Ehrendorfer 1988), it appeared to us that a more thorough analysis of genome-size variation in wild peas could be a useful contribution to an improved understanding of the biological structure of the genus and the ancestry of cultivated P. sativum. In the present work genome size was analysed in 75 accessions of wild peas by ethidiumbromide (EB) flow cytometry, using Feulgen densitometry as a control.
Introduction Material and methods The wild pea species Pisum abyssinicum, P. elatius, P. fidt'um, and P. humile have received relatively little attention by cytogeneticists in recent decades. For instance, no chromosome banding analyses have been done, and only limited evidence for karyotype variation (Conicella and Errico 1990; Errico et al. 1991) and genome-
Commumcated by F. Mechelke M. Baran,~l 9 J. Greflhuber (~,) Institute of Botany, Unlverslt3 of Vienna, Rennweg 14, A 1030 Vienna, Austria W. K. Sx~lecickl Insntute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyfiska 34, 60-479 Poznafl. Poland
Seventy-five seed samples were obtained fi'om the Plsum Gene Bank. Laboratory of Pea Breeding and Collection, Plant Breeding Station, Wiatrowo, 62 100 Wagrov, lec, Poland (see Table 1) Plants were grown and vouchers were made. Vouchers are also deposited at Wiatrowo. A revision by one of the authors tW.K.S ) modified the taxonomic affihation of two accessions as indicated in Table 1. The taxonomic affiliation of the samples as listed in Table 1 adheres to the designation of the gene bank, but is not alwass consistent with all characters given in the artificial classification of Lehmann (1954) Internal standards used were T t m c u m m o n o c o c c u m with flow cytometry and the culmar. P sarivum "Kleme Rheml~inderin" x~qth Feulgen densitometry. Seeds were germinated on plates. In flow cytometry every nuclear ~solation was jointly' done for one individual of the test material and one of T. m o n o c o c c u m . Preparation of the nuclear suspensmn, staining, and conditions of measurement are described in Baranyi and
718 Table 1 Ethidlum-bromide (EBI flow cytometric and Feulgen densitometric data in P. abyssinicum. P. elatius, P. fulvmn, and P. humile. numbered as given by the seed bank entries. The internal standard was T. monococcum for flow cytometry and P. sutivum "Kleine Rheinliinderin" for Feulgen densitometry. EB and Feulgen data are presented as the ratio (%) of Pisum versus the internal standard. For EB data also the re-calibrated ratio(%) of Ptsum species versus P. satwum "Kleme Rheinliinderin' (see Table2) is given. EB d a t a : F o r each accessmn the average ratio (mean) with the Scheff6 groups ( P > 0.05) indicated, its standard deviation (SD, with reference to N~t, the number of seedling pairs tested (N~) and the number of total runs (N,) WT
Origin
are given. Feulgen data:ten telophase nuclei per primary root-up menstem and shde were measured. Up to seven lines plus the standard were jointly processed and measured. The standard deviation (SD) refers to the number of nuclei (N) of the test sample and represents the relanve weaghted standard devmtion obtained by combining the variation of the test material and the standard (for formula see Greilhuber and Ebert 1994). Origin of accessions:Afg. Afghanistan, Aria. Anatolia, Eth. Ethiopm, Ind. India, Isr Israel, Kat. Katmandu, Pal. Palestine, Rus. Russia, Sud. Sudan. Syr. Syria. Tib. Tibet, Tur. Turkey, ? unknown
EB, Pisum spp.
Feulgen, Pisum spp.
no
% of T. monococcum
% of P. sarivum
N~
Mean s~hCff~'~'~
SD
Rel. EB
P. abyssinicum 1 Eth. 2 Eth. 3 Eth. 4 Eth. 5 Sud. 6 Eth. 7a Tlb. 8 ? 9 Eth. 10 Eth. 11 9 12 Eth. 13 Tur. 15 b Ind. 16 ~ 17 ? 18 ? 19 ? 20 Eth. 21 Eth. 22 ~ 23 ~ 24 Eth. 26 Eth. 27 ?
78.50 r-r 73.55 A-D(-G) 78.09 f L 78.86 ~-r 78.92 I-L 78.90 I-L 73.85 t v~-o) 74.15 B-H 76.37 D-J 78.32 ~-L 73.27 A-c 78.24 t-L 73.72 a-v(-G) 73.44 ~-DI-G~ 78.19 I-L 78.411-L 76.93 (v-)G-K 78.60 I-L 79.64 J-N 79.381 L 78.131-L 78.27 I-L 79.71J-M 78.90 ~ L 78.09 I-L
0.68 0.64 1.22 0.56 0.80 0.44 0.77 0.89 0.56 0 89 0.88 1.35 0.60 0.61 1.43 0.47 1 t0 0.92 1.38 2.14 0.54 0.73 0.66 t.00 0.83
108.05 101.24 107.49 108.55 108.63 108.60 101.65 102.06 105.12 107.80 100.85 t07.69 101.47 101.09 107.63 10793 105.89 108.19 109.62 109.26 107.54 107.74 109.72 108.60 107.49
3 3 4 4 3 3 3 3 3 3 4 4 3 3 3 3 3 3 3 4 4 3 5 4 4
P. elarius 101 102 103 104 105 106 107 108 109 I10 111 112 113 114 115 116 117 118 120 121 122 123 124
Aria. Pal. ? Rus. Rus, Pal. Pal. ~ 9 ? Tur. Eth. ? Sud. Eth. ? 9 ~ ? ? ? Ana. ?
73.36 A-c 73.29 A - ~ 74.16 B-H 73.70 a - v ( - a ) 80.29 K-N 76.29 c-J 76.10 c-~ 72.72 A'B 74.07 B c 80.48 K-N 73.22 A-c 74.08 B-H 73.68 A-EI-~) 73.59 A-D~-G) 72.56 a'B 76.45 (D-)E-J 76.64 (E-~-J 77.63 H-r 70.96 A 73 64 ~ v~ G) 83,38 M-N 73.34 ~ c 83.49 N
0.59 ~80 0.73 1.18 0.67 0.79 0.92 0.53 0.59 0.44 0.30 1.06 0.30 0.71 0.43 0.70 0.45 0.61 0.67 0.52 0.36 0.74 0.69
100.98 100.88 102.08 101.45 110.52 105.01 104.75 i00.10 101.95 110.78 100.78 101.97 101.42 101.29 99.88 105.23 105.49 106.85 97.67 10t.36 114.77 100.95 114.92
P.fulcum 301 302
Pal. ?
78.79 I - r 79 27 J-L
1.21 1.23
108.45 109.I1
N~
% of P. satiwm7
N
Mean
SD
9 9 10 9 9 9 9 8 9 9 9 10 7 8 8 6 9 7 8 9 9 9 9 9 11
107 85 99.90 107.35 108.15 104 39 106.89 98.50 100.66 102.93 [07.01 99.52 105.52 99.56 99.76 106.50 111.25 108.09 110.11 111.31 108.05 108.01 107.13 107.61 106.93 103.58
5.62 5.20 4.55 5.35 5.32 5.02 4.46 3.79 4.48 4.33 4.28 4.39 3.97 5.14 4.02 6.27 3.77 6.05 5.51 4.30 4.28 4.80 4.35 4.27 3.94
150 30 30 30 30 30 60 30 30 30 30 30 30 60 30 30 30 30 30 30 30 30 30 30 30
3 3 3 3 4 3 4 4 4 3 3 3 3 4 3 4 3 3 3 3 3 6 3
9 7 8 7 8 8 10 8 9 7 7 8 9 9 9 11 9 9 9 8 7 9 7
99.78 99.77 97.83 98.60 I06.31 102.48 102.67 98.89 102.85 110.80 101.12 101.76 102.26 101.25 100.81 102.63 103.76 105.08 98.24 101.88 110.32 98.89 114.32
5.06 5.21 6.26 4.88 7.56 6.18 6.45 5.92 5 49 5.72 5.09 4.94 5.34 5.27 5.29 5.22 6.17 5.99 5.35 4.80 5.60 4.95 6.43
60 60 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
5 8
9 14
105.73 107.37
6.25 5.35
30 30
719 Table 1 (Continued) WT
Origin
Feulgen, Plsztm spp.
EB, Pisum spp.
no.
% of P. satit um
% of
T. monococcum Mean s~176
SD
Rel. EB
N~
N~
% of P. satiwlm
N
Mean
SD
303 304 11256
? Isr. Tur.
79.74~-~ 79.04 ~-L 79.02 I-L
0.78 1.11 0.68
109.76 108.80 108 77
4 3 4
6 9 9
107.48 104.43 106.82
5.19 5.09 3.99
30 60 30
P. humile 401 402 403 404 405 407 410 411 412 413 415 416 417 418 419 420 421 422 423 424 425 427
Syr. Isr. Pal. Syr. Syr. Ind. Tur. Afg. Afg. Tur. Kit. RUS. Tur. ? Rus. ? ? Afg. Ind. o Tur. Ind.
73.22
,g Springer-Verlag 1996
M . Baranyi 9 J. Greilhuber 9 W. K. gwi~cicki
Genome size in wild Pisumspecies
Received: 22 March 1996 ' Accepted: 12 April 1996
Abstract Genome size was measured in 75 samples of the wild pea species Pisum abyssinicum, P. elatius, P. fuh'um and P. humile by ethidium-bromide (EB) flow cytometry (internal standard: Triticum monococcum) and Feulgen densitometry (internal standard: Pisum sativum "Kleine Rheinl~inderin'). Total variation of EBDNA between samples covered 97.7% to 114.92/o of the P. satit, um value, and Feulgen DNA values were strongly correlated with EB-DNA values (r=0.9317, P < 0.001). Only P.fidt, um was homogeneous in genome size (108.9% ofP. sativum). Wide variation was observed between samples in P. abyssinicum (100.9-109.7%), P. elatius (97.7 114.9%) and P. humile (98.3 111.1% of P. sativum). In view of the world-wide genome size constancy in P. sativum, the present data are interpreted to show that the pea taxa with variable genome size are genetically inhomogeneous and that the current classification is not sufficient to describe the biological species groups adequately. Key words P i s u m 9 Wild peas 9 Genome-size variation 9 Flow cytometry 9 Feulgen densitometry
size variation (Schweizer and Davies 1972; Baranyi and Greilhuber 1995. 1996) is available. Using flow cytometry Baranyi and Greilhuber (1996) demonstrated genome-size constancy in P. sativum accessions of very different origins, including wild samples, landraces and high-bred cultivars, and confirmed the earlier results of Greilhuber and Ebert (1994), which were obtained with Feulgen densitometry. However, Baranyi and Greilhuber (1995, 1996) found larger genomes in some accessions of P. darius (up to 119.5% of the P. sativum value) and P. humile (108.9%), while other accessions of these taxa were not different from P. sativum. P. abyssinicum and P. ofldvUm had about 107% of P. sativum in these studies. In view of the general usefulness of genome-size data in biosystematic work (Greilhuber and Ehrendorfer 1988), it appeared to us that a more thorough analysis of genome-size variation in wild peas could be a useful contribution to an improved understanding of the biological structure of the genus and the ancestry of cultivated P. sativum. In the present work genome size was analysed in 75 accessions of wild peas by ethidiumbromide (EB) flow cytometry, using Feulgen densitometry as a control.
Introduction Material and methods The wild pea species Pisum abyssinicum, P. elatius, P. fidt'um, and P. humile have received relatively little attention by cytogeneticists in recent decades. For instance, no chromosome banding analyses have been done, and only limited evidence for karyotype variation (Conicella and Errico 1990; Errico et al. 1991) and genome-
Commumcated by F. Mechelke M. Baran,~l 9 J. Greflhuber (~,) Institute of Botany, Unlverslt3 of Vienna, Rennweg 14, A 1030 Vienna, Austria W. K. Sx~lecickl Insntute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyfiska 34, 60-479 Poznafl. Poland
Seventy-five seed samples were obtained fi'om the Plsum Gene Bank. Laboratory of Pea Breeding and Collection, Plant Breeding Station, Wiatrowo, 62 100 Wagrov, lec, Poland (see Table 1) Plants were grown and vouchers were made. Vouchers are also deposited at Wiatrowo. A revision by one of the authors tW.K.S ) modified the taxonomic affihation of two accessions as indicated in Table 1. The taxonomic affiliation of the samples as listed in Table 1 adheres to the designation of the gene bank, but is not alwass consistent with all characters given in the artificial classification of Lehmann (1954) Internal standards used were T t m c u m m o n o c o c c u m with flow cytometry and the culmar. P sarivum "Kleme Rheml~inderin" x~qth Feulgen densitometry. Seeds were germinated on plates. In flow cytometry every nuclear ~solation was jointly' done for one individual of the test material and one of T. m o n o c o c c u m . Preparation of the nuclear suspensmn, staining, and conditions of measurement are described in Baranyi and
718 Table 1 Ethidlum-bromide (EBI flow cytometric and Feulgen densitometric data in P. abyssinicum. P. elatius, P. fulvmn, and P. humile. numbered as given by the seed bank entries. The internal standard was T. monococcum for flow cytometry and P. sutivum "Kleine Rheinliinderin" for Feulgen densitometry. EB and Feulgen data are presented as the ratio (%) of Pisum versus the internal standard. For EB data also the re-calibrated ratio(%) of Ptsum species versus P. satwum "Kleme Rheinliinderin' (see Table2) is given. EB d a t a : F o r each accessmn the average ratio (mean) with the Scheff6 groups ( P > 0.05) indicated, its standard deviation (SD, with reference to N~t, the number of seedling pairs tested (N~) and the number of total runs (N,) WT
Origin
are given. Feulgen data:ten telophase nuclei per primary root-up menstem and shde were measured. Up to seven lines plus the standard were jointly processed and measured. The standard deviation (SD) refers to the number of nuclei (N) of the test sample and represents the relanve weaghted standard devmtion obtained by combining the variation of the test material and the standard (for formula see Greilhuber and Ebert 1994). Origin of accessions:Afg. Afghanistan, Aria. Anatolia, Eth. Ethiopm, Ind. India, Isr Israel, Kat. Katmandu, Pal. Palestine, Rus. Russia, Sud. Sudan. Syr. Syria. Tib. Tibet, Tur. Turkey, ? unknown
EB, Pisum spp.
Feulgen, Pisum spp.
no
% of T. monococcum
% of P. sarivum
N~
Mean s~hCff~'~'~
SD
Rel. EB
P. abyssinicum 1 Eth. 2 Eth. 3 Eth. 4 Eth. 5 Sud. 6 Eth. 7a Tlb. 8 ? 9 Eth. 10 Eth. 11 9 12 Eth. 13 Tur. 15 b Ind. 16 ~ 17 ? 18 ? 19 ? 20 Eth. 21 Eth. 22 ~ 23 ~ 24 Eth. 26 Eth. 27 ?
78.50 r-r 73.55 A-D(-G) 78.09 f L 78.86 ~-r 78.92 I-L 78.90 I-L 73.85 t v~-o) 74.15 B-H 76.37 D-J 78.32 ~-L 73.27 A-c 78.24 t-L 73.72 a-v(-G) 73.44 ~-DI-G~ 78.19 I-L 78.411-L 76.93 (v-)G-K 78.60 I-L 79.64 J-N 79.381 L 78.131-L 78.27 I-L 79.71J-M 78.90 ~ L 78.09 I-L
0.68 0.64 1.22 0.56 0.80 0.44 0.77 0.89 0.56 0 89 0.88 1.35 0.60 0.61 1.43 0.47 1 t0 0.92 1.38 2.14 0.54 0.73 0.66 t.00 0.83
108.05 101.24 107.49 108.55 108.63 108.60 101.65 102.06 105.12 107.80 100.85 t07.69 101.47 101.09 107.63 10793 105.89 108.19 109.62 109.26 107.54 107.74 109.72 108.60 107.49
3 3 4 4 3 3 3 3 3 3 4 4 3 3 3 3 3 3 3 4 4 3 5 4 4
P. elarius 101 102 103 104 105 106 107 108 109 I10 111 112 113 114 115 116 117 118 120 121 122 123 124
Aria. Pal. ? Rus. Rus, Pal. Pal. ~ 9 ? Tur. Eth. ? Sud. Eth. ? 9 ~ ? ? ? Ana. ?
73.36 A-c 73.29 A - ~ 74.16 B-H 73.70 a - v ( - a ) 80.29 K-N 76.29 c-J 76.10 c-~ 72.72 A'B 74.07 B c 80.48 K-N 73.22 A-c 74.08 B-H 73.68 A-EI-~) 73.59 A-D~-G) 72.56 a'B 76.45 (D-)E-J 76.64 (E-~-J 77.63 H-r 70.96 A 73 64 ~ v~ G) 83,38 M-N 73.34 ~ c 83.49 N
0.59 ~80 0.73 1.18 0.67 0.79 0.92 0.53 0.59 0.44 0.30 1.06 0.30 0.71 0.43 0.70 0.45 0.61 0.67 0.52 0.36 0.74 0.69
100.98 100.88 102.08 101.45 110.52 105.01 104.75 i00.10 101.95 110.78 100.78 101.97 101.42 101.29 99.88 105.23 105.49 106.85 97.67 10t.36 114.77 100.95 114.92
P.fulcum 301 302
Pal. ?
78.79 I - r 79 27 J-L
1.21 1.23
108.45 109.I1
N~
% of P. satiwm7
N
Mean
SD
9 9 10 9 9 9 9 8 9 9 9 10 7 8 8 6 9 7 8 9 9 9 9 9 11
107 85 99.90 107.35 108.15 104 39 106.89 98.50 100.66 102.93 [07.01 99.52 105.52 99.56 99.76 106.50 111.25 108.09 110.11 111.31 108.05 108.01 107.13 107.61 106.93 103.58
5.62 5.20 4.55 5.35 5.32 5.02 4.46 3.79 4.48 4.33 4.28 4.39 3.97 5.14 4.02 6.27 3.77 6.05 5.51 4.30 4.28 4.80 4.35 4.27 3.94
150 30 30 30 30 30 60 30 30 30 30 30 30 60 30 30 30 30 30 30 30 30 30 30 30
3 3 3 3 4 3 4 4 4 3 3 3 3 4 3 4 3 3 3 3 3 6 3
9 7 8 7 8 8 10 8 9 7 7 8 9 9 9 11 9 9 9 8 7 9 7
99.78 99.77 97.83 98.60 I06.31 102.48 102.67 98.89 102.85 110.80 101.12 101.76 102.26 101.25 100.81 102.63 103.76 105.08 98.24 101.88 110.32 98.89 114.32
5.06 5.21 6.26 4.88 7.56 6.18 6.45 5.92 5 49 5.72 5.09 4.94 5.34 5.27 5.29 5.22 6.17 5.99 5.35 4.80 5.60 4.95 6.43
60 60 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
5 8
9 14
105.73 107.37
6.25 5.35
30 30
719 Table 1 (Continued) WT
Origin
Feulgen, Plsztm spp.
EB, Pisum spp.
no.
% of P. satit um
% of
T. monococcum Mean s~176
SD
Rel. EB
N~
N~
% of P. satiwlm
N
Mean
SD
303 304 11256
? Isr. Tur.
79.74~-~ 79.04 ~-L 79.02 I-L
0.78 1.11 0.68
109.76 108.80 108 77
4 3 4
6 9 9
107.48 104.43 106.82
5.19 5.09 3.99
30 60 30
P. humile 401 402 403 404 405 407 410 411 412 413 415 416 417 418 419 420 421 422 423 424 425 427
Syr. Isr. Pal. Syr. Syr. Ind. Tur. Afg. Afg. Tur. Kit. RUS. Tur. ? Rus. ? ? Afg. Ind. o Tur. Ind.
73.22
