nellar DNA diversity in this important group of the genus is still missing; .... enzymes was carried out according to the directions given by the supplier, Takara.
Copyright 0 1984 by the Genetics Society of America
T H E MOLECULAR BASIS O F GENETIC DIVERSITY AMONG CYTOPLASMS OF TRITICUM AND AEGILOPS. 111. CHLOROPLAST GENOMES O F T H E M AND MODIFIED M GENOME-CARRYING SPECIES T. TERACHI, Y. OGIHARA
AND
K. TSUNEWAKI
Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Manuscript received October 6, 1983 Revised copy accepted June 8, 1984 ABSTRACT
The restriction fragment patterns of chloroplast DNAs of all M or modified M genome-carrying Aegilops species, and those of common wheat (Triticum aestivum), Ae. umbellulata and Ae. squarrosa as referants, have been analyzed using eight restriction endonucleases, BamHI, EcoRI, HindIII, KpnI, PstI, SalI, Smal and XhoI. Nine distinctly different chloroplast genomes are evident, and the mutual relatedness among them is estimated based on the number of different restriction fragments. The results lead to the following conclusions. (1) Chloroplast genomes of three Comopyrum species, Ae. comosa, Ae. heldreichii and Ae. uniaristata, are more closely related with each other and are greatly different from those of the Amblyopyrum species, Ae. mutica, and of Ae. umbellulata and Ae. squarrosa. (2) Ae. crassa’s chloroplast genome lies at the center of chloroplast genome diversification, whereas those of common wheat, Ae. squarrosa and Ae. uniaristata are three extreme forms lying far from the center. (3) Chloroplast genomes of three 4x species, Ae. biuncialis, Ae. columnaris and Ae. triaristata, arose from Ae. umbellulata, and that of a fourth 4x species, Ae. ventricosa, arose from Ae. squarrosa. The chloroplast origins of two other 4x species, Ae. ovata and Ae. crassa, remain unsolved. (4) The chloroplast genomes of two Ae. mutica strains are identical, even though their cytoplasms exert quite different effects on male fertility, heading date and growth vigor of common wheat.
S in other plant taxa, Euoenothera (GORDEN al. 1982), Nicotiana A (KUNG,ZHU and SHEN 1982), Lycopersicon (PALMERand ZAMIR 1982) and Brassica (PALMER al. 1983), restriction fragment pattern analyses of e.g.,
et
et
organellar DNAs have been useful as a diagnostic criterion of phylogenetic relationships among species of two related genera, Triticum (wheat) and Aeand TSUNEWAKI 1982; TSUNEWAKI gilops (VEDELet al. 1978, 1981; OGIHARA and OGIHARA1983; BOWMAN,BONNARD and DYER 1983). Here, the term “restriction fragment pattern” means the electrophoretic pattern of restriction endonuclease digests of organellar DNA. According to ZHUKOVSKY(1928), the genus Aegilops in which di-, tetra- and hexaploid species are included consists of six sections, Amblyopyrum, Comopyrum, Cylindropyrum, Polyeides, Sitopsis and Vertebrata. All diploid species of this genus are classified into three groups, C, M and S, based on the results Genetics 108: 681-695 November, 1984.
682
T. TERACHI. Y. OGIHARA AND K. TSUNEWAKI
of genome analyses (LILIENFELD1951). The two sections, Amblyopyrum and Comopyrum, contain four diploid species that are in the M group; they are Ae. mutica (nuclear genome Mt) of Amblyopyrum, and Ae. comosa (M), Ae. heldreichii (M) and Ae. uniaristata (M") of Comopyrum section (KIHARA 1949). Several modified M genomes exist in polyploid species of two other sections; these are Mb, M', MO, M', M" and M' of Ae. biuncialis, Ae. columnaris, Ae. ovata and Ae. triaristata of Polyeides section and Ae. crassa and Ae. ventricosu of Vertebrata section, respectively. The organellar DNAs of the M group species have not been studied, except for the chloroplast DNA of Ae. uniaristatu and Ae. mutica (OGIHARA and TSUNEWAKI 1982). Thus, information about organellar DNA diversity in this important group of the genus is still missing; without this information it is impossible to clarify the maternal lineage of modified M genome-carrying polyploid species. On a wider scale this information is necessary to complete the picture of organellar DNA variation within the Triticum-Aegilops complex. In the present investigation we compared restriction fragment patterns of chloroplast DNA (hereafter ctDNA) isolated from the alloplasmic lines of common wheat which had the cytoplasms of all the aforementioned species and referant species, Ae. umbellulata and Ae. squarrosa, C" and D genome donors to the tetraploid species. MATERIALS AND METHODS Plant materials: Alloplasmic lines of common wheat, not the Aegilops species, were sources of ctDNA. Cytoplasm and nucleus constitutions of 13 alloplasmic and a single euplasmic line of common wheat used are shown in Table 1. All of the alloplasmic lines are self-fertile, except (comosa)-Slm, (hcldreichii)-CS and (mutica>CS which are completely male sterile. Seeds from these lines were propagated by natural out-crossing from a neighbor pollinator. The euplasmic line, (aestivum)-Srg, was used as a control. Chloroplast isolation and ctDNA extraction: Seeds presoaked for 24 hr were sown in 30 X 50 cm wooden flats filled with sterilized soil, the seeding rate being about 1000 seeds/flat. The flats were kept at about 20" during a 17-hr light period and at 15" during the remaining dark period. Seedling leaves were collected at the three-leaf stage, from which chloroplasts were isolated. Leaves regenerated later from the same seedlings were harvested once again and used for the same purpose. The total ctDNA yield per flat was ca. 70 fig, an amount sufficient for about 70 electrophoretic runs, but the yield varied somewhat depending upon the lines used. The seedling leaves harvested were cut to about 3 cm long and homogenized using a Waring blender with a buffer containing 0.44 M mannitol, 50 mM Tris (pH 8.0), 3 mM EDTA, 1 mM 2mercaptoethanol and 0.1% bovine serum albumin. The weight ratio of fresh leaves and buffer was about 1:4. The homogenate was filtrated through four layers of cheesecloth and two layers of Miracloth. The filtrate was centrifuged at 1000 rpm for 5 min. The supernatant was again centrifuged at 3500 rpm for 10 min. The pellet (crude chloroplasts) was dissolved in the aforementioned buffer (about 10 ml of buffer is added to the pellet from 100 gm of fresh leaves) and centrifuged in a Percoll discontinuous gradient (10, 40 and 75%) made with the same buffer. lntact chloroplasts were collected from the 40-75% Percoll interface of the gradient (OGIHARA and TSUNEWAKI 1982). From this chloroplast preparation ctDNA was extracted after the method (1975). When these procedures are used, no DNase treatment of of KOLODNERand TEWARI chloroplasts is required prior to ctDNA extraction. Restriction fragment pattern analysis: Eight restriction endonucleases, all of which restrict at a specific six-base pair sequence, were used. Their names and restriction sites (in parentheses) are as follows; BamHl (GJGATCC), EcoRI (GJAATTC), Hind111 (AJAGCTT), Kpnl (CGTACJC),
CHLOROPLAST DNA OF M-GROUP AEGILOPS TABLE 1 Euplasmic and alloplasmic lines of common wheat employed Cytoplasm donor Code no.
Line
03 04 05 06 07 13 14 29 30 31 32 35 36 52
(umbellu1ata)-CS (squarrosa)-CS (comosa>Slm (he1dreichii)-CS (uniaristata)-CS (mutica-M)-CS (mutica-P>CS (biuncialis)-CS (co1umnaris)-CS (ovata)-CS (triaristata)-CS (crassa)-CS (ventricosa)-CS (aestivum)-Srg
Species
Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae.
umbellulata squarrosa comosa heldreichii uniaristata mutica mutica biuncialis columnaris ovata triaristata 4x crassa 4x ventricosa T. aestivum
Nuclear genome (haploid)
c" D M M MU Mt Mt CUMb CUM' CUM" CUM' DM" DM' ABD
Plasma type"
C" D M M MU Mt Mt' C" C" MO C" D2 D B
Source*
K K K P
M M P
K K K
K M K K
Nucleus donor'
cs cs Slm
cs cs cs cs cs cs cs cs cs cs
Srg
* After TSUNEWAKI (1980) and TSUNEWAKI and TSUJIMOTO (1984).
K: Laboratory of Genetics, Kyoto University; M: S. S. MAAN,North Dakota State University; P: I. PANAYOTOV, Wheat and Sunflower Institute, Bulgaria. CS: Chinese Spring, Slm: Salmon, Srg: Shirogane-komugi (all are common wheat cultivar or strain, belonging to T. aestivum).
PstI (CTGCAJG), Sal1 (GJTCGAC), SmaI (CCCJGGG) and XhoI (CJTCGAG). Digestion of ctDNA with these enzymes was carried out according to the directions given by the supplier, Takara Shuzo Company, Ltd., Kyoto, Japan. The DNA fragments digested by all enzymes, except by EcoRI, were separated by electrophoresis at 1 V/cm for 40 hr, using 0.85% agarose slab gels containing 40 mM Tris, 20 mM sodium acetate and 2 mM EDTA. For EcoRI digests, I%, instead of 0.85%, agarose gels were used. The DNA fragments were made visible by ethidium bromide (0.5 pg/ml) staining and were then photographed under long wave UV light. Differences in the restriction fragment patterns between ctDNAs were determined from the photographs. The molecular weight of each fragment was estimated from its mobility as compared with that of the HindIII-digested XDNA fragments. According to ENGELS(1981), the genetic distance (p) between each pair of ctDNA molecules can be estimated from the equation, # = [c - 2(m - k)]/jc, where c is the total number of cuts (= total number of fragments in case of a circular molecule) in two DNA molecules, m is the total number of cleavage sites, k is the number of differentiated cleavage sites between them and j is the length of a recognition sequence (6 for all eight endonucleases in the present case). Using Table 2 of ENCELS(1981), we estimated k from the total number of fragments produced in both ctDNA molecules (F; being equal to c in the present case) and the number of pairs of fragments identical in length (C). However, extension of his table was necessary to its application in the present data; this was done by T. MARUYAMA, National Institute of Genetics, Misima, Japan. 2)/2. The k's and m's were summed for Similarly, m was obtained from a formula, m = (F + k iall eight endonucleases, on the basis of which the # value for each pair of ctDNA molecules was calculated. Clustering of nine ctDNA molecules was worked out by the UPG method of SNEATH and SOKAL(1973) using this # value.
684
T. TERACHI, Y. OGIHARA AND K. TSUNEWAKI
52 03 04 05 08 07 13 14 52 29 3031 32 35 38 52
9 030405m QI 13 14 5229303l 3235 3 8 9
FIGURE1.-Restriction fragment patterns of ctDNA from the cytoplasms of common wheat (lane 52) and 12 Aegilops species ( 1 3 strains in total) carrying the M or modified M genome. The circle and star mark the differential fragment, unique or missing, respectively, in the Aegilops ctDNA. using common wheat ctDNA as controls. T h e lane number gives the code of each cytoplasm given in Table 1 . a. BamHI; b, EcoRI; c, Hindlll; d, KpnI. RESULTS
T h e restriction fragment patterns of the ctDNAs from 14 cytoplasmic sources a r e shown in Figures 1 and 2. In each gel the common wheat ctDNA (lane 52) was placed as a check in the marginal lanes (in some cases only in o n e margin) as well as in the central lane. In these figures, the DNA fragments unique for or missing in Aegilops ctDNA, as compared with those of common wheat ctDNA, a r e marked with a circle or star. In Tables 2 and 3, the estimated molecular weight and copy number of all restriction fragments raised from the Ae. crussu ctDNA a r e given. In these tables unique fragments for other ctDNAs a r e shown below the broken line along with their molecular weight and copy number. Restriction fragment changes observed between ctDNAs of Ae. crussu and other cytoplasms are collectively shown in Table 4. With the BumHI digests, the following nine patterns were obtained, for which individual cytoplasms a r e indicated by their code number given in Table 1: (1) 52, (2) 03, 29, 30 and 32, (3) 04 and 36, (4) 05, (5) 06, (6) 07, (7) 13 and 14, (8) 31 and (9) 35 (Figure la). From the EcoRI digests, four patterns were produced as follows: (1) 52 and 31, (2) 03, 13, 14, 29, 30, 32 and 35, (3) 0 4 and 36 and (4) 05, 06 and 07 (Figure lb). T h e Hind111 digestion gave
CHLOROPLAST DNA O F M G R O U P AEGILOPS
685
c
FIGURE2.-l’Iie same as Figure 1.
a, fstl; b.
Sall; c, Smal; d. Xhol.
the following seven patterns: (1) 52, (2)03, 13, 14, 29, 30 and 32,(3)04 and 36,(4)05 and 06,(5)07, (6)31 and (7)35 (Figure IC). After KpnI digestion, four patterns were produced as follows: (1) 52, 05,06 and 3 1, (2)03, 13, 14, 29, 30, 32 and 35,(3)04 and 36 and (4)07 (Figure Id). The PstI digests also yielded four patterns, as follows: (1) 52,(2)03,04, 13, 14, 29, 30, 31, 32, 35 and 36, (3)05 and (4)06 and 07 (Figure 2a). whereas the Sal1 treatment produced the following three patterns: (1) 52, 03, 13, 14, 29, 30, 31, 32 and 35, (2)04 and 36 and (3)05, 06 and 07 (Figure 2b). After the SmuI digestion the following seven patterns were obtained: (1) 52, (2)03, 13, 14, 29, 30 and 32,(3)04 and 36,(4)05 and 06,(5)07, (6)31 and (7)35 (Figure 2c), whereas the Xhol treatment produced six patterns as follows: (1) 52 and 31, (2)03, 29, 30 and 32,(3)04 and 36,(4)05 and 06,(5)07 and (6)13, 14 and 35 (Figure 2d). CtDNAs from the cytoplasms of a diploid species, Ae. umbellulutu (03),and three tetraploid species, Ae. biuncialis (29),Ae. columnuris (30)and Ae. triaristutu (32).produced identical fragment patterns with each of the eight restriction endonucleases. Similarly, ctDNAs from the cytoplasms of a diploid species, Ae. sguurrosu (04).and a tetraploid species, Ae. ventn‘cosu (36),revealed identical patterns. The ctDNAs from two Ae. muticu cytoplasms (13 and 14) also produced identical patterns. Chloroplast genomes of the 14 cytoplasms are thus classified into nine types based on their restriction fragment patterns. OGIHARA and TSUNEWAKI (1 984)
686
T. TERACHI, Y. OGIHARA AND K. TSUNEWAKI TABLE 2 Estimated molecular sizes (kbfl) of individual BamHI, EcoRI, Hind111 and Kpnl restriction fragments of Ae. crassa ctDNA and those of unique restriction fragments from other ctDNAs (below the broken line) ~
BaaHI
Fragment
KPnI
Hind111
EcoRI
kbp
copies
NO.
Fragment
kbp
No. copies
Fragment
kbp
copies
NO
Fragment
kbp
B1 B21 B3 B4 B5 B6 B71 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19
13.2 10.5 8.0 7.5 7.0 6.6 5.8 5.3 5.15 4.8 4.55 4.2 3.55 3.35 3.25 3.05 3.00 2.3 1.82
1 1 1 1 1 1 1 2
El E2 E3 E4 E5 E6 E71 E8 E9 E10 Ell E12 E13 E3s E7
13.0 7.0 4.4 3.7 3.1 2.8 3.4 2.46 2.32 2.26 2.12 1.68 1.40 3.9 2.7
2 1 1 2 1 2 1 2 2 1 2 2 1 1 1
K1 K2 K31 K4 K5 K6 K7 K8 K9 K10 K11 K2s K3 K3s
18.0 15.8 15.6 11.8 10.8 8.85 8.3 7.4 6.3 4.6 1.9 15.4 15.4 15.3
9.6 9.45 6.8 5.5 3.8 2.7 1.68
13.4 10.1 9.0 8.0 6.7 7.1 6.0 5.9 5.8 5.0 3.61 3.16 2.72 2.64 2.29 2.16 1.78 13.0 9.8 6.7 5.9 3.45
1 1 2 1 1 2 1 2
B2 B2s B5s B7 B12s B17b B20
H1 H21 H2 H3 H3s H4 H51 H5 H61 H7 H8 H9 H10 H11 H12 H13 H14 HIS H2s H4s H4s’ H8s
1
1 1 1 1 1 2 2 1 1 1
1 1 1 1 1 1 2
I
--------_-_
1
1 2 1 1 1
No. copies
1
1 1 1 3 1 1 1 1 1 3 1 1 1
_ _ I -
2 1 1 1 1 1 1 1
classified these chloroplast genome types as follows: type Id, cytoplasm 35; type 3, cytoplasms 03, 29, 30 and 32; type 4, cytoplasms 13 and 14; type 6, cytoplasm 31; type 7, cytoplasm 52; type 9a, cytoplasms 04 and 36; type 10, cytoplasm 07; type l l a , cytoplasm 05; and type l l b , cytoplasm 06. These designations of chloroplast genomes will be used here. From the data presented in Table 4, the number of changes in restriction fragment pattern between each pair of the nine chloroplast genome types was calculated (Table 5). Their total and average numbers are shown in Table 6. Both the insertion of a nucleotide segment into (or its deletion from) a restriction fragment and the gain (or loss) of a restriction site were considered as single restriction fragment changes. In the same table, the genetic distance, fi, between each pair of chloroplast genomes (= ctDNA molecules), which was
687
CHLOROPLAST DNA OF M-GROUP AEGILOPS
TABLE 3 Estimated molecular sizes (kbp) of individual PstI, Sall, Smal and Xhol restriction fragments of Ae. crassa ctDNA, and those of unique restriction fragments from other ctDNAs (below the broken line) ~~
Sal1
Pstl
NO
Fragment
kbp
copies
P1 P2 P3 P4 P5 P6 P71 P8 P9 P10
33.3 19.6 14.5 12.6 11.0 8.4 8.3 5.6 5.3 5.2
1 1 1 1 1 2 1 1 1 1
PlOS Plls
4.7 1.8
1 1
Xhol
Smal
Frag ment
kbp
copies
S1 S2 S3a S3b S4 s5 S6 s7 S8 S6s S71
27.2 21.8 14.2 13.6 11.8 7.2 6.8 6.2 4.5 6.6 7.1
1 1 1 1 1 3 1 1 2 1 1
No.
-------------
Fragment
kbp
20.8 Sm 1 18.2 Sm2 14.7 Sm3 Sm4 11.5 Sm51 8.8 7.7 Sm6 Sm7 7.3 5.1 Sm8 5.0 Sm8b 4.8 Sm9 SmlO 4.3 Smll 3.4 Sm14 2.0 1.75 Sm15 Sm16 1.2 Sm17 I--. 1.0 Smll 21.1 20.7 Smls 8.5 Sm5 6.5 Sm7s' 6.2 Sm7s" 5.8 Sm7b 4.5 Sm8bs
-----
No. copies
Flagment
kbp
copies
1 1 1 2 1 1 1 1 1 1 2 1 1 2 1 2 1 1 1 1 1 1 1
X1 X2 X31 X3 X4 x5 X6 X7 X8 X9 X10 X11 X12 X13 X11 Xls x4s x5s
18.2 14.9 14.4 13.1 12.6 9.7 9.2 8.6 5.65 3.8 3.2 2.85 2.83 2.4 25.7 17.9 12.2 9.5
1 1 1 1
NO
1
1 1 1 2 1 2 2 1 1
1 1 1 1
calculated by the method described in MATERIALSAND METHODS,is given. A dendrogram showing the phylogenetic relationship between all chloroplast genomes is drawn using these fi values (Figure 3). DISCUSSION
The validity of the use of ctDNAs from alloplasmic wheats to reveal ctDNA variation in Aegilops species: Autonomous replication of organellar DNAs from plants and animals is a well-established phenomenon. FI hybrids or alloplasmic lines of Zea, Nicotiana, Triticum and Brassica possess organellar DNAs characteristic of the maternal parent or cytoplasm donor (LEVINGSand PRING1 9 7 6 ; PRINGand LEVINGS1 9 7 8 ; CONDE,PRINGand LEVINGS1 9 7 9 ; FRANKEL, ScowCROFT and WHITFELD1 9 7 9 ; KUNC et al. 1 9 8 1 ; VEDELet al. 1 9 8 1 ; PALMERet al. 1 9 8 3 ) , although in Nicotiana a few exceptional cases have been reported (FRANKEL,SCOWCROFT and WHITFELD1 9 7 9 ; KUNG et al. 1 9 8 1). Furthermore, restriction fragment patterns of ctDNAs prepared directly from nine Triticum and DYER1 9 8 3 ) are identical with and Aegilops species (BOWMAN,BONNARD
T. TERACHI. Y . OCIHARA AND K . TSUNEWAKI
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689
CHLOROPLAST DNA OF M-GROUP AEGILOPS
TABLE 5
The number of ctDNA restriction fragment changes observed among nine chloroplast genomes BamHI: chloroplast genome
EcoR1: chloroplast genome
Id
3
4
6
7
Id 3 4 6 7 9a 10 Ila llb
2 4 3 3 3 2 2 1
0 4 1 3 3 2 2 1
0 0 2 4 2 4 3 3
1 1 1 2 3 3 3 2
1 1 1 0 3 3 3 2
Id 3 4 6 7 9a 10 Ila Ilb
Id
3
4
6
7
9a
10 l l a I l b
1 1 1 1 1 3 3 2
1 1 1 2 2 2 2 1
1 1 2 4 3 3 2 2
0 0 1 3 2 2 1 1
0 0 1 3 2 2 1 1
1 1 1 2 2 3 2 2
1 1 1 0 4 5 4 4
1 1 1 1 1 4 3 3
2 2 2 1 1 2 1 1
1 1 1 1 1 1 2 2 2 2 2 2 0 0 - 0 1 -
Id 3 4 6
7 9a 10
Ila Ilb
Id
3
4
6
7
9a
SmaI: chloroplast 10 I l a l l b genome
0
0 -
0 0
0 0
0 0
1 1
1 1
KpnI: chloroplast genome
SalI: chloroplast genome
PstI:
chloroplast genome
9a
HindII1: chloroplast 10 I l a I l b genome
1 1
1 1
0
0
-
0
0
1
1
1
1
0 1 0 1 2 1
0 1 0 1 2 1
0 1 0 1 2 1
1 0 1 2 1
0 1 2 3 2
1 1 1 2 1
1 1 2 1 0
1 1 2 0 1
1 1 2 0 0 -
Id 3 4 6
7 9a 10
Ila Ilb
1 1 1 0 0 1 1 0
1 1 1 0 0 1 1 0 -
XhoI: chloroplast genome Id
3
4
6
7
9a
10 l l a l l b
1 1 2 4 1 2 1 1
1 0 1 4 2 3 2 2
0 1 1 4 2 3 2 2
1 2 1 5 2 4 3 3
1 2 1 0 5 4 4 4
2 3 2 1 1 3 2 2
2 3 2 3 3 4 1 1
1 2 1 2 2 3 1 0
1 2 1 2 2 3 1 0 -
TABLE 6
Total and average number of ctDNA restriction fragment changes observed among nine chloroplast genomes (upper-right half) and their genetic distance, Ga (lower-left half) Chloroplast genome Chloroplast genome
Cytoplasm belonged (code no.)
Id
35 Id 3 03, 29, 30, 32 30 4 13, 14 36 6 31 53 7 52 77 9a 04, 36 66 10 07 77 Ila 05 53 llb 42 06 Average genetic 54.3 distance (p X lo4)
a ivalue multiplied by
3
5
-
19 36 77 78 101 71 59 58.9
4
6
7
9a
10
6 5 35 77 60 95 59 59 55.0
10 7 7
15 15 15
12 13 11
14 15 16 18 21 21
10 59 78 65 95 107 82 100 83 95 63.5 83.8
11
17 119 95 83 80.5
Ila
llb
11
9 10
-
12 12 15 19 18 7
35 30 82.4
12 63.4
10' is given in the table (for example, 67 means
-
11 13 17 16 5 2 57.9
6 = 0.0067).
Average no. of fragment changes
10.3 10.3 10.4 11.4 16.1 14.9 14.6 12.0 10.4
690
T. TERACHI, Y. OGIHARA AND K. TSUNEWAKI
1la
I
1 11b
1
10 Id
3 4 6 9a
1 I
1
I
9
8
7
x~o-~
I
I
1
6 5 4 3 2 Genetic distance (p)
1
0
1
I
1
1
t
FIGURE3.-A dendrogram showing the phylogenetic relationships among nine chloroplast genomes identified in M and modified M genomecarrying species of Aegilops.
those of ctDNAs extracted from alloplasmic lines of common wheat carrying and OGIHARA1983). These facts valcytoplasms of those species (TSUNEWAKI idate the use of ctDNAs isolated from the cytoplasms of alloplasmic common wheat to study ctDNA variation among Aegilops species. Among our stocks of alloplasmic common wheats, we have at least one self-fertile line with cytoplasm from each of all of the Triticum and Aegilops species, except Ae. comosa, Ae. heldreichii and one strain of Ae. mutica (code no. 14) (TSUNEWAKI 1980; TsuNEWAKI and TSUJIMOTO 1984). On the intraspecij-ac variation of ctDNA: Using four restriction endonucleases, BamHI, EcoRI, PstI and SalI, OGIHARA and TSUNEWAKI (1982) found no differences in the ctDNA among two emmer and three common wheat accessions, among three species, T. araraticum, T. timopheevi and T. rhukovskyi, among three Ae. triuncialis strains, between two Ae. biuncialis strains or between two Ae. crassa strains. On the contrary, they found two fragment differences between t w o Ae. speltoides strains (one is “aucheri”)and between t w o Ae. triuncialis strains. Using the same four restriction endonucleases, BOWMAN, BONNARD and DYER(1983) found differences in one to five fragments among the ctDNAs of three einkorn species. They also noticed changes in six fragments in the ctDNA of t w o Ae. speltoides strains. No differences in ctDNA were recognized among four common wheat cultivars and the wild emmer wheat or between T. araraticum and T. timophemi. In similar studies with seven restriction enand OGIHARA ( 1983) found no differences between donucleases, TSUNEWAKI emmer and common wheat and between T. monococcum and T. urartu in their ctDNA. On the other hand, differences in six fragments were revealed between two strains of Ae. speltoides (one is aucheri). In the present investigation, only one source of ctDNA was used for each species, except Ae. mutica. The ctDNAs from two different Ae. mutica cytoplasms were not different. All of these in-
CHLOROPLAST DNA OF M-GROUP AEGILOPS
691
formations indicate that some intraspecific variation in the ctDNA does exist in certain Triticum and Aegilops species, although in general it is much smaller than the interspecific differences; it is evident from the fact that the average number of restriction fragment differences observed among different species and or species groups was 4.2, 8.1, 8.6 and 12.2 in the reports of OGIHARA TSUNEWAKI (1982), BOWMAN, BONNARD and DYER(1983), TSUNEWAKI and (1983) and the present investigation, respectively, as compared with OGIHARA zero to two, zero to six, and zero to six fragment differences observed within (1982), BOWMAN, a species (or species group) by OGIHARA and TSUNEWAKI BONNARDand DYER(1983) and TSUNEWAKI and OGIHARA (1983), respectively. Diversity among chloroplast genomes: A circular DNA molecule containing n base pairs consists of n six-base pair sequences, of which two neighboring ones overlap in their five base pairs. In each of the n six-base pair sequences, 46, namely, 4096 kinds of sequences are possible, and all endonucleases used in the present investigation recognize only one of them. Thus, at a given six-base pair sequence position, there is a 8/4096 chance for determining the base sequence by using eight endonucleases. This probability is the same for all n six-base pair sequence positions and is determined simply by the number of endonucleases used and the number of base pairs included in their recognition sites, being independent of genome size. Thus, in the present case, about 0.2% of the entire base pair sequence has been revealed. The chloroplast genome size of Ae. crassa is estimated to be about 135 kilobase pairs (kbp) (Tables 2 and 3). Its ctDNA has given 25, 20, 27, 15, 13, 15, 21 and 19 identifiable fragments by BamHI, EcoRI, HindIII, KpnI, PstI, M I , SmaI and XhoI digestion, respectively. This indicates that we have identified 6 X 155 base pairs, i.e., 0.7% of the entire chloroplast genome. Therefore, we may say that about 0.2-0.7% of the chloroplast genome has been examined for sequence differences in the present investigation. As shown in Table 6, both the average genetic distance @) and the average number of restriction fragment changes of each of the chloroplast genomes, as compared to eight other chloroplast genomes, are smallest for the type Id chloroplast genome (ctDNA of Ae. crassa), whereas those of three chloroplast genome types, 7, 9a and 10, are the largest three of all. Similar results were reported earlier, in which restriction fragment patterns of ctDNAs from 29 Triticum and Aegilops species were studied using four restriction endonu1982). Furthermore, we obcleases (Table 6 of OGIHARA and TSUNEWAKI served a very close resemblance between ctDNAs of Ae. crassa and four other species, T. monococcum (plasma type A), Ae. bicornis (Sb),Ae. sharonensis (S') and Ae. kotschyi (S"). These facts suggest that the type Id chloroplast genome, corresponding to the D2 plasma type, is centrally located, whereas type 7, 9a and 10 chloroplast genomes are peripherally located within a spectrum of chloroplast genome diversification. From the data of Table 6 and Figure 3 we see that the chloroplast genomes of three Comopyrum species form a cluster, although the relationship between Ae. comosa and Ae. heldreichii is much closer than those between them and Ae. uniaristata. The cluster formed by these three species is distantly located from all other chloroplast genomes, including that of Ae. mutica, the sole species of
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Amblyopyrum section. Thus, it is evident that relatedness of Comopyrum species to Amblyopyrum species is as distant as that between Comopyrum and Polyeides species (represented by Ae. umbellulata). Origin of the cytoplasm of the tetraploid species carrying the modijied M genome: Chloroplast genomes of Ae. biuncialis, Ae. columnaris and Ae. triaristata are identical with that of Ae. umbellulata and are greatly different from the chloroplast genomes of all Comopyrum species. According to MUKAIand TSUNEWAKI ( 1 975), TSUNEWAKI ( 1 980) and TSUNEWAKI and TSUJIMOTO (1984), the cytoplasms of these four species cause variegation (variegated yellowing of leaf color) in midwinter and growth depression in most common wheats, haploidy and haplo-diplo twinning in t w o particular common wheats and complete male sterility in about half of the common wheats tested; they are classified into the same C" plasma type. On the other hand, the cytoplasms of Ae. comosa and Ae. heldreichii cause growth depression and complete male sterility in all common wheats without inducing variegation, haploidy and twinning. Although the cytoplasm of Ae. uniaristata does not cause variegation, growth depression or male sterility in most common wheats, it does cause male sterility in three of 12 common wheats tested. Based on these facts we previously concluded that Ae. umbellulata is the cytoplasm donor to Ae. biuncialis, Ae. columnaris and Ae. triaristata. However, based on similar studies of cytoplasm-substituted wheats, MAAN(1975, 1978) and PANAYOTOV and GOTSOV(1975, 1976) proposed that Ae. uniaristata or Ae. comosa are the cytoplasm donors of Ae. biuncialis, Ae. columnaris and Ae. triaristata. The present results clearly verify our previous conclusion. Ae. ventricosa has a chloroplast genome identical with that of Ae. squarrosa but different from those of the Comopyrum species. The cytoplasms of Ae. squarrosa and Ae. ventricosa exert no phenotypic effects, neither beneficial nor deleterious, upon common wheat and are classified as D plasma types; cytoplasmic genomes of Ae. comosa, Ae. heldreichii, Ae. uniaristata and Ae. mutica exert various phenotypic effects. We previously concluded that Ae. ventricosa received its cytoplasm from Ae. squarrosa but not from the M' genome donor (TSUNEWAKI, MUKAIand ENDO1978; TSUNEWAKI 1980). The same conclusion was reached by PANAYOTOV and GOTSOV(1976) and MAAN (1978). The present results support this conclusion. Ae. ovata has a nuclear genome similar to those of Ae. biuncialis, Ae. columnaris and Ae. triaristata, namely, the C" and a modified M genome, MO. T h e cytoplasm of Ae. ovata causes complete male sterility in a great majority of wheat cultivars, and great delay of heading in all common wheats, without impairing growth vigor. Various morphological and physiological characters are more or less influenced by this cytoplasm through a prolonged growth period. The effects of Ae. ovata cytoplasm are greatly different from those of Ae. umbellulata cytoplasm, as mentioned before. Thus, we have concluded that the cytoplasm of Ae. ovata was derived from the MO genome donor (MUKAI and TSUNEWAKI 1975; TSUNEWAKI 1980). On the contrary, MAAN (1975, 1978) and PANAYOTOV and GOTSOV(1976) proposed that Ae. mutica and Ae. caudata were the cytoplasm donor, respectively. However, the chloroplast genome of Ae. ovata is unique, being distinctly different from those of all Ae.
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Tve S6l5 Sphr FIGURE4.-Selfed seed fertility spectra of the cytoplasms of Ae. mutica M and P strains tested with 12 common wheat nuclei (after TSUNEWAKI and TSUJIMOTO 1984). a and b, Cytoplasm of M and P strains, respectively. Tve, P168, CS, N26, Slm, JF, Sk and S615: T. aestivum var. erythrospermum, strain P168, cv. Chinese Spring, cv. Norin 26, strain Salmon, cv. Jones Fife, cv. Selkirk and cv. S-615, respectively. Sphr, Cmp, Splt and Mch: T. sphaerococcum, T. compactum, T. spelta and T. macha, respectively.
umbellulata (the C" genome donor) and Comopyrum or Amblyopyrum species. No diploid species with a chloroplast genome closely related to that of Ae. and ovata has been found in the Triticum and Aegilops genera (OGIHARA TSUNEWAKI 1982; present result). Even Ae. umbellulata and Ae. mutica, whose chloroplast genomes are closest to that of Ae. ovata among all of the species tested, showed seven ctDNA restriction fragment changes from that of Ae. ovata. These facts may suggest a very ancient origin of this species, compared to those of other tetraploid species of the same Polyeides section. Ae. crassa's chloroplast genome differs greatly from those of all diploid species studied here. T h e cytoplasm of Ae. crassa does not affect the phenotype of common wheat except for the induction of extreme pistillody in some cultivars under long-day growth conditions (SASAKUMA and OHTSUKA 1979; TSUNEWAKI 1980). In these respects, this cytoplasm resembles that of Ae. squarrosa, and we, therefore, suggested that the cytoplasm donor of Ae. crassa was Ae. squarrosa and designated its plasma type D2, meaning that it is a subtype of 1980). the D plasma type of Ae. squarrosa and Ae. ventricosa (TSUNEWAKI However, the present ctDNA data do not support this suggestion, in that the chloroplast genome of Ae. squarrosa and Ae. crassa differ by 12 restriction fragment changes. At the same time, no Comopyrum or Amblyopyrum species possesses a similar chloroplast genome, which is to say that we do not yet know the origin of the Ae. crassa cytoplasm. Possible intraspecijk dgerentiation of the mitochondrial genome in Ae. mutica: T h e chloroplast genomes of two Ae. mutica strains have identical ctDNA restriction fragment patterns. According to TSUNEWAKI and TSUJIMOTO (1984), however, their cytoplasms are different. As shown in Figure 4, the mutica-M cytoplasm (code no. 13) causes complete male sterility in only three of 12 common wheats tested, whereas the mutica-P cytoplasm (code no. 14) induces
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complete male sterility in all wheats. In addition, the mutica-M cytoplasm delays heading about 10.4 days, whereas the mutica-P cytoplasm does not affect the heading date at all (at most, a 0.1-day delay). Plant height and dry matter weight are also affected by the former cytoplasm, showing 2 and 10% increase from those of the control, respectively, whereas they are decreased by 6 and 9%, respectively, by the latter cytoplasm. Thus, we designated the plasma type of Ae. mutica-M and -P as Mt and Mt2, respectively (TSUNEWAKI and TSUJIMOTO 1984). T h e discrepancy between the ctDNA data and cytoplasmic effects can be due to still unravelled ctDNA differences, because we studied only 0.20.7% of ctDNA sequence. However, we prefer to postulate that other cytoplasmic, genetic systems, most probably the mitochondrial genome, differ between the two cytoplasms of Ae. mutica, and that it affects male gametophyte development, heading date and plant vigor as well. T o clarify this, the variation of mitochondrial DNAs among Triticum and Aegilops species is under investigation. This is contribution no. 469 from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University. T h e work was supported by a Grant-in-Aid for Scientific Research (no. 56440001), from the Ministry of Education, Science and Culture, Japan. We are greatly indebted to S. S. MAAN,North Dakota State University, and I. PANAYOTOV, Wheat and Sunflower Institute, Bulgaria, for supplying the original seed stocks of five alloplasmic common wheats carrying the cytoplasms of Ae. heldreichii, Ae. uniaristuta, Ae. mutzcu (two lines) and Ae. crussa, from which the present materials have been bred by repeated backcrosses. We wish t o express o u r thanks to H. TSUJIMOTO and S. ENOMOTOfor their technical assistance. We also thank to T. MARUYAMA and S. TSUJIfor their help on the statistical treatment of the data. Our greatest appreciation is d u e t o VAL W. WOODWARD,University of Minnesota, for his continuous encouragement and valuable suggestions for improving the manuscript. LITERATURE CITED
R DT. A. DYER,1983 Chloroplast DNA variation between species BOWMAN, C. M., G. B ~ N N Aand of Triticum and Aegilops: location of the variation on the chloroplast genome and its relevance to the inheritance and classification of the cytoplasm. Theor. Appl. Genet. 6 5 247-262. CONDE,M. F., D. R. PRINGand C. S. LEVINGS,1979 Maternal inheritance of organelle DNA's in Zeu mays-Zeu perennis reciprocal crosses. J. Hered. 7 0 2-4. ENGELS,W. R., 1981 Estimating genetic divergence and genetic variability with restriction endonucleases. Proc. Natl. Acad. Sci. USA 78: 6329-6333. FRANKEL,R., W. R. SCOWCROFT and P. R. WHITFELD,1979 Chloroplast DNA variation in isonuclear male-sterile lines of Nicotiana. Mol. Gen. Genet. 169 129-135. CORDON, K. H. J., E. J. CROUSE,H. J. BOHNERTand R. G. HERRMANN, 1982 Physical mapping of differences in chloroplast DNA of the five wild-type plastomes in Oenothera subsection Euoenothera. Theor. Appl. Genet. 61: 373-384. KIHARA,H., 1949 Genomanalyse bei Triticum und Aegilops. IX. Systematischer Aufbau der Gattung Aegilops auf genomanalytischer Grundlage. Cytologia 14: 135-144. KOLODNER,R. and K. K. TEWARI,1975 T h e molecular size and conformation of the chloroplast DNA from higher plants. Biochim. Biophys. Acta 404: 372-390. KUNG,S. D., Y. S. ZHU, K. CHEN, G. F. SHENand V. A. SISSON, 1981 Nicotiana chloroplast genome. 11. Chloroplast DNA alteration. Mol. Gen. Genet. 183: 20-24. KUNG,S. D., Y. S. ZHU and G. F. SHEN, 1982 Nicotiana chloroplast genome. 111. Chloroplast DNA evolution. Theor. Appl. Genet. 61: 73-79. LEVINGS,C. S. and D. R. PRING, 1976 Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize. Science 193: 158-160.
CHLOROPLAST DNA OF M-GROUP AEGILOPS
695
LILIENFELD, F. A., 1951 H. Kihara: genome-analysis in Triticum and Aegilops. X. Concluding review. Cytologia 1 6 101-123.
MAAN,S. S., 1975 Cytoplasmic variability and speciation in Triticinae. pp. 255-281. In: Prairie, edited by M. K. WALI.University of North Dakota Press, Grand Forks. MAAN,S. S., 1978 Cytoplasmic relationships among the D and M genome Aegilops species. Proc. V Int. Wheat Genet. Symp. 1: 231-260. MUKAI,Y. and K. TSUNEWAKI, 1975 Genetic diversity of the cytoplasm in Triticum and Aegilops. 11. Comparison of the cytoplasms between four 4x Aegilops Polyeides species and their 2x relatives. Seiken Ziho 25-26 67-78. OGIHARA, Y. and K. TSUNEWAKI, 1982 Molecular basis of the genetic diversity of the cytoplasm in Triticum and Aegilops. I. Diversity of the chloroplast genome and its lineage revealed by the restriction pattern of ctDNAs. Jpn. J. Genet. 57: 371-396. OGIHARA,Y. and K. TSUNEWAKI, 1984 The diversity of chloroplast DNA among Triticum and Aegilops species. Proc. VI Int. Wheat Genet. Symp., pp. 407-413. PALMER, J. D., C. R. SHIELDS,E. B. COHENand T. J. ORTON,1983 Chloroplast DNA evolution and the origin of amphidiploid Brassica species. Theor. Appl. Genet. 6 5 181-189. PALMER, J. D. and D. ZAMIR,1982 Chloroplast DNA evolution and phylogenetic relationships in Lycopersicon. Proc. Natl. Acad. Sci. USA 7 9 5006-5010. PANAYOTOV, I. and K. G o ~ s o v ,1975 Results of nucleus substitution in Aegilops and Triticum species by means of successive backcrosses with common wheat. Wheat Inform. Serv. 4 0 2022. PANAYOTOV, I. and K. GOTSOV,1976 Interactions between Aegilops cytoplasms and Triticum species. Cereal Res. Commun. 4 297-306. PRING,D. R. and C. S. LEVINGS,1978 Heterogeneity of maize cytoplasmic genomes among malesterile cytoplasms. Genetics 8 9 121-1 36. SASAKUMA, T. and I. OHTSUKA,1979 Cytoplasmic effects of Aegilops species having D genome in wheat. I. Cytoplasmic differentiation among five species regarding pistillody induction. Seiken Ziho 27-28: 59-65. SNEATH,P. H. A. and R. R. SOKAL,1973 Numerical Taxonomy. pp. 573. Freeman & Company, San Francisco. TSUNEWAKI, K. (Editor), 1980 Genetic Diversity ofthe Cytoplasm in Triticum and Aegilops. pp. 290. Japan Society for Promotion of Science, Tokyo. TSUNEWAKI, K., Y. MUKAIand T. R. ENDO,1978 On the descent of the cytoplasms of polyploid species in Triticum and Aegilops. Proc. V Int. Wheat Genet. Symp. 1: 261-272. TSUNEWAKI, K. and Y. OGIHARA,1983 The molecular basis of genetic diversity among cytoplasms of Triticum and Aegilops. 11. On the origin of polyploid wheat cytoplasms as suggested by chloroplast DNA restriction fragment patterns. Genetics 104: 155-1 71.
TSUNEWAKI, K. and H. TSUJIMOTO,1984 Genetic diversity of the cytoplasm in Triticum and Aegilops. Proc. VI lnt. Wheat Genet. Symp., pp. 1139-1 144. VEDEL,F., F. QUETIER,Y. CAUDERON, F. DOSBAand G. DOUSSINAULT, 1981 Studies on maternal inheritance in polyploid wheats with cytoplasmic DNAs as genetic markers. Theor. Appl. Genet. 5 9 239-245. 1978 Study of wheat phylogeny by EcoRI VEDEL,F., F. QUETIER,F. DOSBAand G. DOUSSINAULT, analysis of chloroplastic and mitochondrial DNAs. Plant Sci. Lett. 13: 97-102.
ZHUKOVSKY, P. M., 1928 A critical systematical survey of the species of the genus Aegilops L. Bull. Appl. Bot. Genet. PI. Breed. 18: 417-609. Corresponding editor: M. T. CLECG
