Copyright 0 1986 by the Genetics Society of America

Chloroplast DNA Diversity in Populations of Wild and Cultivated Barley

D. B. Neale,' M. A. Saghai-Maroof,* R. W. Allard, Q. Zhang and R. A. Jorgensen4

Department of Genetics, University of Cal$ornia, Davis, Cal$orniu 95616 Manuscript received June 22, 1988

Revised copy accepted September 6, 1988

ABSTRACT Chloroplast DNA (cpDNA) diversity was found within and among populations (245 accessions total)

of wild barley, Hordeum vulgare L. ssp. spontaneum Koch from Israel and Iran. Three polymorphic restriction sites (HindIII, EcoRI, BclI) which define three distinct cpDNA lineages were detected. One lineage is common to populations in the Hule Valley and Kinneret of northern Israel, and in Iran. The second lineage is found predominantly in the Lower Jordan Valley and Negev. The distribution of the third lineage is scattered but widespread throughout Israel. Sixty two accessions of cultivated barleys, H. vulgare L., were found, with two exceptions, to belong to just one cpDNA lineage of wild barley, indicating that the cpDNA of cultivated barley is less variable than its wild ancestor. These results demonstrate the need for assessing intraspecific cpDNA variability prior to choosing single accessions for phylogenetic constructions at the species level and higher.

H IGHER plant chloroplast genomes are highly conserved in both gene organization and nu-

cleotide sequence across a broad array of taxa (PAL- MER 1985a, 1985b, 1987; BIRKY 1987; ZURAWSKI and CLECC 1987). The small amount of diversity observed within cpDNA genomes, relative to nuclear genomes, led PALMER and ZAMIR (1 982) and others to suggest that cpDNA restriction fragment variants could be useful in constructing molecular phylogenies and the method has subsequently been applied to many taxa, e.g., PALMER, JORCENSEN and THOMPSON (1 985) and SYTSMA and GOTTLIEB (1986). Although sample sizes per species were small in the early phylogenetic stud- ies, there were indications that cpDNA polymor- phisms might exist at the intraspecific level in some species e.g., in Pisum sativum (PALMER, JORGENSEN and THOMPSON 1985), in Nicotiana debneyi (SCOWCROFT 1979), in Pelargonium zonule holt (METZLAFF, BORNER and HACEMANN 198 l), in Aegilops speltoides (BOWMAN, BONNARD and DYER 1983), in Zea mays (DOEBLEY, RENFROE and BLANTON 1987) and in Hordeum vulgare ssp. vulgare and its wild progenitor H . vulgare ssp. spontaneum (CLECC, BROWN and WHITFELD 1984). There subsequently have been several studies of intra- specific variability in plant chloroplast DNA. Among 100 individual plants from 2 1 populations of Lupinus texensis in central Texas, BANKS and BIRKY (1 985) found 88 plants with the same cpDNA restriction

' Present address: USDA Forest Service, Pacific Southwest Forest and

Berkeley, California 94701. Range Experiment Station, Institute of Forest Genetics, P.O. Box 245,

tute and State University, Blacksburg, Virginia 24061. ' Present address: Department of Agronomy, Virginia Polytechnic Insti-

Republic of China. ' Present address: Huazhong Agricultural University, Wuhan, People's

Oakland, California 94608. ' Present address: Advanced Genetic Sciences, 6701 San Pablo Avenue,

Genetics 1 2 0 1105-1 1 10 (December, 1988)

fragments and variable types among the remaining 12. WAGNER et a l . (1987) surveyed 153 individuals of Pinus contorta and 1 15 individuals of P. banksiana from numerous populations in Canada; six cpDNA variants were found in each of the two species. HOL- WERDA, JANA and CROSBY (1986) sampled 15 acces- sions of H. vulgare and 13 of H. spontaneum from throughout the Middle East and found a minimum of five different cpDNA genotypes. However, within population sample sizes were very small in each of these studies and cpDNA genotypic frequencies con- sequently could not be determined.

Within population genetic variation has been stud- ied in both wild and cultivated barleys by using mor- phological variants UAIN and ALLARD 1960), by allo- zyme polymorphisms (ALLARD, KAHLER and WEIR 1972; BROWN, ZOHARY and NEVO 1978; BROWN et al . 1978; NEVO et a l . 1979; KAHLER and ALLARD 1981; BROWN and MUNDAY 1982), by storage protein poly- morphisms (DOLL and BROWN 1979) and by ribosomal DNA variants (SAGHAI-MAROOF et al. 1984) (reviewed by ALLARD 1988). These studies demonstrate genetic variability within and among populations of both wild and cultivated barley for a variety of nuclear DNA encoded characters. The cpDNA of Hordeum appears to be strictly maternally inherited, thus cpDNA poly- morphisms reflect maternal lineages and the genetic structure of barley populations reflects gene flow due to seed dispersal alone and not gene flow due to pollen dispersal (TAKAHATA and MARUYAMA 1981 ; BIRKY, MARUYAMA and FUERST 1983; TAKAHATA 1983).

This paper reports a study to determine the extent of cpDNA diversity within and among populations of wild and cultivated barleys based on a survey of 245 accessions of H . vulgare ssp. spontaneum in Israel and

1106 D. B. Nedle et al.

Ir in. cpDNA diversity was also investigated in 62 varieties of domesticated barley (H. vulgare).

MATERIALS AND METHODS

Plant populations: Two hundred forty-five accessions of H . vulgare ssp. spontaneum from 25 populations in Israel and 5 populations in Iran were obtained from the U.S. Depart- ment of Agriculture (USDA) World Barley Collections. The locations of 20 Israeli populations are shown in Figure 1; many of the collection sites were the same as those of NEVO et al. (1 979). Precise localities for the remaining 5 collection sites in Israel were not available; these could be located only to their geographical region (populations 10, 16, 18, 22 and 23; Table 1). In addition, 5 accessions of ssp. deficiens, 5 accessions of ssp. distichum, and 6 accessions of ssp. irregulare were obtained from the USDA collections and 46 accessions of ssp. vulgare (parents of composite crosses I1 and V) were obtained from the University of California collections.

DNA extraction, Southern blots and DNA hybridiza- tions: Total DNA was extracted from 0.75 g of freeze dried leaf tissue following procedures described in SAGHAPMA- ROOF et al. ( 1 984). Approximately 1 .O p g of each DNA was digested with each of several restriction enzymes and frac- tionated on 0.7-1.0% agarose gels in TAE buffer (MANIA- TIS, FRITSCH and SAMBROOK 1982). DNAs were transferred to Biotrans nylon membranes (ICN, Irvine, CA) and then baked for 2 hr at 80".

Following several hours of prehybridization (1 00 pg/ml salmon sperm DNA), blots were hybridized with 32P-labeled (nick translation) cpDNA probes. Three different sources of probes were employed: ( 1 ) total cpDNA purified from 2. mays, (2) a PstI cpDNA library from Petunia hybrida (PALMER et al. 1983), and (3) several PstI cpDNA clones from barley (DAY and ELLIS 1983). Hybridizations were conducted at 65 O for 24 hr after which filters were washed twice at room temperature in 2X SSC, 0.1% SDS and then followed by two additional 45-min washes at 65". Filters were then exposed to X-ray film (Kodak XAR-5) with intensifier screens for 24 hr at -70".

RESULTS

Preliminary survey of cpDNA RFLP variation in barley: A set of 64 barley accessions (32 H. vulgare ssp. spontaneum, 32 H. vulgare ssp. vulgare) were cho- sen to screen for restriction enzymes revealing poly- morphisms in barley cpDNA. DNAs from these 64 accessions were each digested with 16 restriction en- zymes (BamHI, BclI, BstNI, CfoI, ClaI, EcoRI, EcoRV, HindIII, NcoI, NruI, PstI, SmaI, SphI, SstI, SstII, and XbaI) and blots were hybridized with total cpDNA from maize. Based on these assays, enzymes were classified into one of three categories: (1) mono- morphic with fewer than 20 bands (NruI , PstI, SmaI , SphI, SstI, SstII); (2) apparently monomorphic with more than 20 bands (BamHI, BstNI, CfoI, EcoRV, NcoI, XbaI) ; and (3) polymorphic, (BclI , ClaI , EcoRI, HindIII). Bcll, ClaI, EcoRI, and HindIII digests had 24,25,27 and 22 detectable bands, respectively, when hybridized with total maize cpDNA. Although there were detectable differences in band patterns among barley DNAs digested with these four enzymes, we

were unable to deduce the type of mutational event which led to the band pattern differences.

BclI, Clal, EcoRI and HindIII blots were then stripped of the labeled maize cpDNA probe and re- hybridized sequentially with each of several PstI cpDNA clones from petunia. Each petunia clone hy- bridized only to homologous barley fragments, thus reducing the number of bands observed on autoradi- ographs. Three point mutations (Figure 2) and a single insertion/deletion sequence were detected in this manner. The BclI, EcoRI, and HindIII point mutations were in sequences homologous to petunia clones P8, P6 (map locations of petunia clones are shown in SYTSMA and GOTTLIEB 1986), and barley clone PHvcP3 (DAY and ELLIS 1985), respectively. PHvcP3 is homologous to petunia P10 except that the petunia clone did not hybridize to the 0.5-kb HindIII fragment of the barley DNAs. The small insertion/ deletion sequence was found in only one DNA for EcoRI and ClaI digests hybridized to petunia P18. All barley restriction fragment length polymorphisms (RFLPs) were thus within the large single-copy region of the barley chloroplast genome or within petunia sequences homologous to this region.

RFLP variation in H . vulgure: One of the initial objectives of this study was to study temporal variation in composite crosses of H. vulgare ssp. vulgare. We consequently examined the parental lines from which composite crosses I1 and V had been synthesized for the most polymorphic enzymes, BclI, EcoRI, and HindIII. All 46 parents surveyed lacked the BclI, EcoRI or HindIII polymorphic restriction sites, with the single exception of the cultivar Glabron, which had the BclI site. The parents of composite crosses I1 and V are known to carry a representative sample of genetic variability from throughout the world which suggests that cpDNA may be nearly invariant world- wide. The study of temporal variation in composite crosses was consequently abandoned.

Members of three other diverse types within H. vulgare (distichum, deficiens, and irregulare) were also examined for BclI, EcoRI and HindIII polymor- phisms. We were unable to obtain accessions of a fourth type agriocrithon. None of the 16 accessions from distichum, deficiens, and irregulare had the EcoRI or HindIII sites; however, one of the five accessions of distichum had the BclI site, previously detected only in H. vulgare ssp. spontaneum.

RFLP variation in H . vulgure ssp. spontuneum. All three restriction sites (HindIII, EcoRI and BclI) were polymorphic in wild barley cpDNA; thus eight multi- restriction site genotypes are possible. The HindIII and EcoRI sites were always present (+) or absent (-) simultaneously but all three sites were not found in any individuals, thus only the three cpDNA genotypes [- - -1, [- - +], and [+ + -1; in the order HindIII,

Chloroplast DNA Diversity in Barley 1107

FIGURE 1 .-Distribution of H . uulgure ssp. spontuneum populations sampled in Israel. Numbers correspond to population numbers given in Table I . Frequency pie diagrams are not shown for populations 7-1 1 , 18, 22 and 23 because sample sizes were <5. The location of population 16 was somewhere within the Central Mountain Ridge Region but its exact location was not known. The sy* lbols + and - refer to the presence or absence of restriction sites in the order Hindlll. EcoRI, and Bcl l .

1108 D. B. Neale et al.

TABLE 1 substitutions and one insertion/deletion. This low Multirestriction site cpDNA genotypes for 245 H. vulgare ssp. level of diversity is comparable to that observed in spontaneum DNAs sampled at 25 locations in Israel and 5 in two previous studies of cpDNA variation in wild and

Iran domesticated barley (CLEGG, BROWN and WHITFELD

Geograph- Popula- 1984; HOLWERDA, JANA and CROSBY 1986). CLEGC,

cpDNA genotypeb ical re- tion BROWN and WHITFELD (1984) found a single CDDNA ion" No. Location _" - - + + + - N

A B B B C C C C C C C D D D D D E E F F F F F G G H H H H H

1 2 3 4 5 6 7 8 9

10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mt. Hermon Rosh Pinna Gadot Tabigha Mt. Meron Damon Western Galilee Lower Galilee Mt. East Upper Galilee Region 1 1 Mt. Carmel Bar Giyyora Eiyzariya Judean Foothills Jerusalem Mt. Region 12 Bor Mashash Region 16 Bet Shean Mehola Jericho Region 8 Region 4 Atlit Ashqelon Shahabad-Ilam-A Qazvin Ilam-Meheran Shahabad-Ilam-B Unknown

0 0 9 9 5 0 0 5 7 1 0 8

10 0 0 10 7 1 0 8 2 11 8 21 0 0 1 1 0 0 4 4 0 0 2 2 0 0 2 2 0 0 1 1 0 0 9 9 1 13 1 15 1 0 4 5 2 1 11 14 2 6 5 1 3 0 9 0 9 0 0 1 1 0 13 0 13 0 18 0 18 0 9 0 9 0 3 0 3 1 1 1 3 0 0 12 12 0 12 8 20 4 0 0 4 2 0 0 2

13 1 0 14 8 0 1 9 1 0 0 1

245

a Letter designations for regions follow those proposed by BROWN et al. (1978), with the addition of one region for Iranian populations. A, Golan Heights; B, Hule Valley and Kinneret; C, Galilee and Carmel Mountains; D, Central Mountain Ridge; E, Negev; F, Lower Jordan Valley; G, Mediterranean Coastal Plain; and H, Iran.

cpDNA genotypes are based on the presence (+) or absence (-) of three restriction sites; HindIII, EcoRI and BclI.

EcoRI, BclI , were found. Although sample sizes were small, there were distinct differences among popula- tions in cpDNA genotype frequencies (Table 1). With a few exceptions, populations in the same region were more similar than populations in different regions, e.g., (1) Mt. Meron was more like populations in region B than C, (2) Eyzariya was more like populations in regions E and F than D, and (3) the two populations in region G (Atlit and Ashqelon) were different.

DISCUSSION

We have found cpDNA restriction site variation both within and among populations of wild barley in Israel and Iran, and among cultivated barleys. We surveyed approximately 2000 nucleotides of the bar- ley chloroplast genome and detected three nucleotide

polymorphism among nine accessions of H. vulgare ssp. vulgure and five polymorphisms among 1 1 acces- sions of H. vulgare ssp. spontaneum. They concluded that cpDNA variability is lower in cultivated barley than wild barley, possibly as a result of domestication. In contrast, HOLWERDA, JANA and CROSBY (1986) concluded that there were no differences in cpDNA diversity between cultivated and wild barley based on 15 and 13 accessions, respectively. Our results, based on the three polymorphic restriction sites, support the conclusion of CLEGG, BROWN and WHITFELD (1984) that wild barley is more variable than cultivated bar- ley.

Multirestriction site frequencies were calculated for 19 of the 30 populations where N 2 5 (Table 2). Nine populations were monomorphic and 10 were poly- morphic, and all three genotypes were found in four populations. These results demonstrate that cpDNA variation exists not only within H. vulgare ssp. spon- taneum but also within individual populations. Chlo- roplast DNA lineages do not appear to be randomly distributed in Israel. The [- - -1 lineage is predomi- nant in the area north of the Sea of Galilee and the [- - +] lineage in the Lower Jordan Valley, whereas the [+ + -3 lineage has a scattered distribution over much of Israel (Figure 1). The existence of population level cpDNA variation, such as in wild barley, makes it possible to investigate adaptive patterns of cpDNA variation as well as correlated patterns of variability with nuclear or possibly mitochondrial encoded traits.

The presence of within species cpDNA variation leads to an important point concerning plant phylo- genetic constructions based on cpDNA restriction fragment variation. Studies of this type (reviewed by PALMER 1987) typically sample a single individual accession per species. This is justified based on the general notion of the conservation of cpDNA se- quences. However, as this study shows, it should not be assumed that there is no intraspecific cpDNA var- iation and that phylogenetic studies of closely related species, which utilize cpDNA restriction fragment pol- ymorphisms, should consider intraspecific variability prior to constructing phylogenies.

0 1

This research was supported by National Institutes of Health grant GM 32429. We thank JEFF PALMER and NOEL ELLIS for providing cpDNA clones, DON SLEETER for graphics, RON SEDER- OFF, JEFF PALMER, and two anonymous reviewers for comments on earlier versions of the manuscript, and KEN SYTSMA for help in initiating D.B.N. to cpDNA analysis.

Chloroplast DNA Diversity in Barley 1109

0.5 U I

1 2 3 l o l 2 l 3 l 4 l5 FIGURE 2.-HindIII, EcoRI and BclI digests of 15 accessions of H. uulgare ssp. spontaneum polymorphic fragments are noted. A, Hind111 digests hybridized with PHvcP3 cpDNA clone from barley. Lanes 5.6.9-1 1, 14, 15; 6.1-kbHindII1 fragment. Lanes 1-4, 7, 8, 12, 13; 5.6- and 0.5-kb Hind111 fragments. B, EcoRI

from petunia. Lane 1 lambda HindlII- SmaI size marker. Lanes 6,7, IO-12,15, 16; 2.0-kb EcoRI fragment. Lanes 2-5, 8, 9, 13, 14; 1.1 and 0.9-kb EcoRI frag- ments. C, BclI digest hybridized with P8 cpDNA clone from petunia. Lane 1 IambdaHindIIISmaI size marker. Lanes

ment. Lanes 6, 10, 12, 13; 7.7- and 2.4-

B (Kb) Ip, -

6.7-

4.3- digests hybridized with P6 cpDNA clone

2.3- - I --- 2.0-

2-4, 7-9, 11, 14-16; 9.0-kb BdI frag- - --

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6

C

8.6- 9.4 8.3 6.7

4.3

2.3 2.0

kb BclI fragments.

1 2 3 4 5 6 7 8 9 10 11 12 13 1 4 1 5 1 6

1110 D. B. Neale et al.

TABLE 2

Frequencies of multirestriction site genotypes for 19 populations of H. vulgare ssp. spontaneurn in Israel and Iran

Frequency of multirestric- tion site genotypes

Population No. Location "_ " + + + -

1 Mt. Hermon 1 .ooo 2 Rosh Pinna 1 .ooo 3 Gadot 0.875 0.125

5 Mt. Meron 0.875 0.125 6 Damon 0.095 0.524 0.381

13 Eiyzariya 0.067 0.866 0.067 14 Judean Foothills 0.200 0.800 15 Jerusalem Mt. 0.143 0.071 0.786 16 Region 12 0.154 0.462 0.384 17 Bor Mashash 1 .ooo 19 Bet Shean 1.000 20 Mehola 1 .ooo

24 Atlit 1 .ooo 25 Ashqelon 0.600 0.400 28 Ilam-Meheran 0.929 0.07 1 29 Shahabad-Ilam-B 0.889 0.111

4 Tabigha 1 .ooo

12 Bar Giyyora 1 .ooo

21 Jericho 1 .ooo

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Communicating editor: M. T . CLEGC