region (SAGHAI MAROOF et al. 1984; R . W. ALLARD, unpublished results). Twenty distinct rDNA spacer- length variants (slvs) have been identified in barley;.
Copyright 0 1990 by the Genetics Society of America
Genetic and Molecular Organizationof Ribosomal DNA (rDNA) Variantsin Wild and Cultivated Barley R. W. Allard,' M. A. Saghai Maroof,* Qifa ZhangS andR. A. Jorgensen4 Department of Genetics, University of Calfornia, Davis, Calfornia 95616 Manuscript received April 2, 1990 Accepted for publication July 25, 1990
ABSTRACT Twenty rDNA spacer-length variants (slvs) have been identified in barley. These slvs form a ladder in whicheach variant (with one exception) differs from its immediate neighbors by a 115-bp subrepeat. The 20 slvs are organized in two families, one forming an eight-step ladder (slvs 100-107) in the nucleolus organizer region (NOR) of chromosome 7 and the other a 12-step ladder (slvs 108a-118) in the NOR of chromosome 6. The eight shorter slvs (100-107) segregate and serve as markers of eight alleles of Mendelian locusR m 2 and the 12longer slvs (108a-118) segregate and serve as markers of 12 alleles of Mendelian locus R r n l . Most barley plants (90%) are homozygous for two alleles, including one from each the 100-107 and the 108a-118 series. Two types of departures from this typical pattern of molecular and genetic organization were identified, one featuringcompound alleles marked by two slvs of Rrnl or of R m 2 , and the other featuringpresence in Rrnl of alleles normally found in Rrn2, and vice versa. The individual and joint effects on adaptedness of the rDNA alleles are discussed. It was concluded that selection acting on specific genotypes plays a major role in molding the strikingly different allelic and genotypic frequency distributions seen in populations of wild and cultivated barley from different ecogeographical regions.
E
UKARYOTIC ribosomal RNA genes are organized in large tandemly repeated arrays at one or a few chromosome locations (reviews inLONGand DAWID1980; JORCENSEN and CLUSTER1988; CLECG 1989; DVORAK1989). Each repeat includes a single rDNA transcription unit that codes for the 18S, 5.8s and 26s complex of ribosomal RNA of plant species, as well as an intergenic spacer (IGS) region that separates the transcription units of adjacent repeats. Each IGS region contains an array of tandemly repeated sequences, referred toas subrepeats, that are typically 100-200 base pairs(bp) in length in plants.The length of these subrepeats varies within most species by no more than a few base pairs and the subrepeats are identical or closelysimilarin sequence (e.g. APPELS and DVORAK 1982; ARNHEIM et ul. 1982). However, the number of subrepeats within the rDNA repeats is often quite variable within species. This variation in the number of tandem copies alters the length of the IGS region, leading to differences that can be detected by restriction enzyme and Southern blot techniques. In barley(Hordeum vulgure),restriction enzyme SstI cleaves each of the several thousand rDNA repeats
' Present address: Department of Agronomy and Range Science, University of California, Davis, California 95616. 'Present address: Department of Crop and Soil Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg,Virginia 2406 1. Presentaddress: Department of Agronomy, Huazhong Agricultural University, Wuhan, People's Republic of China. ' Present address: DNA Plant Technology Corporation, 6701 San Pablo Avenue, Oakland, California 94608. Genetics 126: 743-751 (November, 1990)
twice, once on each side of the IGS region, yielding two fragments of DNA from each repeat unit. One of these SstI fragments is invariant (-3880 bp in length) whereas the other varies in length as a result of the differences in the number of subrepeats in the IGS region (SAGHAIMAROOFet al. 1984; R . W. ALLARD, unpublished results). Twenty distinct rDNA spacerlength variants (slvs) have been identified in barley; the shortest is designated spacer-length variant 100 (slv 100) and the longest slv 118. Each variant differs from the immediately adjacent variants by 115 bp so that the series forms a complete ladder from 4625 to 6695 bp in length. One exceptional variant, slv 108a, is intermediate in length between slv 107 and slv 108. Extensive surveys of IGS variability (SAGHAIMAROOF et al. 1984; SAGHAIMAROOF, ALLARDand ZHANG 1990; R . W. ALLARD, unpublished results) have shown that approximately 90% of individual barley plants havetwo different slvs and that suchplants breed true for these two variants (barley is 99% selffertilized). Furthermore, one of the two slvs present is nearly always one of the eight with the smaller number of subrepeats (slvs 100-107) and the other is nearly always one of the 12 with the larger number of subrepeats (slvs 108-1 18). SAGHAIMAROOFet al. (1984) showed from analysis of segregation ratios in an F2 family that slvs 108 and 1 12 are inherited as codominant alleles of a Mendelian locus, which they designated R r n l , and that slvs 104 and 107 are inherited as codominant alleles of a second unlinked locus,
R. W. Allard et al.
744
which they designated R m 2 . They also showed, by wheat-barley addition line analysis, that Rrnl and R m 2 are located in chromosomes 6 and 7, respectively, in association with the nucleolus organizer regions (NORs) of these chromosomes (APPELSet al. 1980).However, occasional barley plants are truebreeding for only one slv, or for three slvs; also, in rare cases, both of the slvs of plants with only two slvs are members of eitherthe 100-107 series, or the 108a- 118 series. In this paper we report the results of Mendelian analyses of inheritance patterns for 16 among the 20 slvs that have been observed in the barley species. The results establish that the 20 slvsof barley are organized in two families, one of which forms a regularly complete ladder of variants (slvs 100-107) associated with the N O R of chromosome 7 and with Mendelian locus R m 2 , whereas the otherfamily forms a ladderof variants (slvs 108a-118)associated with the N O R of chromosome 6 and with Mendelian locus R r n l . The slvs all differ by 1 15 bpwith the exception of slv 108a, which is 73 bp longer than slv 107 of R m 2 and 42 bp shorter than slv 108 of R m l . T h e results also indicate that crossing over and recombination occasionally produce compoundalleles marked by two slvs and that crossing over and recombination mayalsooccasionally lead to presence in Rrnl of alleles normally found in Rrn2, and vice versa. We show further that alleles 107 (Rrn2) and 112 ( R r n l ) behave as widely adapted “wild-type” alleles in wild barley (Hordeum vulgare ssp. spontaneum, henceforth abbreviated H.S.), whereas allele 104 replaces allele 107 as the predominant allele of R m 2 in most populations of cultivated barley ( H . vulgare ssp. vulgare, abbreviated H.V.). MATERIALS AND METHODS
Genetic materials: The cultivated barley (H.V.) parents of the families studied were, with four exceptions, named cultivars. The exceptions were: (1) an unnamed accession, CI 278, obtained from the Cereal Investigations Collection of the U.S. Department of Agriculture, and (2) three singleplant selections from Composite Cross 11, an experimental population of cultivated barley (see SAGHAIMAROOFet al. 1984). Most of the wild barley (H.S.) parents of this study were obtained from the World Barley Collection of the U.S. Department of Agriculture; these parents are designated by Plant Introduction (PI) numbers. Other H . S . plants were from collections made by R. W. ALLARD in Syria, Lebanon and Jordan. Among the 2 1 families studied 11 were from hand-pollinated crosses; each of these 11 families was obtained by sowing seedsfrom a single self-pollinatedFl plant. Selfing was enforced by enclosing spikes in glassine bags. Each of the 10 remaining families was obtained by enforced selfing of single plants of H.S.; these plants were selected for progeny testing because Southern blot signal intensities indicated they wereheterozygous for specific alleles of either Rrnl , R m 2 , or both Rrnl and R m 2 (signal intensities of alleles present in heterozygous condition are generally less
TABLE 1 Tests of “goodnessof fit” to 1:1:1:1:2:2:2:2:4 and 1:Zl ratios of Ft families segregating for four slvs Family No.
1
2
3
Parental phenotype
Atlas 68 X PI 262628 (112,104)(108,106) Atlas 68 X Algerian (1 12,104) X (1 10,107)
N”
6.13 67 0.25 0.13 71
PI296926 X59 CI278 (112,104) (109,106)
4 Atlas 68 X H.S. 5.79(108a,107) (112,104)
X*
valuesb
3.16 0.72 0.99 14.77 4.52 3.57
10.55 53 0.32
5‘
a
H.S. 20.90*** (114,112,107,103)
40 10.20** 7.60*
Number of individuals assayed per family.
x* values listed in order of the two-locus segregation (top), the Rrnl segregation (middle) and the Rm2 segregation (bottom). ‘ Family 5 was obtained by self pollination of a plant of H.S.with phenotype 114, 112, 107, 103. *, **, *** Probability < 0.05, 0.01 and 0.001, respectively.
dense than those of alleles present in homozygous condition). DNA preparation and the detection and designation of slvs: Total cellular DNA was isolated and rDNA slvs were detected as described in SAGHAIMAROOF et al. (1984). Spacer-length variants were also designated as by SACHAI MAROOFet al. (1984) except that 100 was added to each designation. Thus, forexample, slv 1 andslv 15, the shortest and longest variants known in 1984, became slvs 101 and 1 15,respectively. Representative slvs are illustrated in Figures 2 and 3 of SAGHAI MAROOFet al. (1984). RESULTS
Families segregating for four slvs: Five families of this type were studied(Table 1). T h e parents of families 1-4 were typical in that each parent had two slvs, including one from the series 100- 107 and one from the series 108a-118. The F1 hybrid plants all had fourslvs; Southern blot signal intensity was lighter in each of the F, plants than in their homozygous parents.Ninephenotypes were observed in the F:! generation of each family, as reported for family 1 (Atlas 68 X PI 282628) in Table 2. T h e nine phenotypes included two (phenotypes l and 2) with the two parental slvs, two (phenotypes 3 and 4) representing the two recombinant homozygous phenotypes, four (phenotypes 5-8) with three slvs each, representing the mixed single-locus homozygous-heterozygous phenotypes, and one (phenotype 9) with four slvs corresponding to the doubly heterozygous FI phenotype. The two-locus segregation in each of the families is expected to produce the nine phenotypes (genotypes)
Organization Genetic
of rDNA
745
TABLE 2 Analysis of Mendelian inheritancein the Fs of Atlas 68 (112,104) X PI 262628 (108,106) Observed phenotype
Inferred genotype
Rm2 1 2 3 4
6 5 2
112,104 108,106 112,106 108,104
5 112,108,104 6 112,106,104 7 108,106,104 112,108,106 8 9
Rrnl 112,112 108,108 112,112 108,108 112,108 112,112 108,108 5 112,108
112,108,106,104 112,108
4.19104,104 4.19106,106 4.19106,106 104,104
Expected frequency
Expected no.
Observed no.
1/16 1/16 1/16 1/16
4.19
5
8.38 8.38
5 9
8.38
11
104,104 106,104 8.38 106,104 106,106
2116 2116 2116 2116
19 106,104 16.75
4116
Two-locus segregation: x&] = 6.13,0.50 < P C 0.70. R m l segregation: x$, 0.25, 0.80 C P C 0.90. R m 2 segregation: - 0.13, P > 0.95.
I
in proportions of 1:1:1:1:2:2:2:2:4 (Table 2). Tests of “goodness of fit” of observed to expected numbers (Tables1 and 2) gave values C 15.51, P > 0.05, forthefour families derivedfromhand-pollinated crosses. Single-locus segregation ratios forslvs 1 12 us. 108, 112 us. 110, 112 us. 109, and 112 us. 108a, and for slvs 106 us. 104, 107 us. 104, 106 us. 104 and 107 us. 104, in families 1, 2, 3 and 4, respectively, all gave satisfactory fits to 1:2:1 ratios (x& < 5.99, P > 0.05). All of the nine expected phenotypes (genotypes)also appeared in family 5 (Table 1). However, the fit to the expected 1: 1:1:1:2:2:2:2:4 two-locus segregation ratio was poor in family 5 (Tables 1 and 3) due to excesses in genotypic classes 4, 7 and 8, in which slvs 112 and/or 107were homozygous, and todeficiencies in genotypic classes 1, 2, 3,5 and in 6, which genotypic classes 1 14and/or 103 were homozygous. Standardized residuals (SR = [obs. - exp.]/.\/exp.) show that the excess observed in genotype 4 (1 12,112,107,107) was statistically significant. The fits to the expected 1 :2: 1 ratio forthe Rrnl and Rrn2 segregations were also poor (Table 3) primarily due to excesses in the homozygous 112,112 and 107,107 classes and deficiencies in the homozygous 114,114and103,103 classes. Standardized residuals showed that the excesses in genotypic classes 1 12,112 and 107,107were statistically significant. The results reported in Tables 1, 2 and 3 thus support the conclusion of SAGHAI MAROOFet al. (1984) that slvs 1 12and 108 and slvs 107 and 104 are alleles of Rrnl and R m 2 , respectively. They show, in addition, that slvs 1 14, 110, 109 and 108a segregate as alleles of Rrnl and that slvs 106 and 103segregate as alleles of Rrn2. Thus direct segregation tests in families 1-5 establish that slvs 1 14,112, 1 10,109, 108 and108a belongto the NOR gene family of chromosome 6 ( R r n l ) and that slvs 107,106,104and103 belong totheNORgene family of chromosome 7 (Rrn2). The results also
suggest that, when homozygous, therDNA alleles marked by slvs 1 14 and 103 have adverse effects on reproductive capacity and/or viability (ALLARD 1988) under the greenhouse conditions in which the parents and segregating families of this experiment were grown. Families segregating for two slvs: Among the 10 families of this type studied (Table 4) one family (No. 6) was descended from a hand-pollinatedF1 hybrid of Pamela Blue (109,107) X PI 296897 (108,107); the F1 hybridparent of this family was phenotypically 109,108,107. The othernine families (families 7-15) were descended from single plants of H.S. with three slvs. The plants from which families 7-1 5 were descended were chosen for study because their Southern blot signal intensities were light for two among their three slvs, which suggested that each of these plants might be heterozygous for the slvs of either Rrnl or Rrn2. Families 7, 8, 1 1, 14 and 15 are of particular interest because they included five slvs that had not previously been tested for allelism, namely, slvs 118, 117,105,101and 100. Three phenotypic classes appeared in each segregating family and in each case these phenotypes included the two homozygotes and the heterozygotefor the locus thathad been presumed, on the basis of signal intensity, to be heterozygous in the plant from which the family was descended. The segregation patterns reported in Table 4 also show that slvs 118 and 117 segregate as alleles of Rrnl and that slvs 105, 101 and 100 segregate as alleles of Rrn2. This increases the total number of slvs (and rDNA alleles) that have been assigned by direct segregation tests to eight forRrnl and seven for Rrn2, leaving five of the 20 slvs (and rDNAalleles) untested. However, the five untested slv markers (1 16, 115, 1 13, 1 1 1and 102) are all bracketed in the complete ladder by assigned rDNA alleles. It therefore seems likely that slvs 1 16, 1 15, 1 13 and 1 1 1 markalleles of
R. W. Allard et al.
746
TABLE 3 Analysis of Mendelian inheritance in the selfed progeny of a plant 0fH.S. with phenotype 114,112,107,103 (family 5) Observed phenotype
114,107 103,103
Inferred genotype
1 2 2 112,103 112,112 114,1143 114,103 4 112,107 112,112 5 114,112,103 114,1122/16 107,103 6 114,114 114,107,103 1 7 112,107,103 107,103112,112 8 114,112,107 107,107 114,112 9 114,112,107,103 4/16 107,103 114,112
114 1 2 114,112 3 112
107,107 2.50 2.50 103,103
1/16 1/16
107,107 103,103 5.00
1/16 211 6 211 6 2116
x f ~=l 20.90, P < 0.001 One-Locus Segregations 4 114 112 1/4 x& = 10.20, 0.001> P > 0.01 107,107 114 107,103 112 103,103 1/4 X& = 7.60, 0.02 < P < 0.05
114,114 114,l 12 112,112
1 107
2 107,103 3
Expected frequency
103
e Standardized residuals, SR = observed - expected/-; 0.0 1, and 0.00 1, respectively.
.
L
Tests of "goodness of fit" to1:2:1 ratios for families segregating for two slvs
6 7 8 9 10 11 12 13 14
15
Parental phenotypes
N b
Pamela Blue X PI 296897 53 (109,'107) (108,'107) 118,c108~107 28 117,c108:107 6.96* 19 114:108:107 21 112,c108:107 34 112,107,'105' 3.60 20 112,107,'104' 20 110,107,c104' 20 112,104,c101' 26 112,107:100' 21
xb l
3.97 7.91** 6.35* 3.35 0.30 3.28 6.23* 11.86***
* Families 7-15 were obtained by self pollination of H.S. plants with the phenotypes indicated. * Number of individuals assayed in each family. ' Segregating alleles. *, **, *** Significant at probability levels 0.05, 0.02, and 0.01, respectively.
Rrnl and that slv 102 marks an allele of Rm2. In the next section we will see that a segregation test places slv 113 and theallele it marks in R m l . Tests of goodness of fit to expected 1:2: 1 ratios gave x& values < 5.99, P > 0.05 for five of the 10 families in Table 4.However, P was < 0.05 for families 7, 8, 9, 14 and 15, due primarily to deficiencies in the homozygous classes 1 18,118, 117,117,114,114, 10 1 , l O 1, and 100,100(allele 1 14 was also in significant deficiency in family 5). These results suggest that the alleles marked byslvs 118, 117, 1 14, 10 1 and 100, along with allele 103, have adverse effects on
2.50 2.50 +2.85** 5.00
Observed no.
2 1 7
2
Standardized residual"
-0.32 -0.32 -0.95
9 8 8
-1.34 -1.79 +1.79 +1.34 -0.63
10.0 20.0 10.0
18 18
-1.90 -0.45 +2.53*
10.0 20.0 10.0
17 18 5
+2.2 I * -0.45 -1.58
5.00 5.00 10.0
SR values > 1.96, 2.58 and 3.29 are significant at probability levels 0.05,
TABLE 4
Family"
Expected no.
reproductive capacity and/or viability under greenhouse conditions (ALLARD1988); these alleles have also been found to have adverse effects on survival in MAROOFet d . populations of H.S. and in H.V. (SAGHAI 1984; SAGHAIMAROOF, ALLARD and ZHANG 1990). A hybrid with a true-breeding plant with three slvs: Some of the plants with three slvs that have been found in H.V. and H.S. have, on progeny testing,been found to breed true for oneslv and to segregate for the other two slvs, following the pattern of families 7-15. However, other plants with three slvs have bred true for all three of their slvs. A hand-pollinated F1 hybrid between a true-breeding plantwith phenotype 113,112,107, found in PI 220523, and a true-breedingplant with phenotype 108,107found in PI 296897, had four slvs (1 13, 112, 108 and 107). On selfing, an F1 hybrid plant of this cross produced an F2 family (family 16) of 41 individuals, including 19 with three slvs (1 13, 112 and 107), 10 with two slvs (108 and 107) and 12 with four slvs (113, 112, 108 and 107). Thus, although there was no segregation for R m 2 in this family, segregation involving slv 108 and a compoundslv marked by both slvs 1 12 and1 13 occurred in R m l . It follows that the genotype of the PI 296897 parent was 108,108, 107,107 and that of the PI 220523 parent was 113-112, 113-112, 107,107. The 1 13-1 12 allele presumably resulted from unequal crossing over and recombination that produced a compound allele marked by slvs 113 and 112. A test for goodness of fit to a 1:2: 1 ratio gave x&] = 11.00, 0.001 < P < 0.01. The poor fit is due to a
Organization Genetic
747
of rDNA TABLE 5
Analysis of Mendelian inheritance in the Fn(family 17) of PI 220523 (113,112,107) X PI 296897 (108,105,101) Inferred genotype Observed phenotype
Rrn I
1 113,112,107 108,105,101 2 3 113.1 12,105,101 108,107 4
113-112,113-112 108,108 113-112,113-112 108,108
113,112,107,105,101 5 6 113,112,108,107 113,112,108,105,101 7 8 108,107,105,101
113-112,113-112 113-112,108 113-1 12,108 108,108
9
Rm2
107,107 105-101,105-101 105-101,105-101 107,107 7 8.50 107,105-101 107,107 8.50 8 8.50 105-101,105-101 8.50 107,105-101
113,112,108,107,105,101 107,105-101 113-112,108
Expected frequency
Expected
1/16 31/16 1/16 1/16
4.25 4.25 4.25 4.25
no.
2116 2/16 211 6 211 6 4/16
Observed no.
9
2 8 9 10
17.0
12
Two-locus segregation: = 13.12,O.lO C P C 0.20. Rrnl segregation: x@ 2.57, 0.20 C P C 0.30. Rrn2 segregation: - 6.82, 0.02 P C 0.05.
I
significant excess (SR = +2.73, P < 0.01) in the homozygous 113-1 12, 1 13-1 12class and a near significant deficiency in the heterozygous 1 13-112,lOS class (SR = -1.87). This is consistent with evidence (SAGHAIMAROOF,ALLARDand ZHANG 1990; R. W. ALLARD, unpublished results) that alleles 1 12 and 107 are heavily favored by selection in many different environments, particularly when alleles 1 12 and 107 occurtogether in homozygous condition, andthat allele 113 has adverse effects on survival in both wild and cultivated barley. Although allele 113 has deleterious effects when present alone in R m l , it appears that the presence of allele 112 in the compound 113112 allele corrects for the deficiencies of allele 113 under the greenhouse conditions of this experiment. Allele 1 13 thus appears to have behaved as a more or less neutral “hitchhiker” on allele 1 12in the present experiment. A hybrid between parents with three slvs each: Family 17, obtained by selfing a hand-pollinated FI hybrid between PI 220523 (phenotype 113,112, 107, genotype113-112,113-112,107,107) and atruebreedingplantfrom PI 296892 with phenotype 108,105,lO 1,was studied to determine whether slvs 105 and 101 in this plant also behave as a compound allele marked by two slvs. The F1 hybrid (phenotype 113,112,108,107,105,101) produced an F2 family of 68 individuals (Table 5) inwhich ninephenotypes appeared in a ratio of 1:1: 1:1:2:2:2:2:4 and the three expected phenotypes appeared in each the Rrnl and Rm2 single-locus segregations (Table5).Thus slv 105-101 also segregates as a compound two-slv allele that presumably arose through crossing over and recombination. The standardized residual for the homozygous 113-112,113-112,107,107 classin the twolocus segregation is significant (+2.30*)indicating that this class is in excess, as it was in family 16. The standardized residual forthe homozygous 107,107
class in the R m 2 segregation is also significant (+2.18*) suggesting that the 107,107 homozygote is favored over the 107, 105-101 heterozygous and the 105- 011, 105- 011 homozygous classes. A family segregating for slvs 112 and 107:. A few plants that have only a single slv, alwayseither slv 1 12 or slv 107, have been found in low frequency in H.V. but not in H.S. (SAGHAIMAROOFet al. 1984; SACHAI MAROOF, ALLARDand ZHANG 1990; R. W. ALLARD, unpublished data). Two selections (Fa-10, with phenotype 107and F45-58, with phenotype112)from Composite Cross 11, an experimental population of cultivated barley (SAGHAIMAROOFet al. 1984), were chosen as parents for study of Mendelian inheritance in a hybrid between plants each of which have only a single slv. Southern blot signal intensities for slvs 107 and 1 12 were particularly heavy in selections Fa-10 and F45-58,respectively. The F1 hybrid had two slvs, 107 and 112, both with moderate signal intensities, and in the F2 generation (family 18)3,74and9 individuals were observed with phenotypes 107, 112 and 107, and112, respectively. Fits of observed numbers to expected numbers, based on the assumption that either or both Fa-10 and F45-58 are null for one locus, were poor. If it is assumed that selection Fa-10 is genotypically 107,107, 107,107 and selection F4558 is genotypically 112,112, 112,112 for Rrnl and Rm2, respectively, the expected phenotypic ratio in FZ is 1:14: 1 for individuals with only slv 107, with both slvs 107 and 112, andwith only slv 112, respectively. The fit of observed to expected numbers for the 1:14:1 ratio was good ( X ~ Z = , 3.52, 0.10 > P > 0.20). Furthermore, signal intensity was high in all FP plants that had only slv 107 orslv 112; consequently, the genotypes of such plants were presumably 107,107, 107,107 or 112,112, 112,112, the same as the Fa-10 and F45-58 parents; however,signal intensity varied widely among the plants with both slvs (signal
748
R. W. Allard et al.
intensity varied fromlight to moderately light to moderate for approximately 4/16, 6/16 and 4/16 of the individuals that had both slv 107 and slv 1 12). The above two-locus F2 segregation pattern indicates that slv 107 was present in both Rrnl and R m 2 of Fg10 andthat slv 1 12was present in both loci in F45-58. The length and signal intensity of alleles 1 12 and 107 appeared to be the same whether they are located in Rrnl and Rm2. Segregation in a family from the cross(108, 105, 101) X (107): One family (family 19) of this type, PI 296892 (phenotypically 108,105,lO1) X F8-10 [genotypically 107,107107,107)], was studied to obtain additional evidence as to whetherF8-10 carries slv 107 in both Rrnl and Rm2. The F1 hybrid, as expected, had four slvs (108, 107, 105 and 101). However, six rather than thefive phenotypes expected appeared in the F2 generation as follows: the two parental phenotypes (108,105,101and107),the F1 phenotype (108,107, 105,101), two expected recombinant phenotypes (108,107 and 107,105,lO1) and one individualwith anunexpectedphenotype (107,101). It is unlikely, for two reasons, that the unexpected plant with phenotype 107,101 arose from union of a migrant 101 gamete with a 107 gamete descended from the Fg-10 parent: (1) thespikes of the F1 hybrid parent of family 19 were protected from outcrossing by glassine bags and (2) nobarley plants with allele 101 were present in the greenhouse other than descendents of the F1 parent of family 19. It thus appears that the unexpected plant with phenotype 107,lO1 arose from union of a 107 gamete with a 101 gamete produced by breakup of compound allele 105-10 1. Ignoring the unexpected 107,lO1 individual, the expected phenotypic ratio in FBis 1:3:8:3:1 (108, 105-101):(107, 105101):(108,107,105-101):(108,107):(107). Observed = 9.69, 0.02 < P < numbers were 6:17:33:4:2, 0.05 (uncorrected for continuity) and x:3l = 4.94,0.20 < P < 0.30 (corrected for continuity). F:! plants with the two parental classes (108, 105-10 1 and 107) both bred true when they were progeny tested, whereas F2 plants of the 107,105-101 and 108,107 classessegregated giving 1:2:1 ratios; additionally, when signal intensity was takenintoaccount, two phenotypic expressions were discernable within the two latter classes. The results with families 18 and 19thus allow the following deductions: (1) the single-slv phenotype of F8-lO is genotypically 107,107, 107,107, ie., slv 107 occursin both Rrnl and Rrn2; (2) the length and signal intensity of slv 107 is the same whether it is located in its normal position in R m 2 , or whether it is located in R m l ; (3) the length (1 15 bp) and the signal intensity of allele 107apparentlyremained unalteredduringtheperiod it was associated with allele 105 as a component of a compound allele, and also after it became independent upon breakup of the
compound allele; and (4) infrequent episodes of crossing over and recombination apparently can produce compound alleles and also uncouple the components of compound alleles. Crosses to determine the chromosome location of alleles 105 and 107 in plants phenotypically 105,107: As discussed earlier, nearly all plants found in H.S. and H.V. have been true-breeding for two slvs, one from the series 100-107 ( R m 2 ) and one from the series 108a-118 ( R m l ) . However, occasional individuals with two slvs have phenotypes indicating that both of their slvs are from either the100- 107 or the 108a-1 18 series. Examples are plants with phenotypes In this 102,107,105,107,108,109,and111,112. section we report theresults of Mendelian analyses of two crosses that were studied to determine the chromosome locations of slv 105 andslv 10’7 in two accessions, PI 382604 and an unnumberedaccession from Syria. Both accessions were phenotypically 105,107 and the question asked was whether these accessions were genotypically 107,107,105,105 or 105,105, 107,107 for Rrnl and R m 2 , respectively. Both accessions were crossed with Atlas 68 which is known to be homozygous for slv 1 12 in Rrnl and slv MAROOFet al. 1984; also families 104 in R m 2 (SAGHAI 1, 2 and 4 of Table 1). The F1 hybrid of Atlas 68 with PI 382604 had four slvs (112, 107, 105 and 104). The FB family (family 20) of 42 individuals included nine phenotypes, four with two slvs (1 12,104; 107,105;112,105;and107,104),four phenotypes with three slvs (1 12,105,104; 112,107,104; 112,107,105; and 107,105,104) and one phenotype with four slvs (112,107,105and 104).Observed numbers of the above phenotypes were, respectively, 4, 2, 2, 4, 4, 8, 5, 3 and 10. A goodness of fit test to a 1:1:1:1:2:2:2:‘2:4ratio gave xfsl = 4.49, 0.80 < P < 0.90. Segregation for slvs 112 and 107 and for slvs 105 and 104 both fit 1:2:1 ratios (&I = 0.46, 0.70 < P < 0.80 and xfBl= 3.98, 0.10 < P < 0.20). Thus slv 107 is allelic with slv 1 12 andslv 105 is allelic with slv 104. It is therefore clear that slv10’7is located in Rrnl (chromosome 6 ) in PI 382604 rather than in its usual location in R m 2 (chromosome 7). The F1 hybrid of Atlas 68 with the Syrian accession (phenotype105,107)hadthe same four slvs (1 12, 107, 105 and 104) and the same ninephenotypes appeared in the F:! family of 46 individuals (family 21). Observed numbers were 4 , 4 , 1, 3, 8 , 4 , 7 ,5 and 10, giving goodness of fit to a 1:1:1:1:2:2:2:2:4 ratio of xfsl= 4.78, 0.70 < P < 0.80. However, segregation in this case was for slv 112 and slv 105 (x&]= 2.48 (0.20 < P < 0.30) and for slv 107 and 104 (xfq = 2.13, 0.30 < P < 0.50). Thus, in this accession, slv 107 is located in its usual position in R m 2 whereas slv 105 is located in Rrnl ratherthan in its normal position in Rm2.
Genetic Organization of rDNA
DISCUSSION
Extensive surveys have shown that the greatmajority (-90%) of plants inwild and cultivated barley populations are true breeding for two among the 20 known slvs in the species and that oneof the two slvs of typical plants is from the eight shorter slvs (100107) and the other is from the 12 longer slvs (108a118). Analyses of Mendelian segregation patterns in families 1- 15 of the present study establishedthat: (1) slvs 100- 107 typically segregate and serve asmarkers of eight alleles of Mendelian locus R m 2 and slvs 108a1 18 typically segregate and serve as markers of 12 alleles of Mendelian locus Rrnl and, (2) that the 20 known slvs are organized in two families, one constituted of a regularly complete ladder of eight slvs (100107, 4625-5430 bp in length) associatedwith the NOR of chromosome 7 and the other constituted of a ladder of 12 slvs (108a-118, 5545-6695 bp in length, 42 bp shorter when slv 108a is present), associated with the NOR of chromosome 6 . The subrepeats are all 115 bp in length except for slv 108a, the shortest slv of R r n l . Approximately 6% of barley plants have three slvs and approximately 0.5% have four slvs. About half of such plants are heterozygous at Rrnl or R m 2 and all plants with four slvs are heterozygous at both loci; these plants are presumably natural hybrids, or descendents of recent natural hybrids, between parents that have three or four slvs in total. However, about half of the plants with three slvs breed true for all three slvs. Results of Mendelian analyses of segregation patterns infamilies 16 and 17 established that plants that are true-breeding forall three of their slvs are homozygous for one normal alleleof Rrnl or R m 2 and alsohomozygous for a compound allele marked by twoslvsof the alternative locus. These compound allelespresumably arose as a result of crossing over and recombination such that the two alleles segregate as a unit. Progeny tests are required to distinguish three-banded individuals that are heterozygous at one locus from three-banded individuals that carry compound alleles; hence the genotypes of plants with three slvs cannot be determined simply from inspection of the sl phenotype, as has been the case for all of the other genotypes thus far encountered in barley. The results of Mendelian analyses of segregation patterns in families 18-22 suggested that the length and signalintensityofalleles remains the same whether they are resident in Rrnl or R m 2 . Also the 115 bp integrity of the subrepeats appears to have been maintained strictly in both Rrnl and R m 2 . Interestingly the only exception found thus far is slv 108a, theshortest slv of R m l , which is 42 bp shorter than slv 108. Spacer-length variant 108a, although widespread inthe Middle East, usuallyoccurs in much
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lower frequency than slv 108 which suggests that loss of the 42-bp segment was detrimental to adaptedness. Studies of SI variability in both wild and cultivated barley populations (SAGHAIMAROOF et al. 1990; R. W. ALLARD, unpublished results) have shownthat two alleles, 107 of R m 2 and 1 12 of R m l , are present in frequency ( f ) >> 0.50 in the great majority of populations of wild barley. Sixother alleles (106 and 105 of R m 2 and108a, 108, 109 and 110 of R r n l ) are present inlow to moderate frequencies (f = 0.050.20) in populations of H.S. from ecologically unusual sites, whereas the 10 remaining alleles are infrequent or rare inallwild populations.Allele 112 is also predominant worldwide (f = 0.30) in Rrnl in cultivated barley and allele 107 is the most frequent allele (f > 0.50) in the cultivated barleys of Mediterranean climates (R. W. ALLARD,unpublished data). However, on a worldwide basis, allele104 (f = 0.65) rather than allele 107 (f = 0.30) is the predominant allele of R m 2 in H.V. The 17 remaining alleles are all infrequent or rare in H.V. Thus the frequencies of the more common alleles are often very different in wild and cultivatedbarley; the most striking casesinvolveallele 104, which is infrequent (f = 0.03) in wild barley but predominant (f > 0.50) in most populations of cultivated barley, and the moderately frequent alleles of wild barley (106, 105, 108a, 108,109 and 1 lo), which are absent or rare in cultivated barley.Overall, rDNA variability is much larger inwild than in cultivated barley and the arrays of rDNA genotypes present in the two subspecies are very different. These differences are not surprising because wild barley is found in a much wider range of habitats than the habitats that occur in the arable fields to whichcultivated barley is confined and also because the conditions of life in cultivation are very different from conditions of life in the wild. There have been two studies the of effects of natural selection on alleles marked by the slvs of barley. In a study of a large experimental population (715,000 reproducing adults per generation) of cultivated barley that had been grown without conscious selection over a 53 generation period, SAGHAI MAROOF et al. (1984) found that steady directional changes occurred in allelic and genotypic frequencies at loci Rrnl and R m 2 . By generation 23 allele 112 of R m l , which was present in f = 0.89 in the initial generation of this population, had become fixed (f= 1.OO) and all other alleles of this locus had been eliminated. Alleles 104 and 107 of R m 2 were present in f = 0.61 and 0.32, respectively, (close to worldwide frequencies) in the initial generation of the population; however, by generation 23 all alleles of R m 2 , except 104 and 107, had been eliminated and by generation 53, the frequencies of these two alleles had reversed to 0.30 and 0.70. This was interpreted as indicating that the alleles
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marked by slvs 1 12, 104 and 107 were strongly favored over all other rDNA alleles by natural selection under the environmental conditions (Mediterranean climate) in which this experimental population of H.V. had been grown. In the second study of the effects of natural selection, SAGHAIMAROOF, ALLARDand ZHANG (1990) found that the frequencies of alleles and genotypes differed widely from habitat to habitat in 18 wild barley populations from ecologically diverse sites in Israel and Iran. Using discrete log-linear multivariate techniques they showed that two-locus genotypic frequencieswere correlated with nine factors of the physical environment, indicating that the alleles and genotypes marked by the slvs differ in their adaptive properties. Six among the rDNA alleles that are rare (f < 0.01) in H.S. and/or H.V. were included in the present experiment (alleles 118, 117, 114, 103, 101 and 100). In each case segregation ratios were significantly distorted (Tables 1, 3 and 4), suggesting that each of the rare alleles had adverse effects on reproductive capacity and/or viability under greenhouse conditions. This result is consistent withthe results of other studies of lociaffecting rDNA, allozyme, disease resistance, morphological, and other variants; alleles that have adverse effects on survival under conditions of minimal competition in the greenhouse or in experimental gardens haveconsistentlybeen rare in nature andthey have also been consistentlyat a selective disadvantage innatural populations and in populations of cultivated plants (e.g., CLEGG,KAHLERand ALLARD1978; ALLARD1988; GARCIAet al. 1990). The rare rDNA alleles of barley are found, almost without exception, in genotypes that include allele 1 12,107 or 104 and, most often, the association is in a compound allele in which one component is one of the wild-type alleles; this suggests that the survival of rare alleles in populations depends in part on “hitchhiking” on favored wild-type alleles(SAGHAI MAROOF, ALLARDand ZHANG 1990). The combined Mendelian, molecular and ecogeographic analyses of these studiesprovide insights into the mechanisms of evolutionary change in the rDNA families. The homogeneity ofthe subrepeats in barley indicates that recombination either occurs only rarely within the 115-bp subrepeats and/or that such recombination as occurs withinthe subrepeats leads to structural rearrangements that are eliminated by selection. Regardlessofcauses the length of the subrepeats appears to have been maintained strictly at 1 15 bp and the only readily detectable distinguishing feature among the alleles thus far identified are the differences in the number of subrepeats. The wide differences in the frequencies and ecological preferences of the 20 rDNA alleles marked by the slvs in populations of both wild and cultivated barleyare therefore note-
worthy, especially in view the of erratic and apparently capricious relationships between the number of subrepeats and adaptedness. Spacer-length variant 100 lies in an SstI fragment that is 4625 bp in length; the number of subrepeats in slv 100 is unknown but it cannot be >4625/115 = 40. It is clear, however, that slv 101 has one more subrepeat than slv 100, that slv 102 has one more subrepeat than slv 101, and so forth to slv 1 18which has 18 more subrepeats than slv 100. One possible explanation for the differing adaptive properties of the 20 alleles marked by the slvs is that adaptedness stems solelyor largely from the differing numbers of identical subrepeats that are present in the IGS region. Thus, in cultivated barley, slv 104, with four more subrepeats than slv 100, has the optimum number of subrepeats in the series 100-107, whereas in wild barley slv 107, with seven more subrepeats than slv 100, has the optimum number. In Rrnl allele 112 appears to have the optimum number of subrepeats. It is not readily apparent how differences of one or a few apparently identical subrepeats lead to large differences in adaptedness, e.g., why allele 1 12,which has one more subrepeat than allele 1 1 1 and one fewer than allele 113, is much better adapted than its neighbors in the ladder. One alternative explanation for the differences in adaptedness of the 20 alleles is that the 1 15-bp subrepeats, although identical in length inallslvs,may differ in sequence and that slvs 104, 107 and 1 12 carry sequences that are highly favored in selection. Another and possibly more likely alternative explanation is that the transcription units associated with the different slvs differ in sequenceand that thetranscription units of alleles such as 104, 107 and 1 12 carry sequences that are especially favored by selection. These issues might be resolved by sequencing the slvs and transcription units of a selected set of alleles. There is another type of variation inrDNA, namely continuously varying quantitative differences in copy number per haploid genome, that hasrarelybeen MAROOFand ALmeasured precisely. ZHANG, SAGHAI LARD (1990) found in a study of 10 populations of H.S. that thealleles marked by slvs 107 and 112 have, on the average, about 20% more copies than the other slvs, Although copy number was usually low for the rare and/or infrequent alleles, it was conspicuously variable for the frequentalleles. Thus adaptedness in H.S. appears to be less closely correlated with copy number than with the number of subrepeats in the IGS region. We conclude that natural selection acting directly on the rDNA alleles of Rrnl and R m 2 plays a major role in the development and themaintenance of the observed patterns of molecular and genetic organization of rDNA variability inwild and cultivated barley, i e . , that the quality rather than the quantity of rDNA plays a larger role in determining
Genetic Organization of rDNA
ability to live and reproduce in a given environment. We thank M. T. CLEGG,JAN DVORAK, P. D. CLUSTER,SCOTT HAWLEY and SCOTT WILLIAMS for critical readings of an earlier draft of this manuscript. We also thank K. M. SOLIMAN for carrying out the rDNA assays of families 1, 2, 6, 16 and 19. This research was supported in part by grants to R.W.A. from the U.S. Public Health Service (National Institutes of Health Grant GM-32429), the National Science Foundation (Grant BSR 831 10869), and the University of California (MacDonald Endowment Fund).
LITERATURECITED ALLARD, R. W., 1988 Genetic changes associated with the evolution of adaptedness in cultivated plants and their wild progenitors. J. Hered. 79: 225-238. APPELS, R., and J. DVORAK,1982 The wheat ribosomal DNA spacer region: its structure and variation in populations and among species. Theor. Appl. Genet. 63: 337-348. APPELS, R., W. L. GERLACH, E. S. DENNIS,H. S W I and ~ W. J. PEACOCK, 1980 Molecular and chromosomal organization of DNA sequences coding for ribosomal RNAs in cereals. Chromosoma 78: 293-3 11. ARNHEIM,N., D. TRECO, B. TAYLOR and E. EICHER, 1982 Distribution of the ribosomal gene length variants among mouse chromosomes. Proc. Natl. Acad. Sci. USA 79: 4677-4680. CLEGG,M. T., 1989 Molecular diversity in plant populations, pp. 98- 1 15 in Plant Population Genetics, Breeding, and Genetic ReM. T. CLEGG,A. L. KAHLER sowces, edited by A. H. D. BROWN, and B. S. WEIR.Sinauer, Sunderland, Mass. CLEGG,M. T., A. L. KAHLERand R. W. ALLARD, 1978 Estimation
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of life cycle components of selection in an experimental plant population. Genetics 89: 765-792. DVORAK, J., 1989 The evolution of multigene families: the ribosomal RNA loci of wheat and related species, pp. 83-97in Plant PopulationGenetics,Breeding, and Genetic Resources, edited by A. H. D. BROWN,M. T. CLEGG,A. L. KAHLERand B. S. WEIR.Sinauer, Sunderland, Mass. GARCIA,P., M. I. MORRIS,L. E. SAENZ,R. W. ALLARD, M. PEREZ DE LA VEGAand G. LADIZINSKY, 1990 Genetic diversity and adaptedness in tetraploid Avena barbata and its diploid ancestors, A . hiytdu and A . weistii. Proc. Natl. Acad. Sci. USA(in press). JORGENSEN, R. A., and P. D. CLUSTER, 1988 Modes and tempos in the evolution of nuclear ribosomal DNA: new characters for evolutionary studies and new markers for genetic and population studies. Ann. MO Bot. Card. 75: 1238-1247. LONG,E. O., andI. B. DAWID,1980 Repeated genes in eucaryotes. Annu. Rev. Biochem. 49: 727-764. SAGHAIMAROOF,M. A., R. W. ALLARDand Q. ZHANG, 1990 Genetic diversity and ecogeographical differentiation among ribosomal DNA (rDNA) alleles in wild and cultivated barley. Proc. Natl. Acad. Sci. USA (in press). SAGHAI MAROOF, M. A., K. M. SOLIMAN, R. A. JORGENSEN and R. W. ALLARD,1984 Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81: 8014-8018. ZHANG, Q., M. A. SAGHAIMAROOF and R. W. ALLARD, 1990 Effects on adaptedness of variations in ribosomal DNA copy number in populations of wild barley (Hordeum vulgaw ssp. spontuneum). Proc. Natl. Acad. Sci. USA (in press). Communicating editor: B. S. WEIR
