_ 4 :fi:%;‘: "‘1: gig 22.: 1‘ ‘1‘" ,4; .‘L 5 v! .' . 2.“qu .' {5.5; #r 130% .3225" fifiw§ if”: . 1 I. ‘l‘d‘ (“n f .3". f}. u». 2 .i‘“ " a :‘W , .09. 5. -. '. II .,.. 0‘?“qu U,‘ i g: Wf‘a , .. \. ._.-- ‘3'1‘2-3'1 _ ’51:: . ; 'KV 5W 6:}. ,.. ‘5." (g; ‘ . .... ‘-."“ alga: 2f? 3...“ . A a \w ‘ ”’ééflj .5. . .51 .5‘: 353:? - - (Cm-.1, 1" « ""‘:‘§‘L.;{?:;§b fl '6, , x a}: .1 , . J . 3A?“ 35 4! I", -..:’./r.y|; n.‘ .. I _, ' , ’:,~ .;--... z... “ ‘91:... M»:- ' . ' " 33:4. ' j .' .. 2.»! 6% 5? 0‘! LIBRARY we... 5.... um... Michigan State‘ Willing/1211!; {ll/1'IllIfill/lIll/lll/l’l.’f/Ilfill University — 3 00609 9224 This is to certify that the thesis entitled Studies of Chloroplast Transmission and F Hybrid Variegation in the Common Bean Phaseolus Vulgaris presented by Pung—Choo Lee has been accepted towards fulfillment of the requirements for Master's Botany and Plant Pathologj degree in Wen/90,75) . Sea/5,9,9 Major professor Date i/‘J//'->.. . k.)- PP 999 OI. O.- >-»‘O”'“-h-b 1.16 Figure 2. Restriction fragment patterns of chNAs from two bean cultivars and from F1 progeny of reciprocal crosses be- tween them. (A) Ethidium bromide stained gel. HaeIII restriction fragments of chNAs from Swedish Brown (SB), Swan Valley (SV), and from the F1 hybrids of reciprocal crosses (SB x SV or SV x SB), were separated on a 5% polyacrylamide gel. Lambda DNAs were purchased from BRL and digested with PstI and HindIII, respectively. (B) Autoradiograph. chNAs were electrophoretically transferred to a Nytran membrane. Southern hybridization was performed using a 32P-labe1ed probe containing a spinach chloroplast petA (cytochrome f) gene and flanking sequences clone (pBF7) which was generously provided by W. Bottomley, Canberra, Australia. 44 E V Figure 3. Restriction fragment patterns of chNAs isolated from two other bean cultivars and from F1 progeny of reciprocal crosses between them. (A) Ethidium bromide stained gel. HaeIII restriction fragments of chNAs from Mecosta (Me), Tuscola (Tu), and from the F1 hybrids of reciprocal crosses (Me x Tu) or Tu x Me), were separated on a 5% polyacrylamide gel. Lambda DNAs were digested with PstI and HindIII, respectively. (B) Autoradiograph. The chNAs shown in A were electrophoretically transferred to a Nytran membrane. The blot was hybridized with a 32P—labeled probe as in Figure 2. 45 ursfiéfifin o..- d.- fi-.. sagn‘----.¢ Figure 4. Autoradiograph showing the limits of an under— represented DNA type. chNAs isolated from the SV and SB cultivars were digested with HaeIII, were mixed in the ratios indicated, and then were separated on a 5% polyacrylamide gel. Lane 1 and lane 8 contain only the chNA of Swan Valley (SV) and Swedish Brown (SB), respectively. (A) Ethidium bromide stained gel. (B) Autoradiograph. The chNAs shown in A were electrophoretically transferred to a Nytran membrane. Probed with the same DNA as in Fig. 2. 46 DISCUSSION In a recent report, occasional rare transmission of paternal plastids was detected in Aficotjaaa (Medgyesy et a1. 1986) and Epilobjum (Schmitz and Kowallik 1986), which were previously thought to exhibit strict maternal plastid inheritance. The rare paternal transmission was discovered in two different ways. Using Epilobium, Schmitz and Kowallik (1986) pooled leaves and extracted chNA from a large number of progeny (N=200), while Medgyesy et a1. (1986) devised a strong selection regime for Nicotjana by first inducing the growth of callus from the progeny and testing for transmis- sion of a paternal antibiotic resistance marker. Thus, Medgyesy et al. showed that 0.07% of the progeny of an interspecific cross and 2.5% of the progeny of an intraspecific cross had received the plastome marker from the male parent. From the Epilobjum studies, it is not clear if the chNA from the paternal parent is present in each plant in small quantities or in only one plant or a few plants among the offspring as the predominant type of chNA. But, in either case, some level of biparental transmission is evident in both Epilobium and Nicotiana. Conceivably, if other "maternal" plants were tested for transmission of plastids from the male parent in one of these ways, more ex- amples of occasional paternal contribution at a low 47 frequency may be discovered. In the experiments described here, similar molecular techniques were used to reinvestigate the manner of plastid inheritance in crosses of Phaseolus vulgaris. Surprisingly, this plant species which has been reported as having sig— nificant levels of biparental non-Mendelian inheritance (Parker 1934; Kirk and Tilney-Bassett 1978), appears to have predominantly maternal transmission of the RFLPs analyzed. In contrast to the reports on intraspecific variation in chNA in other plants including Nficotiana debneyj (Scowcroft 1979), Zea mays (Timothy et a1. 1979), Pelargoniun zonale bort (Metzlaff et a]. 1981), Lycopersjcon peruvianum (Palmer and Zamir 1982), .Hordeum spontaneum (Clegg et a1. 1984), Pisum sativum and P. bumjle (Palmer et a]. 1985), Lupinus texensis (Banks and Birky 1985), and Oenothera johansen (Johnson et a]. 1988), much less heterogeneity of chNA was detected in the comparisons, and polyacrylamide gel electrophoresis was necessary in order to visualize RFLP differences. The identification of an underrepresented chNA type by the visualization of fragment patterns stained with ethidium bromide has been reported to be possible if this DNA is present at 0.3% or more in an overloaded agarose gel 48 (Schiller et a]. 1982). Southern hybridization expands these limits of detection to as little as 0.02% (Schmitz and Kowallik 1986), thus allowing the detection of a low level of paternal transmission of chNA. However, in this study, even using Southern hybridization, the DNA heterogeneity can be detected with confidence only when the second DNA com- posed l% or more of the chNA mixture (Fig. 4B). Electrophoretic blotting of DNA fragments from polyacrylamide gels to a membrane support was less efficient than the standard method of capillary transfer developed by Southern (1975) and used for agarose gels, particularly for DNA fragments of high molecular weight. Thus, the reduced amount of DNA on the membranes may be one factor accounting for the limitation in visualization. of fragments of low abundance in the mixing experiment. To increase the autoradiographic signal, nucleotides with a high specific activity label ( > 3000 Ci/m mol) were used and X-ray film was exposed for varying lengths of time. Nonetheless, resolution was limited to the ability to detect a second DNA type at the 1% level. Another contributing factor may be that the variable bands lie very close to each other in' the gel. A strong signal from one band makes it difficult to see a very weak signal from the other band. 49 The results using this technique indicated that the chNA RFLPs of the F1 progeny are identical to those of the maternal parent (Fig. 2A; 3A), and that the pooled F1 progeny did not possess chNA from the male parent at the 1% level of detection (Fig. 2B; 3B). Thus, these results are consistent with Whatley’s ultrastructural observations with Phaseolus vulgarjs that plastids are absent from the pollen generative cells, which would result in purely maternal transmission of been plastids. The results do not support the interpretation of Parker’s genetic data that sig- nificant levels of biparental transmission of chloroplasts may occur in reciprocal crosses of Phaseolus vulgarjs. It is unfortunate that the bean cultivars examined are different from the cultivar that Parker used, although all cultivated forms of the common bean are classified as belong to the same species. In order to find RFLPs, it was necessary to use two different cultivars. Actually, Parker’s F1 results showing predominantly maternal inheritance are consistent with those reported here. However, as discussed earlier, other aspects of Parker’s genetic data including the ap- parent absence of sorting-out of the mutant trait and the recovery of sectored F2 plants from totally green F1 plants are inconsistent with the normal inheritance and segrega- tional behavior of plastome mutations. 50 In many ways, Parker’s data are more reminiscent of nuclear plastome mutator genes which have been characterized in several plant species (Redei and Plurad 1973; Potrykus 1973; Epp 1973). These recessive nuclear mutator genes are known to greatly increase the frequency of plastome muta- tions when homozygous. Conceivably, Parker’s original variegated plant could have been homozygous for a plastome mutator allele. The F1 progeny would be heterozygous; therefore the plastome mutator would be inactive, yet the plastome mutations induced in the original plant would be inherited in a typical non-Mendelian fashion. When the F1 plants were self-pollinated, a fourth of the F2 progeny would be homozygous for the plastome mutator allele, and new mutations would occur, giving rise to variegated plants even in those progeny derived from green F1 plants. Another explanation for Parker’s results could be that the F2 variegation represents a separate genetic phenomenon, different from the initial non-Mendelian variegation ob- served in the Pencil Pad Black Wax sport. At least three groups (Zaumeyer 1938, 1942; Wade 1941; Coyne 1966, 1967, 1969) have reported observing hybrid variegation following wide crosses between normal green plants of small-seeded and large~seeded cultivars of the common bean PWaseolus vulgaris. Their work and the results described in chapter 4 51 indicate that a nuclear mutator system is activated in a subset of the F2 progeny (about 6-9% show variegation). Parker’s results differ in that they were not derived from crosses of different bean cultivars since the variegated plant was a sport arising in a commercial field. However, it is possible that the sport arose from contamination of a seed lot by another cultivar. To determine the nuclear or non-Mendelian nature of the variegation observed in the F2 progeny, reciprocal crosses would have been necessary, but were not included in Parker’s study. In the experiments described here, analysis of chNA RFLPs clearly indicated that the transmission of chloroplasts in sexual crosses between several cultivars of the common bean Phaseolus vulgarjs is predominantly by uniparental maternal inheritance. Nonetheless, the pos- sibility of occasional rare transmission of paternal plastids, as has been shown for NHcotjana and Epilobium, cannot be ruled out. CHAPTER 4 F2 HYBRID VARIEGATION OF THE COMMON BEAN PHMSEOLUS VUIGMRIS 52 53 INTRODUCTION In interspecific crosses of Oenotbera (Schoetz and Reiche 1957, Stubbe 1959) and Pelargonium (Metzlaff et a]. 1982), hybrid variegation frequently arises in the F1 gen- eration when plastids are transmitted from both parents in crosses (non-Mendelian biparental inheritance), with one plastid being unable to develop properly in the hybrid nuclear background. In what was initially thought to be an analogous situation, many of the F2 progeny (6-9%; Table 2) in wide crosses of the common bean Pfiaseolus vulgarjs dis— play cell lineage patterns of leaf variegation. This inves- tigation was designed to specifically test whether biparen- tal inheritance and subsequent plastome-genome incom- patibility could be directly responsible for the F2 hybrid variegation in the common bean Phaseolus vulgaris. Reciprocal crosses were made between large seeded and small seeded bean cultivars to produce F1 progeny. These plants were self-crossed to produce the F2 progeny. Using a polyacrylamide gel system, I was able to identify RFLPs in chNA from the parental plant lines. Subsequently, chNA was isolated from the parental plant lines and total cellular DNA was isolated from the F2 variegated progeny. Restriction 54 Table 2. Frequency of variegated F2 progeny from crosses of cultivars of Phaseolus vulgarjs. Plant designations are following: Mecosta (Me), Tuscola (Tu), Swedish Brown (SB), Swan Valley (SV). Most of these data were obtained using F2 seed provided by Dr. James Kelly (JK) of the Michigan State University Bean Breeding Program. F2 data from one of my crosses (PC). 5 Germi- Freq. g: nation 9: Variegated Variegated Cross Source F; Progeny Seedlings Total £ Seedlings MexTu JK 84.7 10 111 9.0% TuxMe JK 34.3 3 49 6.1% SVxSB JK 74.9 8 158 5.1% SBxSV JK 82.5 11(5)* 137 8.0% SBxSV PC 98.7 24 296 8.1% * ()indicates totally chlorotic seedlings. digests were analyzed by gel electrophoresis to determine whether F2 variegated plants contain a mixture of chNA from the two original parents. In addition, I have also performed reciprocal crosses between the variegated plants and the parental bean lines to determine whether non-Mendelian fac- tors are involved in the hybrid variegation of the common bean Phaselous vulgarjs. RESULTS chNA composition of F2 variegated plants Analysis of chNA from the F2 variegated plants them— 55 selves should allow us to determine whether biparental in- heritence is directly responsible for the F2 hybrid variegation. Since the chlorotic sectors composed 25 - 50% of the plant tissue in the variegated leaves sampled, if two different plastid types from the original parents were responsible for the F2 variegation, we would expect to be able to see bands from both types of chNA in the variegated progeny in ethidium bromide stained gel. However, Figure 5A indicates that the chNA restrction patterns of F2 progeny were identical to those of original maternal parents. These were confirmed by Southern hybridization using a heterologous probe (pBF7) which had homology to one of the restriction fragment polymorphisms. (Fig. SB). Inheritance of the variegated trait If the F2 leaf variegation occurs because non-Mendelian mutations are induced in the bean plants, when reciprocal crosses are made using the variegated F2 progeny and the green parental line, differences in the frequencies of variegated offspring will be apparent. Table 3 shows data from reciprocal back crosses of one set of F2 variegated plants to the parental lines. All progeny of the back cross populations are green and there are no differences in reciprocal crosses. 56 saxsm anxsa ggxam amass A as E am 5 as an fi an fl an Figure 5. chNA composition of F2 variegated plants. (A) Ethidium bromide stained gel. HaeIII restriction fragments of chNAs from Swedish Brown (SB) and Swan Valley (SV), and total cellular DNAs from the F2 variegated plants of reciprocal crosses (SB x SV or SV x SB ), were separated on a 5% polyacrylamide gel. The lambda DNA was digested with HindIII. (B) Autoradiograph. The DNAs shown in A were electrophoretically transferred to a Nytran membrane. The blot was hybridized with a 32P-labeled probe as in Figure 2. 57 Table 3. Inheritance of variegation. Variegated F2 progeny from the cross Swedish Brown x Swan Valley (line 5 of Table 2) were used in reciprocal crosses with either Swedish Brown (SB) or Swan Valley (SV) cultivars. Variegated plants were scored after the primary leaves were fully expanded (about 2 weeks after planting). Female Male 5 Germi- i figggg i Variegated Total £ Parent Parent nation Seedlings Seedling Seedlings Var. F2 SB 96.4% 53 0 53 SB Var. F2 . 100% 44 0 44 Var. F2 SV 87.5% 77 0 77 SV Var. F2} 100% _ 14 0 14 DISCUSSION Many of the F2 progeny in wide crosses of the common bean Phaseolus vulgaris display cell lineage patterns of leaf variegation (Zaumeyer 1938, 1942;, Wade 1941; Coyne 1966, 1967, 1969). A similar hybrid variegation occurring in F1 plants called "plastome-genome incompatibility" is well characterized in Oenotbera (Stubbe 1959) and Pelargonium (Metzlaff et a]. 1982), which have high levels of biparental non-Mendelian transmission of plastids. In Phaseolus vulgarjs, the evidence regarding non-Mendelian inheritance 58 was conflicting: the ultrastructural data of Whatley (1982) indicate that the pollen generative cell may completely lack plastids, which would result in purely maternal transmission of been plastids. However, an earlier report on the trans— mission of a variegated trait in reciprocal crosses was sug— gestive of significant levels of biparental non-Mendelian inheritance (Parker 1934; Kirk and Tilney—Bassett 1978). In other experiments (chapter 3 in this thesis), the analyses of chNA restriction fragment length polymorphisms have _shown that the transmission of chloroplasts in sexual crosses between cultivars of the common bean Phaseolus vul- garis is predominantly by uniparental maternal inheritance. This result supports Whatley’s ultrastructural observations of the generative cell in the common bean. The analysis of chNA from the F2 variegated plants themselves indicates that their chNA restriction patterns are identical to those of the original maternal parents (Fig. 5). Thus the F2 variegated progeny of the common bean do not contain a mixture of chNA from the two original parents at significant levels. Therefore, F2 hybrid variegation of the common bean Phaseolus vulgaris does not appear to be due to biparental inheritance of chloroplasts and subsequent plastome~genome incompatibility. 59 To determine the nuclear or non-Mendelian nature of the variegation observed in the F2 progeny, the manner of in— heritance of the variegated trait in reciprocal crosses was determined. Since all of the progeny of reciprocal crosses were green (Table 3), the pale green sectors of the variegated plants are probably due to the expression of homozygous recessive alleles, which are returned to the heterozygous state through back crossing. The absence of differences in reciprocal crosses allows us to rule out the involvement of non-Mendelian factors in the hybrid variega— tion of the common bean. These genetic data permit the conclusion that F2 hybrid variegation in the common bean is not due to chloroplast mutation, but may be due to mutation(s) of nuclear gene whose product is necessary for chlorophyll biosynthesis or chloroplast development. Zaumeyer (1938, 1942), Wade (1941), and Coyne (1966, 1967, 1969) showed in genetic studies that the F2 variegation was controlled by nuclear genes, but each reported a different pattern of inheritance. Based on their analyses of F3 progeny, Zaumeyer (1938, 1942) and Coyne (1966, 1967) concluded that the character was due to two recessive genes, and Wade (1941) reported that this was determined by any one of three reces- sive genes. However, Coyne’s resu1t(l969) was interpreted to 60 indicate that four loci were involved (Kirk and Tilney- Bassett 1978). By utilizing pooled F1 and F2 bean seed, I have been able to test several theories for F2 hybrid variegation in the common bean Pfiaseolus vulgarjs. However, a precise pedigree is necessary for the accurate assessment of the genetic basis of the variegation. For example, a pedigree would allow one to determine whether variegated progeny ob- served in the F2 generation are derived from the same or different F1 plants. This will give a more informative pic- ture of the timing and nature of the mutational events than one can gain from the pooled F2 data. Furthermore, com- plementation analysis is necessary to determine whether the F2 hybrid variegation in the common bean is due to mutation of a single locus in all plants or whether it represents multiple complementation groups. To determine this, F2 variegated plants derived from crosses of different F1 plants should be crossed with each other. This will allow us to determine if multiple loci are affected, or if a single gene is the target of mutation or altered expression. 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Ligation was fol- lowed by transformation, and then colonies were selected which displayed an Ampr Tets phenotype from among the many transformants. Recombinant plasmids were isolated from selected colonies using the mini prep method. They were digested with a restriction enzyme and were run on the agarose gel to compare the size of recombinant plasmid. This method is specially useful for the comparison of blunt end ligation (i.e. HaeIII and NruI), because one cannot recut a vector DNA and an insert DNA in that case. The size of vec- tor DNA was about 4.4 kb and that of insert DNA was about 3.0 or 1.1 kb. Most recombinant plasmids analyzed in these experiments were smaller than the original vector DNA. Even though transformants showed an Ampr Tets phenotype, they did 70 not contain insert DNAs. They may show an Ampr Tets phenotype, not because target DNA was inserted in Tetr region in the plasmid, but probably because they were par- tially deleted in the Tetr gene region in the plasmid. Similar results were encountered using the method described above. Since the variable fragments were usually very faint in the polyacrylamide gels, I could not see the DNA bands in the agarose mini gel after eluting them from the polyacrylamide gel using various methods. Therefore, the only possible explanation for these results is that the variable fragments might be degraded or lost during the eluting procedure. Since I was unable to directly clone the variable frag- ments from bean cultivars, shotgun cloning was undertaken using the entire chNA of Swedish Brown digested with HaeIII and inserted into the NruI site of pBR322 vector. Using the colony hybridization methods, 22 signals out of 500 colonies were positive. Even though I could not directly clone vari- able chNA fragments, the same fragments could be labeled and used as probes for colony hybridization. Recombinant plasmids were isolated from 22 colonies, and were analyzed on the gel as described previously. A 7.4 kb recombinant plasmid was chosen as a candidate clone, since the size of vector DNA was 4.4 kb and one of the fragments of interest 71 was 3.0 kb. The 7.4 kb recombinant plasmid was amplified, labeled, and hybridized to 3 chNA filter of parental lines. However, the result of Southern analysis did not show any polymorphisms, although the recombinant plasmid contained a slightly smaller DNA fragment than the variable fragment in the parental lines. Various clones from the available Oenotbera and spinach chloroplast genomic library were used as heterologous probes for Southern analysis to find RFLPs in the parental lines. The variable fragment which was eluted from the HaeIII 3.0 fragment of been chNA was hybridized to filters of Oenotbera chloroplast genomic library. Seventeen positive signals from the Southern analysis were detected. Plasmids were isolated from the Oenotbera chloroplast genomic library which showed homology with the HaeIII 3.0 kb fragment of Swedish Brown. They were labeled and used as heterologous probes to the bean chNAs of parental lines. All the results of Southern analysis did not show any RFLPs. Similar experi- ments were conducted with the BamHI 1.1 kb fragment from Swan Valley, with similar results. Only a spinach chloroplast petA (cytochrome f) gene clone recognized one of RFLPs of interest in the bean cultivars. After the variable fragments were eluted from the 72 polyacrylamide gel using the method by electrophoresis onto the Whatman 3MM paper, they were able to be labeled and used both as a homologous probe and as a heterologous probe. However, they could not be cloned into pBR322 vector. These results may be interpreted to indicate that the variable fragments of bean chNA in the polyacrylamide gel may have lost their discrete restriction site ends during the elution procedures for unknown reasons. "11111111111111“