Israel Journal of Plant Sciences
Vol. 55
2007
pp. 159–169
Induced mutagenesis to augment the natural genetic variability of melon (Cucumis melo L.) Yaakov Tadmor,a,* Nurit Katzir,a Ayala Meir,a Ayelet Yaniv-Yaakov,a Uzi Sa’ar,a Fabian Baumkoler,a Tamar Lavee,a Efraim Lewinsohn,a Ari Schaffer,b and Joseph Burgera a Institute of Plant Sciences, Agricultural Research Organization, Newe Ya’ar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel b Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel (Received 25 February 2008 and in revised form 17 March 2008)
ABSTRACT Induced mutagenesis of agricultural crops creates new variation in genes, including those affecting agriculturally important traits such as plant morphology and fruit quality. Melon (Cucumis melo L.) is a diploid species (2n = 24) with a small genome, estimated as 450 Mb, but with relatively high levels of sequence and fruit shape polymorphism. We treated seeds of ‘Noy Yizre’el’, a ‘Galia’ melon-type parental line, with the chemical mutagen EMS (ethyl methane sulfonate). The resulting M1 plants were self-pollinated to produce about 3,000 M2 families, segregating for the induced mutations that we regard as a “mutation library”. Phenotypic analyses revealed newly induced variation, mostly governed by single recessive mutations, affecting different plant organs, including cotyledon, leaves, flowers, and fruit, at different growth stages, from emergence to mature fruit. Several mutations show phenotypic similarities to mutations found in other plant species. Further studies are required to determine whether the same gene had been mutated in both species, indicating functional homology. This mutation library is an important source for new traits. Some of the identified mutants have already been incorporated into our breeding program. Moreover, the melon mutation library serves as an essential infrastructure for the discovery of important genes, for the annotation of unknown sequences, and for phenotypic and genetic comparison with mutation libraries of other plant species. Keywords: Cucumis melo L., EMS mutagenesis, mutation library, functional homology
INTRODUCTION Breeding of agricultural crops is based on the selection of desired genotypes from a pool of a genetic reservoir that includes the available genetic variation that has traditionally originated from naturally existing variation within the crop under study. Novel variation can be created, by reshuffling of the genetic material through sexual hybridizations, or induced, by mutations or transgenesis. Eighty years ago, Muller (1927), in Drosophila, and Stadler (1928), in barley (Hordeum vul-
gare), showed that ionizing radiation can mutate genes and, as a result, cause the appearance of known mutant phenotypes as well as new phenotypes that were not developed ‘naturally’ during the evolution process. Hermann Joseph Muller received the Nobel Prize in 1946 for the discovery of the production of mutations by Xray irradiation. Both Muller (1927) and Stadler (1928) discussed the potential of induced mutagenesis for *Author to whom correspondence should be addressed. E-mail:
[email protected]
© 2008 Science From Israel / LPPltd., Jerusalem
160 breeding. Stadler was skeptical about the potential of induced mutagenesis to really improve agricultural crops’ performance, due to the recessive nature of most mutations obtained by irradiation and his belief that recessive mutations are deleterious knockouts of a gene (Stadler, 1930). Since then, mutational breeding has been further developed and today provides a major contribution to agricultural crops (Ahloowalia and Maluszynski, 2001; Ahloowalia et al., 2004). Additionally, recently highthroughput reverse genetics methods, such as TILLING (targeting induced local lesions in genomes; Henikoff et al., 2004), have been developed for the screening of large collections of mutants. Such large collections of induced mutations in a common genetic background are termed ‘mutation libraries’ because ideally these collections include mutations in every gene. Melon (Cucumis melo L.) is an important horticultural crop across wide areas of the world. Geographic and cultural differences led to diverse consumption habits reflected in the many fruit types that are commercially available. Sweet types are consumed as a dessert, while the immature fruits of the non-sweet types are eaten raw, pickled, or cooked. Additionally, a special type of melon, fragrant melon or pocket melon, is generally cultivated for ornamental or aromatic uses. These different uses, coupled to different cultivation regimes, revealed large morphological variation in fruit characters such as size, color, and taste (Bates and Robinson, 1995; Burger et al., 2006). This variation has been utilized by modern melon breeders for the development of new cultivars and varieties. This variation is also reflected at the DNA level, and indeed melon’s genetic variation has been utilized to create genetic linkage maps and for the isolation of genes (Baudracco-Arnas and Pitrat, 1996; Wang et al., 1997; Oliver et al., 2000; Danin-Poleg et al., 2002; Périn et al, 2002; Silberstein et al., 2003; Monforte et al., 2004; Gonzalo et al., 2005). Nevertheless, new sources of genetic variation are needed. Modern competitive markets are creating a demand for new original and attractive melon types, and the advantage of near isogenic lines (NIL) for comparative genomic approaches, for gene discovery and cloning, and for the improvement of agricultural products requires rich new sources of genetic polymorphism. Additionally, several melon genomic projects are generating a vast amount of sequence data, but some of it is annotated only by sequence similarities to known genes and, for the most part, no functional role has been assigned to the many genes identified (http://cucurbit.bti.cornell. edu/ and http://www.melogen.upv.es/genomica/melon/). Reverse genetics is an approach to discover the function of a known gene. An ‘induced mutations reverse genetics’ procedure includes the screening of a large mutated population for a mutation in the gene in question. Once a Israel Journal of Plant Sciences 55
2007
mutation is identified, the role of the gene can be deduced based on the mutated altered phenotype. The utilization of reverse genetics is gaining increasing efficiency with the development of high throughput sequence screening technologies such as TILLING (Henikoff et al., 2004) or 454 analyses (Barbazuk et al., 2007). Ethyl methanesulfonate (EMS) is a widely used chemical mutagen. EMS has high mutagenicity and low mortality and its application is simple. The chemical principle of EMS mutagenesis is based on the ability of EMS to alkylate guanine bases, which results in base pairing of the alkylated guanine with a thymine base in the next DNA replication, causing primarily G/C to A/T transitions, which ultimately may result in an amino acid change or transcription termination. A set of mutants in the same genetic background is considered a ‘mutation library’. Optimally, such a library will include mutations in all the genes when at this stage this library is considered as saturated. With EMS one can reach saturation with irreversible mutations in the genome without having to screen a large number of individual mutants. Moreover, EMS mutagenesis not only generates loss-of-function mutants, but can also generate dominant or gain-of-function versions of a gene. The following manuscript describes the development of a mutation library in melon, utilizing EMS as the mutating agent, and the initial phenotyping of M2 families demonstrating the potential of EMS mutagenesis to create new infrastructure for breeding and for gene discovery in melon. MATERIALS and METHODS Plant material Melon is an insect-pollinated annual, and thus hand pollination is required to maintain and ensure self-pollination. We chose to mutate the breeding line ‘Noy Yizre’el’, an andromonoecious powdery mildew-resistant cultivar that serves as the female parent of ‘Galia’. ‘Galia’ is an Israeli-bred melon F1 hybrid that serves today as a prototype (Karchi, 2000). EMS mutagenesis Seeds of ‘Noy Yizre’el’ were treated with EMS (Sigma M-0880) as described by Menda et al. (2004). Seeds were put in water in an Erlenmeyer flask for 12 hours while bubbling air with an aquarium pump. After 12 hours, EMS was added for an additional 12 hours, after which seeds were thoroughly washed with running water and dried on paper towels. After this treatment, seeds remained viable for at least six months. To define the EMS concentration that yields LD15, a dose known to yield a high mutation rate with minimum sterility at the M1 and M2 generations, we treated
161 seeds with water, 0.1% EMS, 0.2% EMS, 0.5% EMS, 1% EMS, 1.5% EMS, 2% EMS, and 3% EMS. Dried treated seeds were planted in HISHTIL trays (8 ´ 16) in a 1:1 peat: vermiculite soil mixture. Plantlets emerged in all EMS concentrations, however, we noticed significant delayed emergence, up to 30 days after sowing, positively correlated with EMS concentration. We chose the day of 100% emergence in the water treatment as our scoring date and selected 1% EMS, since it had 85% emergence on this date. Once EMS concentration was defined we treated 5,000 seeds with 1% EMS to create M1 seeds. M1 plants were planted in the greenhouse and were self-pollinated to create M2 families, which were phenotyped (Fig. 1).
Fig. 1. Phenotyping at the plantlet stage. M2 family NYM 1035 segregates for albinism.
Phenotyping Phenotyping has been done by visual inspection of the plants under two conditions: plantlets were phenotyped in HISHTIL trays in the greenhouse while mature plants and fruits were phenotyped in the field. Sixteen plantlets and 12 mature plants from each M2 family were phenotyped in the greenhouse and in the field, respectively. Each M2 family was documented by receiving a unique ID to which digital pictures and seeds location were linked. Plantlet phenotyping included the following major descriptors and sub-descriptors: (1) cotyledon color —white, pale yellow, pale green, green (wild type), dark green; (2) cotyledon size—very small, small, normal (wild type), large; (3) cotyledon number—none, split one, two (wild type), one split and one normal, three; (4) plantlet size: very small, dwarf, normal (wild type), giant. Whole plant phenotyping included the following major descriptors and sub-descriptors: (1) leaf color —white, pale yellow, pale green, green (wild type), dark green; (2) leaf size—very small, small, normal (wild type), large; (3) leaf structure—wiry, narrow, normal (wild type), asymmetrical; (4) plant size—very small, dwarf, normal (wild type), large; (5) plant structure—lying, bushy; (6) stem structure—curly (yes/no), branching (high, normal, low), internodes (short, normal, long); (7) flower—size (normal, large, small), color (yellow, white, pale yellow, dark yellow), petal number, male:female ratio (normal, high, low), male sterility, female sterility; (8) fruit—color (rind of young fruit and rind and flesh of mature fruit), size (normal, small, large), shape (globular, elliptical, elongate), rind netting
Fig. 2. Dominant rind mutation appeared at the M1 stage. Fruit of plant number 2601 (right) as compared to Noy Yizre’el wildtype fruit. This altered phenotype segregates as a dominant trait in NYM 2601 M2 family. Tadmor et al. / Induced mutations in Cucumis melo
162 (few (wild type), no netting, heavily netted). All mutants were phenotyped under similar conditions, i.e., heated greenhouse for plantlets and field conditions for mature plants. The families’ ID, pictures and phenotyping data by the different descriptors, creates a ‘phenotypic catalog’ (Menda et al., 2004)
vided into to five major groups: plantlet phenotype, leaf, plant structure, flower, and fruit. Plantlet mutations
Mutagenizing ‘Noy Yizre’el’ seeds with 1% EMS gave rise to visible altered phenotypes in less than 0.1% of the plants analyzed at the M1 stage. Most of the altered phenotypes were not inherited by their offspring, however, we could detect several dominant mutations, for example, M1 plant no. 2601 had an altered phenotype of the fruit rind (Fig. 2). This phenotype segregated as a single dominant gene trait in the Noy Yizre’el mutant M2 family 2601 (NYM 2601). Still, most of the M1 plants exhibited a wild-type phenotype corroborating again the recessive nature of most EMS induced mutations. In general, we did not succeed in fertilizing ~5% of the M1 plants, most probably because of sterility. Close to 10% of the families segregated for a mutated phenotype at least in one of the descriptors we have used. Menda et al. (2004) reported ~50% EMS mutated families out of the 6,000 M2 families phenotyped. In each descriptor we phenotyped mutants, however, the plantlet mutants group was significantly larger due to the larger number of families screened, the relatively large number of plantlet descriptors, and due to the detailed phenotyping of small morphological changes at this stage. Thus far, we have created 3,000 M2 families based on the line ‘Noy Yizre’el. 1,800 families were screened for visual mutations at the plantlet level, while 600 families were phenotyped at mature stage, i.e., including mature fruit phenotype. The descriptors we have used are di-
Mutations at the plantlet stage started to show up at emergence, where several families exhibited uneven germination. This phenomenon needs further study since it could be due to lethal homozygous mutations or caused by seed quality. After emergence we could detect pigmentation, plantlet size, and cotyledon structure mutations. The most abundant mutation at this stage has been dwarfism (Fig. 3); 40 families out of the 1,800 families phenotyped at this stage (~2%) had two to eight dwarf plantlets, indicating that there are many loci in the melon genome that may cause dwarfism if mutated. One family, NYM (Noy Yizre’el Mutant family) 776, segregated for a ‘giant’ phenotype with one recessive gene. A quarter of the plantlets in this family emerged the same day as their other family members, however, they grew faster and taller as plantlets and as mature plants (Fig. 4). We could stabilize the homozygous recessive ‘giant’ phenotype and the wild-type phenotype. Comparison of two sets of isolines indicated significant height difference between the giant families and the wild-type and Noy Yizre’el samples (Fig. 5). Sixteen families segregated for albinism (Fig. 1). Three types of albinism were observed: (i) albino plantlets with no pigments at all; (ii) those that did not accumulate chlorophyll, however, accumulated some amount of carotenoids, and thus had yellow color; and (iii) those that had yellow-green color due to the accumulation of small amounts of chlorophyll and carotenoids (Fig. 6). Additional mutations observed at the plantlet level included, but were not limited to, ‘multi cotyledons’, ‘fused cotyledons’, elongated leaves, round leaves, indented leaves, and shoot meristemless (Fig. 7).
Fig. 3. Dwarfism segregating in M2 family NYM 482.
Fig. 4. NYM 776 exhibited a ‘giant’ phenotype at both plantlet (left) or mature plants (right) stages.
RESULTS and DISCUSSION
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Fig. 5. Means comparison of plantlets heights (n > 12). NYM 776-1, 2, 3, and 4 are lines stabilized by self pollination to present the ‘giant’ (1 and 2) and wild-type (3 and 4) phenotypes. Levels not connected by the same letter are significantly different (p < 0.01).
Fig. 6. Albinism in the mutation library (from left to right): yellow-green albino, M2 family NYM 82; yellow albino, NYM 15; and white albino, NYM 380.
Leaf mutations
Plant structure mutations
Leaf mutations are grouped into mutations affecting mature plant leaf size, leaf color, and leaf morphology. In the Noy Yizre’el mutation library, families carrying mutations with altered leaf size are limited to reduced leaf size, such as has been observed in NYM 112 (Fig. 8). We could not detect any family segregating for dark leaves, however, several families segregated for light-green or yellowish leaves, including NYM 152. Families segregating for altered leaf morphology included those which segregated for ‘curly’ edges (NYM 65; Fig. 8), ‘clover-type’ leaves (NYM 770; Fig. 8), or round leaves (NYM 1140; Fig. 8).
Several families segregated for mutations affecting plant structure. The most impressive mutated phenotype was NYM 86, which had a DUMPY phenotype, including short internodes and altered leaves phenotype (Fig. 9). NYM 1200 also had short internodes, which resulted in dwarf plantlets, and short mature plants with normal size and shape leaves (Fig. 9). Other examples of plant structure mutations include those affecting plant sizes, such as the ‘giants’ (Fig. 4) or dwarfs. Interestingly, many mutations affecting plant structure affected also the reproduction of the plant due to flower sterilities. Tadmor et al. / Induced mutations in Cucumis melo
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Fig. 7. Additional plantlet mutations: (A) NYM 553 exhibited multi-cotyledons phenotype; (B) NYM 905 exhibited fused cotyledons phenotype; (C) NYM 137 exhibited round leaves; (D) NYM 587 exhibited elongated leaves; (E) NYM 951 segregated for indented leaves; and (F) NYM 567 segregated for shoot meristemless phenotype.
Fig. 8. Mutations altering leaf morphology, size, or color: (A) NYM 65 segregated for ‘curly’ leaves; (B) NYM 112 segregated for small leaves; (C) NYM 152 wild-type (left) vs. mutated yellowish leaves (D); (E) NYM 770 segregated for clover-type leaf; (F) NYM 1140 segregated for round leaves. Israel Journal of Plant Sciences 55
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Fig. 9. Mutations affecting plant morphology. (A) to (F) NYM 86 segregated for dwarfism at the plantlet stage and developed DUMPY plants as a mature plant. (G) to (I) NYM 1200 also segregated for dwarfism at the plantlet stage due to short internodes. The mutated plants developed short internode mature plants with normal size and shape of leaves.
Fig. 10. Mutations affecting flower morphology. (A) Flowers of NYM 137 (left) are smaller than the wild-type (right) and the mutant female flower does not develop normal petals. (B) NYM 537 develops different flowers. (C) to (E) NYM 543 carries a recessive mutation causing reduced pigmentation in a pleiotropic manner; pale green-yellowish plantlet (D), pale greenyellowish plant (E), and white light yellow flowers (C). Tadmor et al. / Induced mutations in Cucumis melo
166 Flower mutations Mutations affecting flower morphology, flower color, and flower size were noted. Since fruit set of analyzed plants occurred in the field where pollination is assisted by bees, we could not score male sterility. Examples of
Fig. 11. Mutations altering fruit shape and color. (A) (from left to right) Fruits of wild-type Noy Yizre’el, miniature fruit of NYM 137, and elongated fruit of NYM 72. (B) Young fruits of mutated NYM 152 compared to wild type. (C) Mature fruit of wild-type NYM 152 (left) compared to mutated fruit (right). (D) Young fruit of NYM 1265. (E) Mature fruits (rind and flesh) of mutated NYM 1265 fruits (right) as compared to wild-type fruit from the same family. (F) Rind of mutated fruit of NYM 1058 as compared to wild-type rind. (G) Flesh of mutated fruit of NYM 1058 as compared to wild-type flesh. Israel Journal of Plant Sciences 55
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flower mutations include NYM 137 with small male and female flowers, while the female flower also has undeveloped petals; NYM 537, which developed the altered shape of a flower; and NYM 543, which suffers from yellowish phenotype and has pale yellow flowers (Fig. 10).
167 Fruit mutations The most interesting mutations, from the breeder’s point of view, are those affecting the fruit phenotype without adverse effects on other plant traits. Examples of fruit mutations include those affecting fruit shape, such as NYM 137 with miniature fruit and NYM 72 with elongated fruit as compared to the round wild-type fruit (Fig 11A). NYM 152 developed a light green-yellow immature fruit and NYM 1265 developed a light green immature fruit (Fig. 11B and 11D, respectively). NYM 152 developed fruits with no chlorophyll and suffered from yellow young leaves while NYM 1265’s fruits had a significantly reduced amount of chlorophyll when the fruit matured (Fig. 11C and 11E, respectively), but had normal chlorophyll levels in leaves. NYM 1058 had a normal dark green immature fruit, however, when matured the mutated fruit had no chlorophyll, neither in its rind (Fig. 11F) nor in its flesh (Fig. 11G). These mutants demonstrate the ability of induced mutagenesis to alter fruit characteristics. Moreover, some of the fruit mutations were specific to the fruit without any adverse effect on other plant performances.
synthesis (Fig. 9); and several families segregate for the appearance of three cotyledons, similar to the twn1 mutation in Arabidopsis (Vernon et al., 2001) or the ‘poly cotyledons’ of tomato (Madishetty et al., 2006). We haven’t yet determined whether orthologous genes were mutated to cause these phenotypic similarities. If so, such genes share functional homology. Summary and conclusions Domestication has brought about the production of elite crop varieties, but has caused a narrow genetic basis to cultivated crops. The genetic reservoir of a defined cultivated species was partially accumulated during the evolution of its ancestors, and the other part has been accumulated during domestication or while cultivated. Exploiting advanced genetic tools to trace back traits that were left behind in exotic germplasm, including ancestral species and primitive varieties, has brought great advances in our understanding of plant morphol-
Pleiotropism Utilizing defined descriptors for phenotyping as early as at emergence and, finally, with mature plants, enables the estimation of the pleiotropic effects of each mutation. Approximately half of the M2 families that were scored to have an altered phenotype in one of the descriptors had at least another descriptor indicating the pleiotropic effect of the visual mutations. This number is very similar to the estimated number of pleiotropic mutations induced in tomato either by EMS or by fast neutron irradiation (Menda et al., 2004). For example, in the NYM 264 family segregated for dwarfism at the plantlet stage, these dwarf plants developed to dwarf mature plants with small leaves, short internodes, and complete sterility (Fig. 12). Phenotypic similarities to known mutants in other species Interestingly, many of the melon mutant families identified displayed a phenotype that has been reported and in several cases has been studied at the gene level in other species. For example, NYM 1358 segregates for a ‘glabrous’ phenotype similar to that described in Arabidopsis (Fig. 13; Szymanski et al., 2000); NYM 86 segregates for short stature, reduced axillary branching, and altered leaf morphology, which has phenotypic similarities with the tomato ‘dumpy’ (dpy) mutant (Koka et al., 2000) and the Arabidopsis BAS1 mutant (Neff et al., 1999), both deficient in the brassinosteroids bio-
Fig. 12. NYM 264 M2 family segregated for dwarf plants at the plantlet stage (A). This mutation caused the development of miniature mature plants (B) with small yellowish leaves and sterile flowers indicating the pleiotropic effect of this mutation. Tadmor et al. / Induced mutations in Cucumis melo
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Fig. 13. NYM 1358 segregated for trichomeless (‘glabrous’) phenotype (from left to right and from top to bottom): wild-type vs. mutated meristems, leaves, stems, male flowers, petals, and female flowers
ogy and potential agricultural performance. Indeed, the extended utilization of such genetic material promoted improvement of crop compositional quality and field performance (Zamir, 2001; Fernie et al., 2006). This long process of increasing variation within a crop can be sped up utilizing induced mutagenesis coupled to modern screening tools. Advanced modern approaches utilize mutated plants not only for breeding purposes but also as a mutation library representing the induced variation of a species in a near isogenic background. Such mutation libraries can be utilized for both forward and reverse genetics studies as well as for the identification of new genes. Additionally, we have screened our mutation library only by visual phenotyping under field or greenhouse conditions. A more detailed screening of the mutation library will likely bring about the identification of mutants with altered metabolomic patterns as well as resistance to abiotic stress, pests, and pathogens and better fit to intensive agricultural conditions, adaptations to new locations, or carrying nutritional or environmental assets. Based on our study and on other similar studies, we can summarize the advantages of an EMS mutation library. It enables the analysis of single gene mutations Israel Journal of Plant Sciences 55
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in a NIL background and it serves as infrastructure for the identification of developmental genes, for the identification of mutations in a gene of interest by TILLING or by direct sequencing analyses, for the identification of a beneficial new variation in an elite background, and for comparative biology studies. Since the “mutation library” described here was generated on an important melon breeding line, ‘Noy Yizre’el’, some of the mutations characterized have immediate economical potential as they can be readily introduced in elite agricultural varieties. ACKNOWLEDGMENTS Contribution number 105/2008 from the Israeli Agricultural Research Organization. This work has been part of the activity of the ARO ‘Center for the improvement of fruit quality in the Cucurbit family’. We wish to acknowledge all the granting agents who support our activities, especially those who partially supported the creation of our melon mutation library. These include the ARO Director’s Fund, grants from the Chief Scientists of the Israeli Ministries of Science and of Agriculture, MAGNET-BioTov of the Israeli Ministry of Industry,
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