major deletion in F results in clingstone melting flesh (CMF), and critical deletions in ... market industry uses mostly FMF and CMF fruit, while the peach canning ...
EndopolygalacturoflaSe Genotypic Variation in Prunus C.P. Peace', A. Callahan 2 , E.A. Ogundiwin', D. Potter', T.M. Gradziel', F.A. Bliss' and C.H. Crisosto' 'Department of Plant Sciences, University of California, Davis. CA, USA 2USDA .-ARS Appalachian Fruit Research Station, Kearneysville, WV, USA Keywords: allelic diversity, Melting tiesh, Freestone, fruit softening, peach Abstract Softening is an integral component of ripening for most fruit. The cell wallmetabolizing enzyme, endopolygalactu ronase (endoPG), is involved in fruit softening of many species. In peach and nectarine (Priiniis persica), maximum expression of endoPG coincides with the climacteric peak and period of rapid softening known as the "melting phase" that is characteristic of most fresh-market cultivars. We previously identified endoPG as the gene controlling the major fruit firmness and texture traits of illeltingflesli (M) and Freestone (F) in peach. There appear to be at least two copies of the gene at the same locus, and we hypothesize that one copy controls M and another F, where the three main phenotypes observed are due to major deletions in one or both gene copies. Smaller changes in endoPG are suspected to result in quantitative differences in fruit firmness and texture. Other Prunus fruit crops, such as apricot, plum, and cherry, also have traits resembling M and F, and given the close synteny within Pr,,nus, endoPG may play an important role in fruit quality in each of these crops. Therefore, diverse germplasm was surveyed with PCR tests for endoPG to confirm the role in Primus fruit softening, and to identify further alleles in cultivated and related wild species that may convey useful new quality characteristics. A microsatellite associated with endoPG was the most polymorphic region of the gene using PCR amplification, allowing a detailed examination of endoPG genotypic diversity in Prunus. Sequence analysis of other parts of the gene is complementing our insights into the diversity and function of this important fruit quality gene. INTRODUCTION Softening is an integral component of ripening for most fruit. The fruit of peach and nectarine (Prunus persica [L.] Batsch) are highly perishable, with a shelf life of only a few days at room temperature. This perishability is primarily due to rapid softening after harvest, which results from a coordinated interplay between various enzymes (Trainotti et al., 2003 Brummell et al., 2004a). An all-too-common practice used to extend shelf life is to harvest earlier, providing the market with fruit that are firmer but not fully developed. Such fruit are often of inferior quality, with poorer flavor and less intense skin and flesh. Another means of delaying the softening process is to place fruit in cold storage, although many stone fruit cultivars develop symptoms known collectively as internal breakdown (Crisosto et at., 1999). Understanding the genetic control of the fruit softening process has the potential to redress these fruit quality problems. Endopolygalacturonase (endoPG) is a cell wall-metabolizing enzyme known to be involved in fruit softening of many plants (Hadfield and Bennett, 1999). In peach and nectarine, maximum expression of endoPG coincides with the climacteric peak and period of rapid softening known as the "melting phase" that is characteristic of most fresh-market cultivars (Lester et al., 1994). EndoPG was identified as the gene controlling the major fruit firmness and texture traits of Melting flesh (M) and Freestone (F) (Peace et al., 2005a). The enzyme is also involved in the development of mealiness, an important symptom of internal breakdown (Brummell et al., 2004b), but mealiness does not occur in clingstone non-melting varieties presumably due to the lack of a functional endoPG enzyme (Peace et al., 2005b). There appear to be at least two copies of the endoPG gene at the F-M locus (Callahan et al., 2005), and we hypothesize that one copy controls M and another F, Proc. IS on Biotechnol. Temp. Fruit Crops & Trop. Species Eds. R.E. Litz and R. Scorza Acta Hon. 738. ISHS 2007
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where deletions in one or both copies explain the three major phenotypes observed. In this scheme, two functional copies provide the freestone melting flesh (FMF) phenotype, a major deletion in F results in clingstone melting flesh (CMF), and critical deletions in both gene copies lead to varieties with clingstone non-melting flesh (CNMF). The fresh market industry uses mostly FMF and CMF fruit, while the peach canning industry relies on CNMF fruit. The freestone non-melting flesh combination, which would be useful to both the fresh and canning industries, does not appear to exist, but may be possible. Quantitative variants among the major phenotypes are observed - some stable and others strongly influenced by environmental conditions. The role of endoPG in determining such variants is not known. Genes controlling Mendelian traits (where one of the phenotypic extremes typically results from a loss-of-function mutation) are excellent candidates to explain quantitative variation in similar traits, thought to arise from minor sequence differences in such genes (Robertson et al., 1989). Therefore, smaller changes in the endoPG gene(s) may result in quantitative differences in fruit firmness, texture, and stone adhesion amongst peach and nectarine varieties. Other Primus fruit crops, such as apricot (P armeniaca), plum (P domestica and others), and cherry (sweet, P avium: sour, P cerasus), also have traits resembling Mand F, and given the close synteny within Prunus (Dirlewanger et al., 2004), endoPG may play an important role in determining fruit quality in each of these crops. The next logical step for understanding the role of this gene in Przinzis fruit softening is to determine the extent of genotypic variation in Prunus and relate this to phenotypic variation. In the present study, genotypic diversity within each of the qualitative F-Mphenotypes was assessed in available germplasm. MATERIALS AND METHODS The Prunus varieties (cultivars, selections, and accessions) surveyed for the present study consisted of 109 peach and nectarine cultivars and selections, 20 accessions of peach originating as seedlings from Pakistan and accessions of closely related species (P davidiana, P mira, P terganensis, and P kansuensis), 15 hybrid selections of peach x almond (P dulcis) and peach x almond-related species (P webbii, P argentea, and P scoparia), 18 cultivars of sweet cherry (P aviu,n), 6 cultivars of sour cherry (P cerasus), 8 cultivars of apricot (P armeniaca), and I plum accession (P doinestica). Leaf samples for most of this germplasm was obtained from: Kearney Agricultural Center (Parlier, CA, USA), the canning peach and almond breeding programs of UC Davis (Davis, CA, USA), orchards of California peach and nectarine growers (USA), Dr. W.R. Okie (Byron, GA, USA), and the USDA National Clonal Germplasm Repository (Davis, CA, USA). Varieties were chosen to represent diverse origins and wide phenotypic diversity for softening changes during ripening and degree of flesh adhesion to the stone in ripe fruit, where such information was provided by breeders, patents, and Okie (1998). For most varieties, DNA was extracted in duplicate from leaf samples, according to Peace et al. (2005a). Of the remaining varieties, cherry DNA was provided by Dr. Amy lezzoni (MSU, East Lansing, Ml, USA), and apricot DNA was provided by Dr. Maria Luisa Badenes (IVIA, Valencia, Spain). DNA profiles for endoPG were obtained using PCR primers that flank a microsatellite within the 5' non-coding region of a peach EST for endoPG, and performing PCR, polyacrylamide gel electrophoresis, and marker visualization as described by Peace et al. (2005a). Six control samples were included in each gel of 48 lanes, consisting of the cultivars 'Dr. Davis', 'Georgia Belle', 'Suncrest', 'Babcock', 'Mayglo', and 'Tree 308' (a CMF seedling of 'Georgia Belle' with an unknown pollen parent) that represented a wide range of P persica alleles of known sizes. Other than the controls, all samples were run in duplicate in adjacent lanes. Phenotypic information was combined with size of the most intense band to label the endoPG microsatellite alleles of P persica. Alleles were assigned to groups that were based on the three major alleles of known functionality, i.e., F (where F- is associated 640
with the FMF phenotype), f(where ff or ff1 confers a CMF phenotype), or fl (where fIfi L'i\es CNMF fruit), according to variety phenotypes and consensus among the varieties. "here multiple bands occurred, certain ones were designated as representative of the allele and others were considered accessory. For example, Georgia Belle' is known to be heterozygous for an F allele and the null allele (Peace et al., 2005a), and displayed sets of bands at 237, 233, 205, and 201 bp. The 237 and 205 bp bands were observed in most other FMF varieties, while the 233 and 201 bp were also seen in CNMF varieties. While all four sets of bands therefore collectively represented one allele of the F functional group, only the 237 and 205 bands were diagnostic of this allele the allele was labeled as F:n. Another FMF cultivar, Fantasia', had all of these bands and another at 211 bp. The CMF cultivar Mayglo' displayed only the 211 bp allele - this allele was therefore associated with the f functional group and labeled as f 211 . From consensus among varieties of which bands represented distinct alleles, the endoPG genotype (allelic combination) was assigned to each variety. Fantasia', to continue the previous example, was thus recorded as F 205 f211 . As the null allele (complete absence of endoPG genes at the F-M locus, and thus no amplified product) is known to exist in P persica at least (Peace et al., 2005a), varieties exhibiting only one allele could have been homozygous for that allele or heterozygous with the null allele. Certain other allelic combinations were also indistinguishable in appearance, though specific genotypes could be elucidated for many varieties by examining their pedigrees. The null allele was assigned to the fl functional group, and labeled flnu . For other Prunus species, phenotype and inheritance of sets of bands were not necessarily established, so no attempt was made here to assign specific alleles to the observed sets of bands. Two calculations were made to determine allelic frequencies in peach/nectarine. The first assumed Hardy-Weinberg equilibrium amongst all varieties to determine the null allele frequency and elucidate ambiguous cases. The second calculation assumed no crossing between melting and non-melting types (thus all varieties with a single F or f allele were assumed to be homozygous), and random mating conditions within all CNMF types except the Californian canning cultivars (which were assumed to be homozygous for the fl, () , allele as none of these cultivars were homozygous for f111). This latter calculation provided lower estimates of frequency for f1 201 and fi null, and an upper estimate for other alleles. Expected Heterozygosity (He) was calculated for ,the endoPG microsatellite, from both sets of frequency data, using the formula H = 1 where p i is 1th the frequency of the allele. RESULTS AND DISCUSSION
The endoPG microsatellite exhibited a high degree of allelic diversity across the
Prunus germplasm tested (some of which is displayed in Fig. 1). Within peach and
nectarine germplasm (excluding interspecific hybrids), ten alleles were observed, three being associated with the F functional group (labeled as F205, F 207 , and F231 ), five with the f group (f209, f211, f.23 , f227 , and f 29), and two with the fl group (f1 201 and fl .,.11) (Fig. 2). The 109 peach and nectarine varieties were chosen a priori to contain a fairly even mix of the three major phenotypes, and therefore the functional groups were represented in roughly equal proportions. Within each of the groups, two alleles had a high frequency (>10%) (Fig. 2). For the F group, the most common alleles were F 2115 and F 231 . For the f , were group, f211 and f229 were most common. Both fl group alleles, f1 2111 and fI null common, particularly if varieties exhibiting only one visible allele were considered to include heterozygotes with these fl alleles (Fig. 2, "B" marker frequencies). The four rare alleles (F2117, f213 , f227 , and f229) were seen only in combination with common alleles even the two varieties displaying f 213 only were in fact both f213 fl, as determined from pedigree analysis. The Expected Heterozygosity for this microsatellite was 0.83 and 0.84, based on the two calculations of allelic frequencies (random mating across all varieties and restricted random mating, respectively). F group alleles exhibited three to four sets of bands, f alleles only one, and fl alleles none or two, consistent with a gene copy deletion series giving rise to the three observed F-M phenotypes. 641
Of the 55 possible combinations among the ten alleles, only 20 were obser\ed (Table 1). All combinations among the most common alleles, except F 21 fl2 1 , were seen (Table 1). No allelic combinations were observed between any two rare alleles (Table I) such combinations would be very rare if trees carrying all gametes were allowed to combine freely. Even considering that in peach breeding programs, crossing usually occurs only within melting flesh varieties or within non-melting flesh varieties, no allelic combinations were noticeably absent in the germp!asm surveyed. All but one of the absent combinations were expected in less than one variety among the 109 surveyed, the exception being the f21 I tli i combination, for which only 1.8 cases were expected. In peach and nectarine varieties, endoPG genotype was diagnostic of phenotype, providing further evidence for its practical value in marker-assisted selection for Melting flesh and Freestone, where these traits could be determined at the seedling stage in breeding programs (Peace et al.. 2005a). The endoPG genotype also helped clarify the phenotype for several varieties. A few cases of mismatched endoPG genotype and reported phenotype were encountered, but our own (blind) classifications during the 2005 summer season confirmed that phenotypes indeed matched genotypes. For example, in the peach cultivar 'Springcrest' and some of its mutants, 'Maycrest' and 'Spring Lady', an FMF genotype was observed (F2o5F:/F2of12o I /F2 n), although these cultivars are reported as CMF (Okie, 1998). Very early season cultivars such as these can be difficult to categorize phenotypically and often include "semi-freestone" or "semi-clingstone" qualifiers (W.R. Okie, pers. commun.). Our observations classified these fruit as semiclingstone FMF, with the freestone character appearing very late in fruit ripening although some dinginess was still retained. Another potential cause of mismatched endoPG genotype and F-M phenotype is interaction with other loci controlling similar traits. For example, the S/nov hard (Hd) locus masks the Melting /lesh trait under normal ripening conditions (Haji et al., 2005), and from our observations, the Freestone phenotype is also hidden in stony hard fruit. Several stony hard cultivars were analyzed in the present study, including 'Yumyeong', and their endoPG genotype identified their underlying F-M phenotype. The endoPG test is therefore very useful for determining the true F-M phenotype of peach and nectarine varieties, particularly where other factors confuse accurate phenotyping. An interesting question is whether specific alleles or allelic combinations have qualitative or quantitative effects on fruit softening and texture characters beyond the three major F-M phenotypes. In the P persica gerrnplasm surveyed here, no clear relationships were seen for "semi-" characters (Table 1). Further research is underway to compare genotypes with quantitative measures of fruit softening. Besides the ten alleles of known major phenotypic effect in peach and nectarine, numerous other alleles were observed when the endoPG microsatellite was screened on other Prunus germplasm (Fig. I and 2). Species that are closely related to P persica (including almond and other members of subgenus Ami'gdahis) are potential sources of new genetic variation for breeding (Gradziel, 2002, 2003 Dirlewanger et al., 2004). The allelic diversity identified here for this functional gene among such germplasm indicates that breeding for novel softening characteristics in peach and nectarine may be achieved using the endoPG microsatelljte to identify genetic variation at the locus and facilitate its introgression into elite cultivars. EndoPG genotypic variation was also observed within apricot and sour cherry (Fig. I and 2), but not sweet cherry. While the number of varieties surveyed for these other crops was small, results so far are consistent with a general role for endoPG in firmness and flesh adhesion in Prunus fruit. This possibility is being further pursued in our research. ACKNOWLEDGEMENTS We thank Dr. Amy lezzoni, Dr. Marisa Badenes, Dr. W.R. Okie, Clay Weeks, and peach and nectarine growers of California for their generous donations of or invaluable help in obtaining, leaves or DNA extracts of cultivars, breeding selections, and germplasm accessions and Daniel Edge-Garza for DNA extraction. Funding was provided by 642
the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2005-35300-15463. Literature Cited Brumniell, D.A., Dal Cm, V., Crisosto, C.H. and Labavitch, J.M. 2004a. Cell wall metabolism during maturation. ripening and senescence of peach fruit. J. Exp. Bot. 55:2029-2039. Brummell, D.A., Dal Cm, V., Lurie, S., Crisosto, C.H. and Labavitch, J.M. 2004b. Cell wall metabolism during the development of meatiness in cold-stored peach fruit: association of meatiness with arrested disassembly of cell wall pectins. J. Exp. Bot. 55:2041-2052. Callahan, A.M., Scorza, R., Bassett, C., Nickerson, M. and Abeles, F.B. 2004. Deletions in an endopolygalacturoflaSe gene cluster correlate with non-melting flesh texture in peach. Func. Plant Biol. 31:159-168. Crisosto, C.F1., Mitchell, F.G. and Ju. Z. 1999. Susceptibility to chilling injury of peach, -1118. nectarine, and plum cultivars grown in California. HortScience 34:1116 Dirlewanger. E., Graziano, E., Joobeur, T., Garriga-Caldere, F., Cosson, P., Howad, W. and Arus, P. 2004. Comparative mapping and marker-assisted selection in Rosaceae fruit crops. Proc. NatI. Acad. Sci. USA 101:9891-9896. Gradziel, T.M. 2002. Almond species as sources of new genes for peach improvement. Acta Hort. 592:81-88. Gradziel, T.M. 2003. Interspecific hybridizations and subsequent gene introgression within Prunus subgenus Amvgdalus. Acta Hort. 622:249-255. Hadfield, K.A. and Bennett, A.B. 1998. PolygalacturonaseS many genes in search of a function. Plant Physiol. 117:337-343. Haji, T., Yaegaki, H. and Yamaguchi. M. 2005. Inheritance and expresiion of fruit texture melting, non-melting and stony hard in peach. Scientia Hort. 105:241-248. Lester, D.R., Speirs, J., Orr, G. and Brady, C.J. 1994. Peach (Prunus persica) endopolygalacturonase eDNA isolation and mRNA analysis in melting and nonmelting peach cultivars. Plant Physiol. 105:225-23 1. Okie, W.R. 1998. Handbook of peach and nectarine varieties: Performance in the southeastern United States and index of names. U.S. Department of Agriculture, Agriculture Handbook No. 714. Peace, C.P., Crisosto, C.H., Garner, D.T., Dandekar, AM.. Gradziel, T.M. and Bliss, F.A. 2005b. Genetic control of internal breakdown in peach. Acta Hort. (in press). Peace, C.P., Crisosto, C.H. and Gradziel, T.M. 2005a. EndopolygalacturOnaSe a candidate gene for Freestone and MeliingJlesh in peach. Mol. Breed. 16:21-31. Robertson, D.S. 1989. Understanding the relationship between qualitative and quantitative genetics. p.81-87. In: T. Helentjaris and B. Burr (eds.), Development and Application of Molecular Markers to Problems in Plant Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Trainotti, L., Zanin, D. and Casadoro, G. 2003. A cell wall-oriented genomic approach reveals a new and unexpected complexity of the softening in peaches. J. Exp. Bot. 54:1821-1832.
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Tables I! I
Table 1. Summary of allelic combinations for endoPG observed in peach and nectarine. Because some allelic combinations were expected to give the same banding pattern as others, only 43 different banding patterns were possible. Alleles listed in bold were those with the highest observed frequency. Phenotypes: FMF = freestone melting flesh; CMF = clingstone melting flesh; CNMF = clingstone non-melting flesh; sc = semi-clingstone: sf = semi-freestone; parentheses indicate that only some varieties with this allelic combination have the "semi-" phenotype. Frequencies were the proportion of varieties with each allelic combination across the species (n = 109) or within FMF + CMF varieties (n = 80) and CNMF varieties (n = 29). Example cultivars in parentheses are seedlings of a mapping population derived from 'Dr. Davis' and 'Georgia Belle', where the trees listed were the result of outcrossing to unknown pollen parents. Allelic combination Phenotype Frequency Frequency Example cultivar in species in phenotype F 205 F 205/F 205 11 201 /F 205 171 null (sc)FMF 10% 14% Georgia Belle' scFMF 1% 1% 'Crimson Baby' F 205 F 27 F 205 F 231 /F231 f1 201 (sc)FMF 7% 10% 'Redhaven' (sc)FMF 8% 11% 'Fantasia' FMF 1% 1% (Tree 309) F205111 13 (sc)FMF 3% 4% 'Suncrest' F 205 1`229 F 231 F231 /F 231 flnu ll (sc)FMF 6% 9% 'Loring' FMF 1% 1°/0 'Babcock' F231111 09 (sc)FMF 2% 3% 'Earliglo' F 231 f211 FMF 5% 6% 'Elberta' F 231 1`229 CMF 2% 3% Spring Bright' f209 f211 CMF 12% 16% Mayglo' 1721 J2 11 42 11 fl null CMF 1% 1% 'August Gb' f211 f227 (sf)CMF 6% 8% 'Fairlane' f2ii f22 CMF 2% 3% (Tree 308) f23 f213 /f23 f1 null CMF 1% 1% (Tree 379) 23 201 1` 0 (sf)CMF 6% 8% 'Chinese Cling' f229f229/f229f1 null CMF 1% 1% (Tree 114) f229 f1 01 1 201 f1 201 /11 201 f1 0011 CNMF 20% 76% 'Andross' 1 CNMF 6% 24% 'F1a9-20C' f1f1,
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Figures
241239237233 231229225223221219-
1'.
1 2 3 4 5 6 7 8 9 10 11 241 —233 —225
2152132112092072052012OO-99_ 197— 189
Fig. 1. A "stairway" of endoPG microsatellite alleles in Prunus, as observed on a silverstained polyacrylarnide gel following PCR amplification. Cultivars/accessions for each lane are as follows, which are peach unless otherwise noted: I = 'Dr. Davis', 2 = 'Junecrest', 3 = 'Crimson Baby', 4 = 'Babcock', 5 = 'Fairlane', 6 = 'Nemaguard', 7 = 'xWebl' (peach x P webb/i F 1 ), 8 = Valenciano' (apricot), 9 = 'Jubileum' (sour cherry), 10 = 'xArgl' (peach x P argentea), 11 = 'Montmorency' (sour cherry). Band sizes (in bp) are indicated to the left (and some to the right) of the gel picture, with the position of a 200 bp size marker also shown with an arrow.
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Prunus persica 241 F
Other Prunus species
207 F205 F235 f 229 f 227 f 213 f 271 f 209 f1 20 fl75
2 3 4 5 6 7 8 9 10 11 12 13 14 —
237 233 — — — — — 229 — —
—
—
— 217 213 209
— — - —
—
— —
— — — —
05520 —
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—
—
— —
— — — — —
2115 — —
201 - -: — .-
—
— —
—
— — —
197
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—
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P. pe,isico allele frequencies —
89
F207 F205 F231 f 229 f 227 f 213 f211 f 239 f1 2 f1,7
A
14% 11%
B
20% 11% 14% 12%
12% 10% l% 2% 17% 1% 17% 25% 01%
-
-156
3% 21% 1% 17% 10% — — —
-ISO
Fig. 2. Schematic of endoPG microsatellite alleles, as observed on silver-stained large polyacrylamide gels, and their frequencies in 109 peach and nectarine varieties, and endoPG genotypes observed for this marker in some other Prunus species. Band sizes (in bp) are indicated to the left and right of the diagram, and occur in 2 bp intervals due to differences in copy number of a CT repeat. Alleles were scored for the position of their most intense band. Allele frequencies were calculated assuming equal probabilities of any two alleles combining (A), or assuming random mating only within melting flesh types and within CNMF types excluding Californian canning peaches (B). Other Prunus species are as follows: I = peach relative (Prunus sp. Tho Muang' seedling) 2 = peach relative (P kansuensis) 3 peach relative (P davidiana); 4 = peach relative (P niira); 5 = almond (P duIcis) 6 = almond relative (P argentea); 7 = apricot (P armeniaca 'Harcot'); 8 = apricot (P armeniaca 'Goldrich'); 9 = apricot (P armeniaca 'Lito') 10 = apricot (P armeniaca 'Canino'); 11 = plum (P domestica); 12 = sweet cherry (P aviu'fl Bing'); 13 = sour cherry (P cerasus 'Surefire'); 14 = sour cherry (P cerasus 'Erdi Botermo').
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