Development and application of molecular marker ... - Springer Link

Euphytica 77: 71-75,1994. 9 1994Kluwer Academic Publishers. Printed in the Netherlands.

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Development and application of molecular marker linkage maps in woody fruit crops N.F. W e e d e n l, M. H e m m a t 1, D . M . L a w s o n 1, M. L o d h i 1, R . L . B e l l 2, A . G . M a n g a n a r i s 3, B T R e i s c h 1, S.K. B r o w n 1 & G.-N. Ye 1 1 Department of Horticultural Sciences, Cornell University, Geneva, NY, U.S.A.; 2 USDA, ARS, Appalachian Fruit Research Station, Kearneysville, Wg, U.S.A.; 3 National Agricultural Research Foundation, Pomology Institute, 59200 Naoussa, Greece

Key words: Apple, grape, marker assisted selection, pear, RAPDs, Pyrus communis

Summary Extensive linkage maps, consisting primarily of molecular markers, are being developed for apple, pear, and grape varieties. The intrinsically high heterozygosity of outcrossing perennial species is utilized to produce segregating populations directly from a cross between varieties. Nearly complete linkage maps have been generated for the apple varieties 'Rome Beauty' and 'White Angel'. The map for 'Rome Beauty' contains 161 molecular markers, while that for 'White Angel' has 251 markers. Maps for the pear varieties, 'Bartlett' and NY10353, also are being developed. Linkages conserved between the pear and apple genomes have been identified. In grapes, maps for four varieties are available, the most extensive being those for 'Cayuga White' and 'Aurore'. The apple maps have been used to investigate the genetic basis of morphological and physiological characters. A gene controlling the presence of anthocyanins in the skin of the fruit is located on linkage group 3. Genes controlling early bud break, branching habit, and production of root suckers have also been identified and mapped.

Introduction Marker assisted selection has proven to be a useful tool in many annual crops. However, the technique has not been used extensively in woody perennials despite its advantage in eliminating undesirable genotypes before planting in the field. A dearth of genetic markers in perennial fruit crops is one important reason this approach has not been applied. Very few monogenic morphological polymorphisms have been identified in any perennial species. Isozyme loci, although excellent markers (Manganaris & Alston, 1987, 1988; Manganaris, 1989) are not numerous enough to satisfy the demands of the breeder/geneticist. Thus, the development of DNA marker technology has been followed with considerable interest by fruit breeders. A major goal of ours has been the identification of genetic markers in apple, grape, and pear. We believe these markers will benefit the breeding programs directly through marker assisted selection and

indirectly by facilitating a better understanding of some characters being manipulated. We are particularly interested in a "user friendly" approach. That is, a method that is relatively easy to apply, permitting breeders to incorporate the techniques into their own programs without the need to collaborate with a molecular biology laboratory. In the following sections we describe our approach, present genetic tools being developed, and report how these tools have been used to elucidate, at least partially, the genetic basis of specific traits.

The exploitation of highly heterozygous genomes Early speculations that apple was highly heterozygous have been confirmed convincingly by isozyme studies (Chevreau et al., 1985; Weeden & Lamb, 1985, 1987; Manganaris, 1989). This high level of heterozygosity has greatly facilitated genetic studies in apple because

72 each variety becomes, in effect, an FI, and the progeny produced from an open pollinated tree will segregate for all the loci heterozygous in that tree. For genetic studies it is convenient to make a controlled cross between two varieties, thereby specifying and limiting the number of alleles segregating in the following generation. Genetically, this type of arrangement may be referred to as a "double pseudotestcross". The terminology is awkward, but the cross is familiar to all fruit breeders, being the most commonly used cross in most breeding programs. In such crosses, alleles segregate in a 1:1 ratio unless both parents are heterozygous at a locus in which case a 3 : 1, 1 : 2 : 1 or l : 1 : 1 : 1 ratio is expected. As in any test cross, dominant markers are just as valuable as codominant markers because for a 1 : 1 segregation, phenotype equals genotype. This last consideration is particularly important in our approach, for we rely heavily on dominant DNA polymorphisms generated by amplification with arbitrary primers (RAPDs in the terminology of Williams et al., 1990). Although the dominance of RAPD markers does not appear to be a significant disadvantage in the double pseudotestcross format, other factors such as degree of polymorphism, distribution in the genome, or poor reproducibility may limit their value. Early in our mapping experiments we attempted to establish the merits and limitations of RAPDs relative to other commonly used markers. Our studies in apple, grape and pear indicate that a large number of RAPDs can be identified quickly among varieties and conveniently scored (Table 1). On average, three heterozygous markers were identified in each of the varieties examined. As these varieties represent a wide selection of germplasm, the results suggest that 150 to 300 polymorphic markers can be identified in most varieties using relatively few (50 to 100) short primers. If the RAPDs are randomly distributed across the genome, this number of markers should be sufficient to permit the tagging of important characters segregating in any cross. To confirm the genetic basis of these polymorphisms and to examine their distribution on the nuclear genome we have analyzed RAPD segregation patterns in four double pseudotestcross populations, and compared these segregation patterns with those of segregating allozyme and RFLPs. The crosses investigated included one apple ('Rome Beauty' x 'White Angel'), one pear ('Bartlett' x NY10353) and two grape ('Aurore' x 'Cayuga White' and 'Horizon' x I11 547-1) (Table 2). Extensive linkage maps are being developed for each of the varieties. The most thorough-

ly analyzed population is that from 'Rome Beauty' • 'White Angel'. The maps assembled for these two varieties appear to cover the majority of the nuclear genome, for nearly all the RAPD markers, as well as all the segregating isozyme loci and RFLPs, could be included on just over 20 linkage groups (Hemmat et al., 1994). Using several analytical techniques, we established that nearly all the RAPDs scored reflected true genetic polymorphism and that our error rate in scoring RAPD segregation averaged about 3%, ranging from undetectable for the intensely staining fragments to 8% for faint fragments (Weeden et al., 1992). Markers heterozygous in both parents were used to identify homologous linkage groups in the two parents. Fourteen of the linkage groups identified in 'Rome Beauty' could be paired with their counterparts in 'White Angel'. Our results in pear and grape are very similar to those in apple, except that an extensive error analysis has not been performed for these data and that, in pear, the number of linkage groups has yet to coalesce to a figure close to the haploid chromosome number. Although RAPDs have yet to prove useful for identification of linkages conserved between genera, results on isozyme loci indicate that the linkage between Aat-p and Gpi-chas been maintained in both apple and pear.

Use of markers in genetics and breeding of apple Once an extensive set of genetic markers is available for a variety, the tagging of any heterozygous locus becomes relatively trivial. We were able to find RAPD markers within 10 cM of 27 of the 31 known genes (isozymes, known DNA probes, and powdery mildew resistance) segregating in the 'Rome Beauty' x 'White Angel' population. Further saturation of the maps of 'Rome Beauty' and 'White Angel' is feasible but may be only of academic interest because neither variety is being used currently in our breeding effort. However, the other three crosses are integral parts of grape and pear breeding programs and continue to be investigated intensely. The identification of markers near genes involved. in the expression of more complex characters such as fruit quality, tree habit, and flowering time is of great potential for breeding programs. The set of segregating markers available in the 'Rome Beauty' x 'White Angel' populations were used to help elucidate the genetic basis of certain Of these complex characters (Table 3). The approach used to identify the major

73 Table 1. Genetic polymorphismin apple, pear, and grape as revealed by arbitrary primers

Variety Apple White Angel Rome Beauty Pea..__zr Bartlett NY 10353 Grape Aurore Cayuga White Horizon II1547-1

No. primers tested

No. RAPDs detected

Polymorphismsper primer

62 62

227 162

3.66 2.61

70 70

237 221

3.39 3.15

105 105 81 81

274 270 276 290

2.60 2.57 3.41 3.58

Table 2. Number of segregatingmarkers identified in single varieties and number of linkage groups identified by joint segregationanalysis

Variety Apple White Angel Rome Beauty Pear Bartlett NY 10353 Grape Aurore Cayuga White Horizon I11547-1

Number of segregatingmarkers Numberof linkage groups 253 156

24 21

252 232 299 299 276 290

genes was basically the same for each character and can be exemplified by our analysis of fruit skin color. The fruit skin color of both ' R o m e Beauty' and 'White Angel' is red, with the former variety exhibiting stripes and the latter a solid, uniform red. In the progeny we observed a variety of color patterns on the fruit surface, including a yellow skinned fruit totally lacking red pigmentation. These skin color patterns were grouped in various ways and for each group performed a joint segregation analysis with alleles at each of the marker 'loci'. Although the pattern or hue of red pigmentation did not appear to cosegregate with any of the markers, the red/yellow polymorphism displayed a clear correlation with several markers. Approximately one fourth of the trees produced yellow fruit (65 trees producing some red pigment in fruit skin, 24

24 22 20 25

trees lacking red pigment in the fruit skin). This ratio suggested the action of two genes or a single locus heterozygous in both parents. In either case, absence of red pigmentation would be the genetically defined (homozygous recessive) phenotype, whereas presence of red pigmentation could be due to one of several genotypes. Thus the segregation at marker loci initially was examined only within the trees producing yellow fruit. Two RAPD markers displayed a major distortion in their segregation ratio within the yellow ~ fruiting group: $27c from White Angel (17 " - " , 1 "+") and $45b from Rome Beauty (13 " - " , 1 "+"). Both markers gave segregation ratios near 1 : 1 for the entire population. These results suggested that both ' R o m e Beauty' and 'White Angel' possessed genes affecting the red/yellow dimorphism. Consulting the

74 Table 3. Markersdisplaying linkage with major genes controllingmorphologicalor physiological characters in apple

Character

Marker

Linkage group

Genesymbol

Terminal bearing Initial bud break Leaf expansion Bloom time Rootsucker formation

P27e Prx-c P27e no correlation with a P < 0.001 124e

5 6 5

Tb Rbb Tba

1

Rs

a Time of leaf expansion appears to be a pleiotropic effect of Tb.

Table 4. Linkage between Idh-2 and fruit skin color in a 'Rome Beauty' x 'White Angel' progeny Idh-2 genotype of tree

No. of trees with red pigment in fruit

No. of trees lacking red pigment in fruit

bb ab aa

2a 44 23

22 I 0

a Red coloration very light blush.

maps for each parent, we found that both markers had been placed on the respective linkage group 3 and that a tightly linked isozyme locus, I d h - 2 , was heterozygous in both parents. Joint segregation analysis o f l d h - 2 and fruit skin color is presented in Table 4. The high correlation between the two segregation patterns (X 2 = 77.0, P < 0.00001) indicates that in the population examined, the presence of red pigmentatin in the skin of the fruit is controlled by a single locus, here designated Ry to conform to the terminology used by Alston & Watkins (1973). Both ' R o m e Beauty' and 'White Angel' are heterozygous at this locus and therefore display red pigmentation in the fruit skin. The hue and distribution of red pigmentation appear to be controlled by factors unlinked to I d h - 2 . At present, these results can be applied only to the two parents; however, the results of Schmidt (1988) suggest that Ry is the primary locus governing fruit skin color in many apple varieties. Similar analyses demonstrated that branching habit, timing of initial bud break, and production of rootsuckers were primarily controlled by single loci, each heterozygous in White Angel (D.M. Lawson, M. Hemmat, and N.E Weeden, 1995). Although many other characters were investigated for which the genetic basis could not be elucidated (Hagens, 1992), it is clear that'marker driven genetic analysis represents a powerful tool for finding tags for apparently complex

traits. We currently are applying this approach to the pear and grape populations and continuing our analyses in apple. Present results indicate that this approach has excellent prospects for permitting breeders of perennial fruit to use marker assisted selection in their programs.

Acknowledgements We thank R.C. Lamb, R.D. Way, J.N. Cummins, and H.S. Aldwinckle for their advice, interest, and support on our studies in apple. The work in grape was funded in part by a United States-Israel B A R D Project (US 1888-90) and that in pear by a USDA Specific Cooperative Agreement (# 5 8 - 1 9 3 1 - 2 - 0 4 6 ) .

References Alston, EH. & R. Watkins, 1973. Apple breeding at East Mailing. Proc. Eucarpia Fruit Section, Canterbury: 14-29. Chevreau, E., Y. Lespinasse & M. Gallet, 1985. Inheritance of pollen enzymes and polyploid origin of apple (Malus x domestica Borkh.) Theor. Appl. Genet. 71: 268-277, Hagens, D.M, 1992. Genetic studies in Malus: Integration of isozymes, DNA markers and morphologicaltraits. MS Thesis, Cornell University. 55 pp. Hemmat, M., N.E Weeden, A.G. Manganaris& D.M. Lawson, 1994. Molecular marker linkage map in Malus. J. Hered. 85:4-11.

75 Lawson, D.M., M. Hemmat & N.E Weeden, 1995. The use of molecular markers to analyze the inheritance of morphological and developmental traits in apple. J. Amer. Soc. Hort. Dci., in press.

Manganaris, A.G., 1989. Isoenzymes as genetic markers in apple breeding. Ph.D. Dissertation, University of London. 430 pp. Manganaris, A.G. & F.H. Alston, 1987. Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple 1. The gene Got-l, a marker for the S incompatibility locus. Theor. Appl. Genet. 74: 154-161. Manganaris, A.G. & EH. Alston, 1988. The acid phosphatase gene ACP-1 and its linkage with the endopeptidase gene ENP-1 and the pale green lethal gene l in apple. Acta Hort. 224: 177-184. Schmidt, H., 1988. The inheritance of anthocyanin in apple fruit skin. Acta Hort. 224: 89-97.

Weeden, N.E & R.C. Lamb, 1985. Identification of apple cultivars by isozyme phenotypes. J. Amer. Soc. Hort. Sci. 110: 509-515. Weeden, N.E & R.C. Lamb, 1987. Genetics and linkage analysis of 19 isozyme loci in apple. J. Amer. Soc. Hort. Sci. 112: 865-872. Weeden, N.F., G.M. Timmerman, M. Hemmat, B.E. Kneen & M.A. Lodhi, 1992. Inheritance and reliability of RAPD markers. In: Applications of RAPD Technologyto Plant Breeding, pp. 12-17. Proc Joint Plant Breeding Symposia, Minneapolis, MN. Crop Science Soc Amer, Madison, WI. Williams, J., A.R. Kubelik, D.J. Livak, J.A. Rafalski & S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531-6535.