Genetic mapping of Japanese plum

Genetic mapping of Japanese plum

(Parte 1 de 3)

Genetic mapping of Japanese plum

Crop Breeding and Applied Biotechnology 5:29-37, 2005 Brazilian Society of Plant Breeding. Printed in Brazil

Genetic mapping of Japanese plum

Eduardo Alano Vieira1, Rubens Onofre Nodari1*, Adriana Cibele de Mesquita Dantas1, Jean-Pierre Henri Joseph Ducroquet2, Marco Dalbó3, and Cristine Vanz Borges1

Received 28 February 2004 Accepted 17 March 2005

ABSTRACT - Genetic linkage maps of two Japanese plum cultivars, Chatard and Santa Rosa, were constructed using the pseudo-testcross mapping strategy and AFLP markers. From the segregating population derived from a cross between these cultivars, 8 plants formed the mapping population, with segregating genes for several traits of agronomical interest, such as the reaction to plum leaf scald. The Chatard map contained 56 markers linked in 1 linkage groups, covering a distance of 905.5 cM with an average distance of 16.2 cM between markers. The map of cultivar Santa Rosa contained 84 markers linked in 14 groups covering a distance of 1349.6 cM and presenting an average distance of 16.1 cM between markers. The maps obtained in the present study can be considered a basic framework and will need to be saturated for a more widespread use in breeding programs for the species.

Key words: Prunus, genetic linkage map, AFLP.

The plum we studied belongs to the genus Prunus,

Rosaceae family. Most of the plum varieties used for commercial production in the world are classified as European (hexaploid; 2n=48) or Japanese (diploid 2n=16) types (Okie and Weinberger 1995). While the term ‘European plum’ is applied to cultivars that belong to the species Prunus domestica, the term ‘Japanese plum’ was originally applied to the species Prunus salicina. However, most Japanese cultivars launched in the past few yearsLaboratótio de Genética e Fisiologia Vegetal, Departamento do Fitotecnia, Universidade Federal de Santa Catarina, C. P. 476, 8.040-900, Florianópolis, SC, Brasil.E-mail: nodari@cca.ufsc.brEmpresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina S.A. (EPAGRI), Estação Experimental Jardim Caiçara, C. P. 81, 8.600-0, São Joaquim, SC, BrasilEPAGRI, Estação Experimental de Videira, C. P. 21, 89.560-0, Videira, SC, Brasil involve a cross between P. salicina and several plum species of the same genus. Consequently, the term ‘Japanese plum’ is currently applied to cultivars of this species and its interspecific hybrids.

The Brazilian commercial production of plums is based on Japanese cultivars since they do not have a high chilling requirement and are able to stand high temperatures in the summer. The cultivars derived from European species are not cultivated commercially in Brazil due to their high chilling requirement (Ducroquet and Mondin 1997).

30 Crop Breeding and Applied Biotechnology 5:29-37, 2005

EA Vieira et al.

MATERIAL AND METHODS Plant material

The construction of genetic maps is considered one of the most important applications of molecular markers, not only in genetic analysis of species but also in plant breeding. Even though they do not provide information at the molecular level of genes, maps are meaningful because they give a background to comparative and evolutionary studies, as well as the understanding of the biological processes and chromosome organization. In practical terms, genetic linkage maps provide a framework for the identification of QTL-associated markers of agronomic interest and are an auxiliary tool in breeding programs for choosing the parent for crosses as well as in markerassisted selection (Lande and Thompson 1990).

Among molecular markers, AFLPs have been widely used in genetic mapping because this technique allows the generation of a large amount of polymorphic markers in a relatively short time. In addition, when the mapping population segregates 1:1 (as the pseudo-test cross), although dominant in nature, the AFLPs are as informative as the co-dominant markers (Hemmat et al. 1994, Grattapaglia et al. 1995).

Genetic maps of species belonging to the genus

Prunus were constructed with the use of molecular markers in almond (Joobeur et al. 2000, Ballester et al. 2001) and peach (Chaparro et al. 1994, Dirlewanger et al. 1998, Lu et al. 1998, Shimada et al. 2000), and also in an interspecific crossing between almond and peach (Foolad et al. 1995). However, no genetic map for plum was found in our literature review.

Among the diseases that attack plum trees, the most important in Brazil is the leaf scald, caused by the bacterium Xylella fastidiosa. This bacterium infects the vases of the plants and is transmitted by insects (sharpshooters) causing the death of susceptible plants. It takes three or more years to manifest the disease in contaminated plants. This is a problem for genetic improvement, since breeders have to wait over three years to evaluate and select resistant genotypes. This situation is a perfect opportunity for the development of techniques that allow genotypic selection at the seedling stage, which would save time and costs in comparison to phenotypic selection.

Objective of this study was to construct genetic linkage maps for plum varieties of interest in the state of Santa Catarina (SC), one for the cultivar Santa Rosa (susceptible to leaf scald) and the other for cultivar Chatard (partially leaf scald-resistant).

The segregating population used for the genetic mapping was derived from a crossing involving the cultivars Chatard and Santa Rosa. It was established in 1993 and originally consisted of 223 plants from which 8 were randomly chosen to form the mapping population. The entire population, transplanted in 1995 to the field at the experimental station of EPAGRI in Videira, state of Santa Catarina, is segregating for several genes, among them those for leaf scald-resistance.

DNA Extraction

The genomic DNA was extracted (Doyle and Doyle 1987, with some modifications) from young leaves taken from plants at the beginning of the vegetative cycle, not only from the parents in the cultivars Chatard and Santa Rosa, but also from the 8 plants of the segregating population.

Analysis with AFLP markers

The AFLP analysis was performed with the

Invitrogen AFLP analysis system I as recommended by the supplier. In addition, specific primers were synthesized to obtain other markers.

Genomic DNA was digested with the restriction enzymes EcoRI (rare cutter) and MseI (frequent cutter), and specific adaptors were connected to the ends of the cleaved segments. Then, the digested and cleaved DNA was preamplified using a pair of primers complementary to the adapters EcoRI and MseI and containing a selective nucleotide at the 3’ end (E + 1 and M + 1). The reaction of selective amplification was set up with additional primers at the end cleaved by EcoRI (with two or three selective nucleotides at the 3’ end) combined with complementary primers at the end cleaved by MseI (with three selective nucleotides at the 3’ end). In the reactions of selective amplification, 30 primer combinations were used (Table 1) in

Table 1. Combinations of primers used in the present study and their respective codes

E = Eco RI and M = Mse I

Primers E-AGC E-ACT E-ACA E-AGG E-ACG E-AAG E-ACC E-AAC E-AC E-A

3a 4a

8a 10a

M-CTA 1c 2c

4c 6c

4d 5d

M-CAG 1e 2e 3e

2f 3f 4f 5f

3g 4g 5g 6g

M-CTT 1h 2h

Genetic mapping of Japanese plum

Analysis with AFLP markers

The mapping population size of 8 used in our study is an in-between value of the 63 and 133 peach plants Dirlewanger et al. (1999) and Shimada et al. (2000), respectively, used to construct genetic linkage maps. In the first one, the authors were able to map QTLs associated to fruit quality traits. In fact, the authors used an existing segregating population established for breeding purposes to develop a genetic linkage map to be used in that breeding program.

The 30 combinations of AFLP primers used in the present study generated 1549 markers, 507 of which were present only in cultivar Chatard, 567 only in cultivar Santa Rosa, and 475 were present in both cultivars. The average number of markers generated by primer combinations was 51.6, which was a value very close to the 46.4 markers found by primer combination in Japanese plum trees by Goulão et al. (2001). However, these values are low in comparison with those brought forth by marker combinations in other species: 74 and 78 in two cultivars Malus x domesticas (Xu and Korban 2000); 61 and 80 in two cultivars Prunus persica (Lu et al. 1998, Cervera et al. 2000); 112 in Eucalyptus (Cervera et al. 2000), and 115 in Pinus (Cervera et al. 2000).

The discrepancy among the average numbers of

AFLP markers generated in the plum tree, which are low in relation to other species may be due to: i) the small size of the plum genome, 0.30 pgc-1 (Arumuganathan and Earle 1991); i) the staining methods: silver nitrate was used in the present study and in that developed by Goulão et al. (2001), and radioactivity in other studies, which has been considered highly efficient.

The average number of markers per primer combinations present in cultivar Santa Rosa was 18.9, while the average number of markers present in cultivar Chatard was 16.9. The t-test performed to compare these averages showed that the differences found between the two were not statistically significant (P > 0.65). Not all bands were used for the mapping, either because they were not clear, or because the segregation was a complex one.

The Santa Rosa cultivar also presented a higher number of segregating markers in the proportion 1:1 (8 markers) compared to Chatard (59 markers). The number of markers per primer combination present in one parental and segregating in the proportion 1:1 found in the population were low: an average of 2.0 markers in Chatard and 2.9 in Santa Rosa, i.e. values (Table 2) lower than 7.5 found in Populus sp (Cervera et al. 2001).

Genetic maps addition to the three primer combinations at the end cleaved by EcoRI, with two selective nucleotides at the 3’ end.

The fragments amplified in the reaction of selective amplification were electrophoretically separated in denaturing gel of polyacrilamide (6%) at a constant power of 60 W for approximately 1h and 40 min. The silver nitrate staining process of gels was used in accordance with Creste et al. (2001).

Data analysis of marker segregation

According to our initial analysis, the amplified fragments were classifiable in three categories: i) present in both parents; i) present in cultivar Chatard and absent in cultivar Santa Rosa and segregating in the proportion 1:1 in the progeny and; i) present in Santa Rosa and absent in Chatard, and segregating in the proportion 1:1 in the progeny. The observed proportions were compared to the expected 1:1 Mendelian proportion by the use of the χ2 test based on the probability of 1% of significance with software Linkage-1 (Suiter et al. 1983).

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