The availability of suitable genetic markers is essential to efficiently select and breed apple varieties of high quality and with multiple disease resistances. Microsatellites (simple sequence repeats, SSR) are very useful in this respect since they are codominant, highly polymorphic, abundant and reliably reproducible. Over 140 new SSR markers have been developed in apple and tested on a panel of 7 cultivars and 1 breeding selection. Their high level of polymorphism is expressed with an average of 6.1 alleles per locus and an average heterozygosity (H) of 0.74. Of all SSR markers, 115 have been positioned on a genetic linkage map of the cross ‘Fiesta’ × ‘Discovery’. As a result, all 17 linkage groups, corresponding to the 17 chromosomes of apple, were identified. Each chromosome carries at least two SSR markers, allowing the alignment of any apple molecular marker map both with regard to identification as well as to orientation of the linkage groups. To test the degree of conservation of the SSR flanking regions and the transferability of the SSR markers to other Rosaceae species, 15 primer pairs were tested on a series of Maloideae and Amygdaloideae species. The usefulness of the newly developed microsatellites in genetic mapping is demonstrated by means of the genetic linkage map. The possibility of constructing a global apple linkage map and the impact of such a number of microsatellite markers on gene and QTL mapping is discussed.

Development and characterisation of 140 new microsatellites in apple (Malus x domestica Borkh.)

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database (
 http://www.hidras.unimi.it
 ) to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.

/pdf/microsatellite-markers-spanning-the-apple-malus-x-domestica-40gn2upv6m.pdf

Microsatellite markers spanning the apple ( Malus x domestica Borkh.) genome

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database (http://www.hidras.unimi.it) to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.

Microsatellite markers spanning the apple (Malus×domestica Borkh.) genome

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.

http://digital.csic.es/bitstream/10261/28864/1/Shulaev_Vladimir_et_al.pdf

Multiple Models for Rosaceae Genomics

EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.

Development and transferability of apricot and grape EST microsatellite markers across taxa

Apple simple sequence repeats (SSRs) were intergenerically applied to the characterization of 36 pear accessions, including 19 Japanese pears (Pyrus pyrifolia), 7 Chinese pears (P. bretschneideri, P. ussuriensis), 5 European pears (P. communis), 3 wild relatives (P. calleryana), and 2 hybrids between P. pyrifolia and P. communis. All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear accessions. Nucleotide repeats were detected in the amplified bands by both Southern blot and sequencing analysis, and nucleotide sequences of pear were compared with those of apple. The differences in fragment size among pear or between pear and apple were, in many cases, due to the differences in repeat number. Interestingly, the DNA sequence of flanking regions in apple was highly conserved in pear. Hybrids from P. pyrifolia×P. communis showed one fragment inherited from each parent in all scorable cases, which suggested that each primer pair amplified fragments originating from the same locus. A total of 79 alleles were detected from seven SSR loci in pear, and all pear varieties except for the mutants could be differentiated. In conclusion, SSRs isolated from apple are highly conserved in pear and could be utilized as DNA markers in the latter genus.

SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Genetic linkage maps of the European pear (Pyrus communis L.) cultivar 'Bartlett' and the Japanese pear (Pyrus pyrifolia Nakai) cultivar 'Housui' were constructed based on AFLPs, SSRs from pear, apple and Prunus, isozymes and phenotypic traits by using their F1 progenies. The map of the female parent Bartlett consisted of 226 loci including 175 AFLPs, 49 SSRs, one isozyme and one S locus on 18 linkage groups over a total length of 949 cM, while that for 'Housui' contained 154 loci including 106 AFLPs, 42 SSRs, two phenotypic traits and the other four markers on 17 linkage groups encompassing a genetic distance of 926 cM. These maps were partially aligned using 20 codominant markers which showed segregating alleles in both parents. Compared with the reports of apple genetic maps, these pear maps were not saturated but were near saturation. Distorted segregation was observed in two and one regions of the genome of Bartlett and Housui, respectively. The position of 14 SSRs originating from apple could be successfully determined in pear maps, which enabled us to compare the two maps. Some SSRs developed from Prunus (peach, cherry) were also mapped. The relationships between pear and the other species belonging to the Rosaceae were discussed based on the position of SSRs.

Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears.

Pear scab (caused by Venturia nashicola) is one of the most harmful diseases of pears, especially Japanese and Chinese pear species. The molecular identification and early selection of resistant plants could greatly improve pear breeding. We have identified the position of the scab resistance gene, designated Vnk in an indigenous Japanese pear cultivar Kinchaku, within the pear genome by using simple sequence repeat (SSR) markers derived from pear and apple. The position of Vnk was identified in the central region of linkage group 1 of Kinchaku. Several amplified fragment length polymorphism (AFLP) markers linked to Vnk were obtained by bulked segregant analysis. Among them, the AFLP marker closest to Vnk was converted into a sequence tagged site (STS) marker. Four random amplified polymorphic DNA (RAPD) markers previously found to be loosely associated with Vnk (Iketani et al. 2001) were successfully converted into STS markers. Six markers (one SSR Hi02c07 and five STSs converted from AFLP and RAPD) showed tight linkages to Vnk, being mapped with distances ranging from 2.4 to 12.4 cM. The SSR CH-Vf2, which was isolated from a BAC clone of the contig containing the apple scab gene Vf, was mapped at the bottom of linkage group 1 in Kinchaku, suggesting that the Vnk and Vf loci are located in different genomic regions of the same homologous linkage group.

Y. Sawamura

Papers

SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears.

Genetic mapping of the pear scab resistance gene Vnk of Japanese pear cultivar Kinchaku

New Japanese chestnut cultivar 'Porotan'.

A method for quickly removing pellicles from chestnuts by deep frying in cooking oil.