Proceedings of the 54th Italian Society of Agricultural Genetics Annual Congress
Matera, Italy – 27/30 September, 2010
ISBN 978-88-904570-0-5
Poster Communication Abstract – 7.14
MAPPING PYROSEQUENCING DERIVED CODOMINANT MARKERS
(EST-SNP AND EST-SSR) IN ASPARAGUS OFFICINALIS
SUNSERI F.*, RICCARDI P.*,**, MERCATI F.*, CIFARELLI R.***, FALAVIGNA A.**,
LEEBENS-MACK J.****
*) Dip. BIOMAA, Università Mediterranea di Reggio Calabria, Via Melissari, 89061 Reggio
Calabria
**) CRA-ORL, Unità di Ricerca per l’Orticoltura, Via Paullese 28, 26836 Montanaso L. (LO, Italy)
***) Metapontum Agrobios, SS Jonica 106 Km 448,2, 75010 Metaponto (MT)
****) Department of Plant Biology, University of Georgia, Athens, GA 30602 (USA)
Asparagus officinalis L., SNP, SSR, genetic map, Puccinia asparagi
The genus Asparagus belong to the family Asparagaceae and consists of around 150 species
found as herbaceous perennials, tender woody shrubs and vines (Ellison and Kinelski, 1986,
HortScience 21:1249), the most important of which is Asparagus officinalis. Cultivated asparagus is
a dioecious plant with a chromosome number of 2n=2x=20 and a haploid genome size of 1,323 Mb
(Bennett and Leitch, 2003, Plant DNA C-values database http://www.rbgkew.org.uk).). Genetic
maps including molecular markers linked to loci of interest are extremely valuable for markerfacilitated breeding programs (Gebhardt and Salamini 1992, Int Rev Cytol. 135: 201-237; Becker at
al. 1995, Mol Gen Genet. 249: 65-73). In asparagus genetic maps were originally based on isozyme
markers with four detectable linkage groups (Maestri et al. 1991, Theor Appl Genet 81: 613-618).
Later, RFLP (Restriction Fragment Length Polymorphism) markers revealing seven identified
linkage groups and a covering length of up to 402 cM (Lewis and Sink, 1996, Genome 39: 622627), and RFLP, RAPD (Random Amplification of Polymorphic DNA) and AFLP (Amplification
Fragment Length Polymorphism) markers were used to identify 10 linkage groups, spanning 721.4
cM, with an average distance between markers of 2.6 cM (Spada et al. 1998, Theor Appl Genet. 97:
1083-1089). The chloroplast DNA was also mapped with RFLPs and some genes encoding
photosynthesis-related proteins, rDNAs and tRNAs were localized (Lee et al., 1996, Theor Appl
Genet. 92: 10-14). In the present study, SNP (Single Nucleotide Polymorphism) and SSR (Simple
Sequences Repeat) markers have been developed and used to new genetic map to be used for
Marker Assisted Selection (MAS) for resistance to Puccinia asparagi. Codominant markers were
developed from GSFLX 454 pyrosequencing of cDNA libraries derived from on two parental
genotypes segregating for Puccinia asparagi resistance (paternal genotype 1770 is partially
resistant and maternal genotype G190 is susceptible) but largely homogeneous throughout the rest
of their genomes. An efficient computational SNPs and SSRs mining pipeline identified 1837
putative SNPs and 1092 SSR within the assembled expressed sequence tags (ESTs). Stringent post
processing reduced this number to 284 putative SNPs and 139 SSRs.
A first panel of 144 SNPs and 60 SSRs for GoldenGate genotyping assay (Illumina) and
multiplexed capillary sequencing strategy were chosen in order to genotyping a BC1 population
segregating for Puccinia asparagi resistance. The large BC1 (239 offsprings) population was
obtained from a backcross between all-male F1 population (Marte), that is partially resistant to P.
asparagi and a susceptible female (G190). BC1 populations were phenotyped after artificial and
natural infections with Puccinia asparagi under greenhouse and field conditions, respectively. This
work will yield a better understanding of the genetic basis of Puccinia resistance and a set of
codominant markers for marker assisted breeding programs.