The Novel ps and ps2 Specific Markers for Selection of Functional Male Sterile Tomato Lines in Breeding Programs and Hybrids Seed Production

Functional male sterility in tomato (Solanum lycopersicum L.), controlled by the ps-2 and ps genes can be utilized in the production of F1 hybrid tomato seed. Two novel cleaved amplified polymorphic sequence (CAPS) markers linked to the ps-2 and ps genes were found in tomato. The C4-30 and C2-21 markers were developed based on the conserved ortholog set II (COSII) sequences C2_At3g20020 and C2_At1g65900 located on tomato chromosomes 4 and 2, respectively. The HinfI-derived PCR restriction product C4-3080 was applicable in the detection of functional male sterile plants. In case of the C2-21 marker, a polymorphism was revealed after digestion of the amplicon with restriction enzyme MboI. Specificity of the DNA markers identified was verified by scoring the tomato parental lines, F2 progeny and F1 hybrids, in which maternal lines possessed the ps or ps-2 gene. C4-30 and C2-21 can be used as the diagnostic tools in tomato breeding programs and in F1 hybrid seed production.


Introduction
Traditional methods of F 1 hybrid tomato (Solanum lycopersicum L.) seed production require manual emasculation of the flowers in the early bud stage.Using female lines possessing the trait of functional male sterility controlled by recessive ps and ps-2 genes can reduce the time and cost of such work (Potaczek & Kubicki, 1986;Atanassova, 1999Atanassova, & 2000;;Staniaszek et al., 2000).Several tomato F 1 hybrids exhibiting functional sterility controlled by the ps gene, have been developed at the Research Institute of Horticulture in Skierniewice, Poland.They have been introduced into practical applications and commercial production (Staniaszek et al., 2000(Staniaszek et al., & 2002;;Kozik & Nowakowska, 2007).In Bulgaria, thirteen F 1 hybrids were developed based on functional sterility controlled by the ps-2 gene (Atanassova, 1999).Significant progress in breeding new tomato F 1 hybrids has been made in the Czech Republic and Moldova (Atanassova, 1999).Unfortunately, the occurrence of selfings can in practice limit the application ps and ps-2 sterility.The percentage of selfings, which depends on the temperature and humidity, is highest under high temperature conditions (Simonov, 1967;Atanassova, 2000).The recessive gene ps-2 (positional sterility-2), described in Czech cultivar Vrbicanske nizke, is responsible for functional male sterility in tomato (Tronickova, 1962;Atanassova 1991Atanassova & 1999Atanassova & 2000)).Plants homozygous for the ps-2 allele exhibit pollen maturation but are sterile for mechanical reasons.The recessive gene ps (positional sterility) confers another type of functional male sterility in tomato.Positional sterility -ps is manifested by flowers with non-splitting anthers, corolla petals accreting to androecium at 2/3 of their length (Potaczek & Kubicki, 1986).This form of sterility has been identified by Larson and Paur in 1940 in the tomato cultivar John Baer (Larson & Paur, 1948;Potaczek, 1984;Atanassowa, 2000).Expression of these two forms of sterility strongly depends on environmental conditions (reviewed in Atanassova, 1999).
The ps-2 gene was localized in map segment T0958 -T0635 on tomato chromosome 4 (Gorguet et al., 2006), whereas the ps gene is closely linked to aw (anthocyanin without) gene on chromosome 2 (Dorossiev, 1976;Tanksley et al., 1992;Atanassova, 2000).Based on single nucleotide polymorphism (SNP), Gorguet and co-workers (2009) developed the ps-2 marker, which was applied towards in the screening of 176 tomato breeding lines, 8 of them were ps-2/ps-2.Using the AFLP method and F 2 segregated population derived from the Bulgarian lines CMC1ps2, Li et al. (2006) indicated three markers E37/M47, E38/M48 and E33/M50 -useful in the identification of fertile lines.Recently, two co-dominant markers: microsatellite SSR450 and CAPS marker developed from the sequence RFLP TG123, were found to be linked with the ps-2 gene (Sha et al., 2010).These markers were successfully tested in 123 F 2 plants (Sha et al., 2010).Staniaszek et al. (2000) described three RAPD markers, OPH04-670, OPAX10-780 and OPW13-1230, linked to the ps locus.OPAX10-780 and OPW13-1230 were specific to the fertile male parent and can be used for distinguishing true hybrids from self-pollination of the female parents.OPH04-670 was of use in the identification of functionally male sterile lines possessing the ps gene.However, co-dominant molecular markers for the ps gene need to be identified.
Here, we report two novel co-dominant diagnostic PCR markers, C4-30 and C2-21, for selection of ps-2 and ps functional male sterile tomato lines, respectively.

Plant Material
The ps-2 male sterile line M 3089 obtained after inbreeding and selection of line Start 24, was kindly provided by B. Atanassova, Institute of Genetics "Acad.Doncho Kostoff", Bulgarian Academy of Science, Sofia, Bulgaria (Kozik & Nowakowska, 2007).M 3089 was crossed with a male fertile line M 3372, which was selected by eight generations of inbreeding of the F 1 hybrid 7-2-92 (Rijk Zwaan B.V., The Netherlands).The single F 1 plant mating was self-pollinated to produce the 131 F 2 progeny.
The second F 2 population of 119 plants was generated from F 1 derived from the highly inbred tomato ps line W 1.8a (ps line) and the fertile line M 4191 (selected from Pearly F 1 hybrid, DAPco B.V., The Netherlands).The ps sterile line W 1.8a was generated from a cross between line PH 1106 (ps) and S. chilense LA 1969 (Potaczek, 1999;Kozik & Nowakowska, 2007).
Table 1.Distribution of the restriction fragments of the marker C4-30 in tomato breeding lines and F 1 hybrids F 1 hybrids/breeding lines Origin C4-30 restriction fragments 80 bp

+ presence of marker -absence of marker
The ps-specific marker C2-21 was tested in six male sterile lines: W 1.

+ presence of marker -absence of marker
Plants were grown in a greenhouse.Young, fresh leaves from parental lines, breeding lines, F 1 hybrids (10 plants per each genotype) and two F 2 populations segregating for the ps-2 and ps were harvested and stored at -70 o C until DNA extraction.

Expression of ps-2 and ps-Deriving Sterility
Expression of ps-2 and ps-deriving sterility was observed on each F 2 plant.Plants were classified functionally male sterile when no selfed seeds were found in the fruits.Segregation data were analyzed by the Chi-square (χ 2 ) test.

DNA Extraction
Total DNA was isolated from frozen young tomato leaves of each plant using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany).DNA concentration and purity was determined spectrophotometrically and by visualization on a 0.8% agarose gel.
PCR was performed in 20 μl of 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl 2 , 0.1 mM of each deoxynucleotide, 0.2 μM of each primer, containing 1U Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) and 30 ng genomic DNA as template.The PCR parameters were: initial denaturation at 94°C for 60 s followed by 40 cycles of denaturation at 93°C for 15 s, annealing at 55°C for 20 s and extension at 72°C for 60 s.A final extension was at 72°C for 5 min.The GeneAmp 9700 thermal cycler was used for DNA amplification.Amplicon C4-30 was digested with restriction enzyme Hinf1 (MBT Fermentas, Lithuania), whereas C2-21 product was treated with MboI (MBT Fermentas, Lithuania).Digestion of PCR products were carried out at 37 o C for 3 h in a 20 µl mixture containing 5U of restriction enzyme, 18µl PCR product and 10x concentrated restriction enzyme buffer.

Identification of the CAPS Marker C4-30 Linked to Locus ps-2
PCR product C4-30 of approximately 1000 bp was amplified in male sterile and fertile plants (not shown).The CAPS fragment of about 80 bp was found in the homozygous male sterile line M 3089 (Figure 1, lane 1), whereas the restriction product of about 190 bp was identified in the homozygous male fertile line M 3372 (Figure 1, lane 2) after HinfI digestion and electrophoresis in 3.5% MetaPhor agarose gel.The segregation of sterile and fertile plants was 35 : 96 in the F 2 progeny, which is in agreement with a ratio of 1: 3 (χ 2 = 0.16, P = 0.69).The marker C4-30 80 was detected in all 35 F 2 sterile plants.In the group of 96 F 2 fertile plants 35 were homozygous for C4-30 190 , whereas both restriction products were observed in 61 plants.The restriction patterns of amplicon C4-30 for 7 F 2 progeny are shown in Figure 1, where lanes 3 and 5 are for homozygous sterile plants, lanes 4, 6 and 9 show homozygous fertile plants and lanes 7 and 8 are for ps-2 heterozygous plants.The marker C4-30 was also of relevance for the detection of ps-2 homozygosity in the sterile (M 3090 and M 3091) and fertile (M 3586 and M 3597) tomato lines (Figure 1, lanes 10, 12, 11 and 13, respectively), as well as for 3 F 1 hybrids: Prekoz, Odysseus and Elina provided by B. Atanassova (Figure 1, lanes 14, 15 and 16, respectively).C4-30 80 and C4-30 190 were present in 10 F 1 hybrids generated in a tomato breeding program at the Research Institute of Horticulture, Skierniewice, Poland.These results were summarized in Table 1.
The presented study indicates that the marker C4-30 shows a high ps-2 -selection specificity.These results show that the marker C4-30 can simplify the screening of functional male sterile lines in tomato breeding programs and hybrid seed production.

Identification CAPS Marker Linked to Locus ps
A 1800 bp PCR marker C2-21, was amplified in all parental lines and F 1 plants (not shown).Polymorphism of this amplicon was revealed after digestion with restriction enzymes MboI and electrophoresis in 1.4% agarose gel.The restriction fragment of 380 bp was found in sterile lines with gene ps (Figure 2, lanes 1, 3, 5), whereas 420 bp long fragment was observed in fertile lines (Figure 2, lanes 2, 4, 6).The heterozygous genotypes F 1 plants were given three fragments, 380, 420 and 580 bp.The third fragment of 580 bp from the heterozygous plants was shown to be a heteroduplex between the two fragments 380 and 420 bp (Figure 2, lanes 7-9).
Restriction products were analyzed in 119 plants of the segregating F 2 population.In the F 2 population, segregation of sterile and fertile plants was 24:95.A chi square (χ 2 ) test confirmed a 1:3 segregation of sterile to fertile individuals (χ 2 = 1.48,P= 0.22).The ps specific restriction fragment of 380 bp was detected in 24 functionally male sterile F 2 plants.Among the 95 F 2 fertile plants 37 were homozygous for C2-21 420 , whereas three restriction products were observed in 58 plants (Figure 2, lines 10-23).
The functional male sterility in tomato controlled by recessive gene ps and ps-2 is the important trait in F 1 hybrids seed production.Identification of functionally male sterile plants is labour -consuming, it is possible only during of flowering time or on based the number of seeds per fruit (Potaczek & Kubicki, 1986;Staniaszek et al., 2000Staniaszek et al., , 2002)).Molecular markers associated with genes ps and ps-2 may be helpful for the identification functionally male sterility.The RAPD AFLP and CAPS markers were used to identify locus ps and ps-2 (Staniaszek et al., 2000;Li et al., 2006;Gorguet et al., 2009).RAPD is relatively simple and easy molecular technique, highly discriminate for genetic polymorphism in tomato, but they are dominant and poor reproducibility (Jones et al., 1997).On the other hand the use of AFLP markers for marker-assisted selection (MAS) is very time consuming and expensive.Because of recombination events different molecular tools should be used for MAS in breeding practice.
The results of this study demonstrate that the new CAPS markers C4-30 and C2-21 may be helpful for the identification of the ps-2 and ps genes, respectively.Both markers are co-dominant, the heterozygous genotype can be distinguished from the dominant or recessive homozygous individuals.They can be used in marker assisted selection of the functional male sterility in tomato plants.