ANALYSES of P-Tst-1 , P-Tst-3 and P-Tst-6 RETROTRANSPOSONS IN CONVENTIONALLY AND ORGANICALLY PRODUCED TOMATOES

Tomato is one of the best-studied cultivated dicotyledonous plants in molecular studies. Mobile genetic elements constitute large parts of plant genomes. Retrotransposons are mobile genetic elements within the genome and constitute more than 60% of the tomato genome. Transposable elements (TE) or transposons are DNA sequences that can alter their position within a genome, cause mutations and change the genetic identity of the cells and genome size. We aimed to analyze potato specific-P-Tst-1, P-Tst-3 and P-Tst-6 retrotransposon movements in tomatoes at different developmental stages (mature seedling, flowering stage and fruiting stage) under different cultural conditions (organic and conventional) by IRAP (Inter-Retrotransposon Amplified Polymorphism) technique. We found polymorphism rates between 1-100% for P-Tst-1, P-Tst-3 and 0-86% for P-Tst-6 . When compared to organically tomatoes, conventionally produced tomatoes showed high polymorphism. Moreover, polymorphism ratios were different at developmental stages. This is the first report to analyze potato-specific retrotransposon movements in tomatoes grown under different conditions. Obtaining findings are expected to understand the evolutionary relationships between tomato and potato, and even the effects of different growing conditions on tomato genome to increase yield in agriculture


INTRODUCTION
Solanum lycopersicum L. (tomato), belonging to Solanaceae family, is one of the most consumed vegetables. This plant is a perennial herbaceous plant but is regularly grown as an annual crop (Al-Remi et al. 2018). Tomato is grown worldwide for local consumption and export purposes in a wide variety of geographical areas in greenhouses or open fields (Knapp 2002;Bergougnoux 2014). Tomatoes are self-pollinated or pollinated by another representative of the same variety, to out breed (cross-pollinated) to create hybrid variety which possesses traits of the two families (Samsulrizal and Yusof 2019). In many parts of the world, the vast majority of greenhouse tomatoes are produced using conventional production systems compared to organic systems. Conventional and organic agricultural systems are different regarding soils, pests, fertilizers, insecticides, fungicides and even economic conditions (Mitchell et al. 2007;Seçgin et al. 2018).
Some sequences in genomes tend to maximize their representation from one generation to the next by replicating themselves at a higher rate than other DNA sequences. These genetic materials named transposons or TEs change their places within the genome (Tufan et al. 2020). Different kinds of transposons show distinct mechanisms to increase their copy number. The most common types of transposons are RNA transposons (Class I elements) which transpose a "copy and paste" mechanism with an RNA intermediate, and DNA transposons (Class II elements) which move via "cut and paste" mechanisms without an RNA intermediate. In contrast, transposon-like elements (Helitrons) are speculated to transpose via a "rolling circle" strategy (Piégu et al. 2015). Most of transposons of any type are non-autonomous elements that lack coding ability and require the functions encoded by autonomous elements of a similar family (Wicker et al. 2007;Todorovska 2007).
TEs can make up most of the genetic information in different genomes (Kidwell 2002;Kaya et al. 2013). Even though they can occasionally provide a selective advantage to their host species (Arvas et al. 2021), they multiply in large portions to self-perpetuate (Kumar and Bennetzen 1999). The number and distribution of TEs are dramatically variable among closely related species and cover a meaningful proportion of many animal and plant genomes (Kidwell 2002;Vitte and Panaud 2005;Marakli et al. 2019). As a result of genome contraction and expansion, the number of transposon families may fall and rise in genomes (Piegu et al. 2006;Lisch 2009). The variations in DNA content are a reflection of a constantly shifting balance between selection at host level to regulate transposon activity and at the level of transposons to increase their copy number (Kalendar et al. 2021).
In this study, we determined the potato-specific P-Tst-1, P-Tst-3 and P-Tst-6 retrotransposon movements in tomatoes at different developmental stages (mature seedling, flowering, and fruiting stage) grown under different conditions (organic and conventional) by using IRAP technique.

MATERIALS AND METHODS
Plant materials and DNA extraction: Solanum lycopersicum L. var. Şencan 9 seeds were provided by Atatürk Horticultural Central Research Institute. During the tomato plant development in a greenhouse, organic fertilizers (cow dung manure and organic NPK-nitrogen, phosphorus and potassium) were provided for organically produced tomatoes. On the other hand, inorganic fertilizers (diammonium phosphate-DAP and ammonium sulphate), insecticides and fungicides were used for conventionally produced tomatoes.
Genomic DNAs (gDNAs) from the youngest leaves of experimental plants were isolated using CTAB method (20). Treatments were named CT, and OT for conventional tomatoes and organic tomatoes, respectively. Each of them was composed of three different plants belonging to three different stages: mature seedling, flowering stage and fruiting stage. Spectrophotometric and electrophoretic analyses were performed to determine the quantity and quality of gDNAs, and then gDNAs were stored at -20˚C until use.

IRAP-PCR
P-Tst-1, P-Tst-3 and P-Tst-6 retrotransposons' movements in organically and conventionally produced tomatoes were determined by IRAP-PCR marker technique. Primer sequences were indicated in Table 1. For this purpose, 18 samples including six different plants were designated as CT1, CT2, CT3, OT1, OT2 and OT3, with three replications (mature seedling, flowering stage and fruiting stage) used as templates for IRAP-PCR.

Polymorphism analysis
Monomorphic and polymorphic bands were determined for each treatment according to band profiles on agarose gels. The similarity ratios among the different samples were calculated using the Jaccard Similarity Coefficient formula (Camirand and Brisson 1990)

RESULTS AND DISCUSSION
IRAP-PCR was performed using gDNAs isolated from organic and conventional tomatoes with 3 replications in different developmental stages. A total of 18 genomic DNA samples were used for templates in IRAP-PCR. PCR products were visualized on 1% agarose gel. The polymorphism rates for P-Tst-1 were calculated by comparing each individual with others. Only clear bands were included in the count. Accordingly, 83 bands were counted between 800 bp and 2500 bp in Fig. 1. Among total band profiles, 45 bands were observed in conventionally produced tomatoes, and 38 were counted in organically produced ones. In the counted bands 22, 34 and 27 bands were observed at the mature seedling, flowering and fruiting stages, respectively. Polymorphism percentages were calculated among samples by comparing each sample with the other samples, resulting in 0-100% polymorphism ( Table 2).
The polymorphism rates for P-Tst-3 were also calculated by comparing each individual with others. 99 bands (56 for conventionally produced tomatoes and 43 for organically produced ones) between 400 and 2000 bp were counted for polymorphism analyses (Fig. 2). Similarly to P-Tst-1, we found 0-100% polymorphism among samples. In the counted bands, 35, 40 and 24 bands were observed at the mature seedling, flowering and fruiting stages, respectively (Table 3).
The other retrotransposon, P-Tst-6, also indicated a polymorphic band profile among samples. In analyses, 112 bands (18 monomorphic and 94 polymorphic) were counted between 400 and 2000 bp. In the bands, 61 were observed in conventionally produced tomatoes, and 51 were counted in organically produced ones (Fig. 3). From the total count, 42, 42 and 28 bands were observed at the mature seedling, flowering, and fruiting stages, respectively. There were 0-86% polymorphism rates among samples (Table 4).
Since tomatoes also shared the same family (Solanaceae) with the potato plant, these potato-specific primers such as P-Tst-1, P-Tst-3 and P-Tst-6 were used to investigate the presence and movement of retrotransposons organically and conventionally grown tomatoes. According to the results, P-Tst-1 was highly polymorphic and a 0-100% of polymorphism ratio was found among samples. This result indicated the existence of P-Tst-1 retrotransposon in tomato and potato, and other Solanaceae species. Additionally, P-Tst-1 retrotransposon is active in the tomato genome similar to the potato genome. Moreover, P-Tst-1 retrotransposon in conventionally produced tomatoes showed higher polymorphism than organically produced tomatoes. This result indicated that applying chemicals to conventional tomatoes increased the activities of P-Tst-1 in tomato genome. Furthermore, the bands of P-Tst-1 retrotransposon were also detected with higher numbers at the flowering stage of tomato plants than at the mature seedling and fruiting stage. Similar to P-Tst-1, P-Tst-3 (0-100%) and P-Tst-6 (0-86%) were also polymorphic in conventionally and organically produced tomatoes. Furthermore, P-Tst-3 and P-Tst-6 retrotransposons had abundant band numbers at the mature seedling and flowering stage of the tomato plant compared to the fruiting stage. Yilmaz et al. (2018) studied herbicide effects on rice and reported that herbicide treatment could enhance retrotransposon activity in rice with increasing herbicide concentration. There are numerous reports showing retrotransposition movements in various biotic and abiotic stress conditions like wounding, pathogen attack, and various chemicals such as herbicide, fungicide and insecticide applications (Dimitrov et al. 2011, Cakmak et al. 2015Finatto et al. 2015;Songstad et al. 2017;Yilmaz et al. 2018;Nie et al. 2019).