Determination of the relationship between some morphological traits of the tomato lines and resistance tomato yellow leaf curl virus disease

Background: Tomato ( Solanum lycopersicum L.) is the most produced and exported vegetable in Turkey. There are many pathogens to limit tomato production by reducing yield and fruit quality. Among them, Tomato Yellow Leaf Curl Virus (TYLCV) causes important economic losses. The most efficient and environmental friendly method against TYLCV is the use of resistant varieties. In this trial, it was aimed to determine some morphological traits which were linked to Ty-1 and Ty-3a genes which confer to TYLCV. A commercial hybrid carrying Ty-1, Ty-3a genes as heterozygous was crossed to a susceptable inbred line from Bati Akdeniz Agricultural and Research Institute (BATEM). Marker assisted selection (MAS) was carried out in F 1 and F 2 generations and biological tests were done for TYLCV resistance in F 3 generation. MAS for Ty-3a and Ty-1 genes were compatible with biological tests.

trial, it was aimed to determine some morphological traits which were linked to Ty-1 and Ty-3a genes which confer to TYLCV. A commercial hybrid carrying Ty-1, Ty-3a genes as heterozygous was crossed to a susceptable inbred line from Bati Akdeniz Agricultural and Research Institute (BATEM). Marker assisted selection (MAS) was carried out in F 1 and F 2 generations and biological tests were done for TYLCV resistance in F 3 generation. MAS for Ty-3a and Ty-1 genes were compatible with biological tests.

Background
Tomato (Solanum lycopersicum L.,) is one of the most economically important vegetables in the World. The world tomato production is over 163 million tons from an area of 4.83 million hectares [1].
3 Furthermore, the gene pyramiding that combines multi-resistant genes in a variety via molecular markers, has been an important component of modern tomato breeding programme. Tomato is very rich in the number of available molecular markers [14], thus marker assisted selection (MAS) in tomato breeding programme have been effectively used for transferring of many disease resistance genes. It has been shown that some of resistant genes were in the linkage with other genes, having epistatic properties [15,16,[17][18]. Therefore, the aim of this research is to determine whether there is a relationship between morphological traits and Ty-1 and Ty-3a, resistant genes against to TYLCV.
Thus it is possible to make a selection via morphological markers linked to the TLYCV genes, as an alternative MAS in resistance breeding.

Segregation of Ty-1 and Ty-3a by Molecular markers
F1 plants which were obtained by crossing Tayfun F1 and BATEM1 pure line, were screened with molecular markers linked to Ty-1 and Ty-3a. F2 and F3 can be obtained by selfing F1 and F2 plants.
Individuals of F2 and F3 population were screened via molecular markers to determine segregation of the Ty-1 and Ty-3a genes.

Ty-1 gene;
Ty-1 gene had two allelic forms based on molecular marker (JB1).The susceptable pure lines had susceptable allel (ty1;ty1) at 400 bp and commercial hybrid was heterozygous form having band at 400 bp for susceptable allel and 500 bp for resistant allel. The susceptable induviduals of F2 and F3 had allel at 400 bp and resistant individuals had allels at both 400 and 500 bp (Fig 1).

Ty-3a gene;
Two different allelic forms for Ty3a were observed in F1 individuals, that susceptable ones had allel at 320 bp and heterozygous individual carried allels at 320 and 630 bp. The individuals of segregating populations carried three different allelic forms at 320 bp for susceptable or at both 320 bp and 630 bp for heterozygous resistant or at 630 bp homozygous resistant (Fig 2). The Ty-3a gene is more preferred due to the fact that the Ty-3 gene provides a wider durability, and many other features are undesirable with the gene. Segregation ratios for resistant and susceptable individuals for F1 were checked with Chi-square test.
The resistant Tayfun F1 was crossed with the susceptable line to obtain F1 plants which segregated resistant and susceptable plants in a 1:1 ratio (Table 1). Accordingto Χ² (Chi-square) analysis, Ty-1 and Ty-3a based on molecular marker show edequal segregation at statistical significance level of 0.05.
The segregation of the Ty-3a gene in F2 population tested by molecular marker was homozygous resistant in 99 individuals and heterozygous resistant in 194 individuals and homozygous susceptable in 107 individuals. Moreover, the 107 susceptable individuals for Ty-3a were also found to be susceptable for the Ty-1 gene ( Table 2). Chi-square analysis was used to determined segregation ratio for Ty-1 and Ty-3a in F2 population. According to results segregation ratio was evaluated the same distribution at statistical significance level of 0.05 (Fig 3 and 4).

Disease Assessment
In the biological test; Tayfun F1 (as resistant hybrid), Batem Ozcelik F1 (as susceptable hybrid) and F3 individuals, inoculated with Whiteflies. There were no symptoms of resistant control varieties while susceptable hybrid showed disease symptoms. Ten F3 individuals which were determined as homozygous resistant by molecular marker for Ty-1 and Ty-3a, were also found to be resistant by

The relationhsip between the characteristics of the lines and disease resistance
In the total 95 F3 individuals were evaluated based on both morphological traits and disease resistance. The 56 individuals were determined as a susceptable, 9 individuals were heterozygous resistance and 30 individuals were homozygous resistant by molecular markers.

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There were statistically significant positive correlation between Ty-3a and Ty-1 genes and lengths of stem at first inflorescence, stem length of internode, leaf attitude, plant habitus, length of inflorescence and status of calix position of petal (Table 3).

Segregation of Ty-1 and Ty-3a by Molecular markers
Ty-1 gene; Although three different allels (allel 1 at 400 pb, allel 2 at slightly larger than 400 pb and allel 3at 500 pb) for TY-1 gene; were identified by [19], in our research the individuals of segregating populations and parents had two allels. Also, allel at 500 bp were dominant over allel at 400 bp.

Ty-3a gene;
The individuals of segregating populations carried three different allelic forms at 320 bp for susceptable or at both 320 bp and 630 bp for heterozygous resistant or at 630 bp homozygous resistant [11]. The Ty-3a gene is more preferred due to the fact that the Ty-3 gene provides a wider durability, and many other features are undesirable with the gene.
Segregation ratios for resistant and susceptable individuals for F1 were checked with Chi-square test.

Disease Assessment
Some breeders could obtain false data in negative and positive results because the they can't calculate the distance of markers P6-25 stated [21]. Especially; emphasized that faulty results could be obtained from the sources transferred from different wild species of tomato to culture tomato.
However, our results of the marker and biological testing were found to be compatible with each other.

The relationhsip between the characteristics of the lines and disease resistance
The length of stem at first inflorescence and stem length of internode directly affects the yield of tomato production. According to [22], the length of internode were determined by the addition effect.
Indeterminate varieties have less cluster than determinate varieties so the yield is decrease in indeterminate varieties. For this reason, breeders have preferred the varieties which have short length of stem at first inflorescence and short stem length of internode. However, in this research we determined that as the stem length of internode and the length of inflorescence in tomato increased, the tomato individuals carried homozygous Ty-3a gene. Also the leaf size and the length of internode were determined to be significant in reduction at susceptable tomato plants [23]. Therefore, it can be stated that the length of internode can be used as selection criteria for resistant breeding programme in tomato.
Leaf attitude was evaluated at three different position (erect, horizontal, drooping) according to UPOV. There was a significant positive correlation between resistance to TLYCV and leaf attitude ( Table 4). The individuals having leaf at dropping position was resistant to TLYCV indicating that these genotypes carried Ty-3a gene. The producers and breeders generally prefer the tomato variety with horizontal leaf position to prevent the transmission of fungal diseases. Because it believes that the horizontal leaf position can allow the plants to get better air. In contrast to this, we determined that the resistant tomato individuals had drooping leaf position.
Plant habitus was evaluated as weak, medium and strong type. The plant habitus of the resistant tomato individuals were weak type. Also the resistant tomato individuals had long length of inflorescence. Although not statistically significant, there was a negative correlation between resistance to TLYCV and fruit characteristics such as fruit weight and fruit length ( Table 3). As the fruit weight decreased, the resistance to TYLCV increased. It was also determined that fruit size was effected with resistance gene and homozygous resistant gene resulted in the middle-small fruit group [15]. Two QTLs for fruit weight were identified on chromosomes 2 and the wild allels were associated As [16][17] pointed, we determined that the resistant individuals had undesirable plant traits such as reduction of the number of fruits per plant and fruit traits. [15] also found a statistically significant and negative correlation between resistance to TLYCV and the number of fruits in the first 4 clusters, the number of branches per plant, the number of fruits per plant and yield per plant. It can be stated that in breeding programs for resistance which don't use marker technology, resistant genotypes can be eliminated due to their undesirable fruit and plant characteristics such as small sized fruit, weak plant habitus, less number of fruit per plant.

Conclusion
Molecular Marker Assisted Selection (MAS) has been successfully used to select resistant genotypes in the breeding study. In this research, we identified 95 individuals of F3 population based on their morphologic traits and their resistance to TLCV by molecular markers linked to Ty-1 and Ty3-a and by biological test. It was determined the 30 individuals having homozygous resistant allels, the 9 individuals having heterozygous allels for both Ty-3a and Ty-1 genes. The 56 individuals were susceptable against to TLYCV. In the our study, it was found that the Ty-1 and Ty-3a genes did not segregated in any individuals and were together in all individuals. Thus, it can be concluded that Ty-3a and Ty-1 genes could be allel genes. The 43 morphological traits were observed in the selected individuals of the F3 population by molecular marker to correlate with resistant allel, Ty-3a. It was found that there was statistically significant correlation between Ty-3a and length of internode, length of stem at first inflorescence, status of calix position of petal, leaf: attitude, length of inflorescence, plant habitus. Also there was negative correlation between fruit weight, fruit length and Ty3-a gene.
Consequently, we identified some morphological markers such as stem at first inflorescence, stem

TYLCV Inoculation and Disease Screening
The seedlings were grown in equal volume of steam-sterilized perlite: peat mix. The experiment was conducted in a completely randomized block with two replicates and ten plants were tested each

Morphological traits:
95 F3 individuals which were selected by molecular markers, were also evaluated based on 43 morphological traits according to UPOV criteria (Table 4).

Data analyses
The correlation between the disease resistance gene and 43 traits were analyzed by using pearson correlation analysis in SPSS 14.0 program.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analysed during this study were included in this published article.     Segregation of F2 genotypes in terms of Ty-1 gene Segregation of F2 genotypes in terms of Ty-3a gene