Variability and selection of tomato Sletr1-2 mutants backcross population to improve yield and fruit quality

Enhancing fruit quality and yield is the goal of creating superior tomato varieties. One area of concern for breeders is fruit shelf life. To produce better offspring, hybridization attempts to combine the traits of two parents, but the results can be unpredictable. Backcrossing can create stable offspring with desired traits and eliminate undesirable characteristics from the parent plants. This study aimed to assess the diversity among advanced backcross strains and to obtain backcross genotypes carrying mutant alleles that have a high level of similarity with the superior parental lines. The study was carried out from April to September 2019 at the Indonesian Vegetable Research Institute’s screen house. In this experiment, two populations of backcrossing comprised of commercial parents of ‘Intan’ and ‘Mutiara’ varieties as well as backcross genotypes including MBC3F1 32, MBC3F1 32.2, MBC3F1 32.3, MBC3F1 37.2, MBC3F1 45.3, MBC3F1 37.1, IBC3F1 34.2, IBC3F1 37.2, and IBC3F1 34 were utilized. A variance analysis was used to determine genotype diversity. The genetic similarity between parents and offspring was determined using cluster analysis, and a t-test was used to compare mean performance values. According to the results, the BC3F1 populations of ‘Intan’, and ‘Mutiara’ exhibited a high degree of phenotypic variability. Cluster analysis and a t-test revealed that the IBC3F1.37.2 and IBC3F1.45.3 genotypes resembled ‘Intan’, furthermore MBC3F1.34 and MBC3F1.34.2 had strikingly similar characteristics to ‘Mutiara’. Therefore, several individual plants carrying the Sletr1-2 mutant allele in these four genotypes can further assemble longer tomato fruit shelf life and high-yielding varieties.


Introduction
Tomatoes (Lycopersicum esculentum L) are an essential source of nutrition and are widely consumed worldwide.However, post-harvest losses are a significant problem in the tomato supply chain.Postharvest losses refer to damage and wastage of crops from the time they are harvested until they reach the consumer.The vegetable distribution system in Indonesia, which is quite long and involves at least five business actors [1], can increase the chance of post-harvest losses to tomatoes.So far, tomato post-harvest yield losses are relatively high, reaching 20% [2] and even 40% at harvest [3].These losses are caused by physiological, pathological, and environmental factors that can impair tomatoes' 1255 (2023) 012012 IOP Publishing doi:10.1088/1755-1315/1255/1/012012 2 quality and shelf life.These losses can have a significant impact on the profitability of tomato farmers, as well as food security and availability for consumers.Finding ways to reduce post-harvest losses is critical to maintaining tomato production and supply sustainability and ensuring consumers' availability of high-quality and nutritious tomatoes.
An approach to this issue is developing tomato cultivars with an extended shelf life.Previous research has shown that genetic mutations that affect specific genes can alter fruit ripening, senescence, and shelf life [4] [5].Hybridization is a promising strategy for developing new tomato varieties with desirable traits, as it involves crossing two genetically different individuals.Using mutants as parent plants in hybridization can introduce these genes into superior types, creating new tomato hybrids with an extended shelf life.
Despite its potential, one challenge with this approach is that unfavourable characteristics of the parent plants can also be passed down to their offspring, which could negatively impact the commercial viability and consumer acceptance of new varieties.These negative traits could include poor fruit quality, disease susceptibility, or decreased yield potential [6].However, by combining traditional breeding methods and modern molecular techniques, it may be possible to develop new tomato varieties with a better shelf life and other desirable characteristics like disease resistance and increased yield potential.
Backcrossing is a plant breeding technique that repeatedly crosses offspring with improved varieties to introduce desirable traits.This method allows breeders to develop new varieties with both parents' best features and retain the improved variety's genetic background.Backcrossing provides reduced time and resources to develop new varieties, improved adaptability and resilience, and enhanced genetic diversity among existing varieties.The backcross in this study was carried out to restore the superior characteristics of commercial parents that changed due to the introgression of the Sletr1-2 mutant allele.Backcrossing was performed several times so that a large proportion of the genome of the recipient parent could be recovered and a small portion of the desired donor parent introgression was retained [7].
On the other hand, backcrossing leads to diversity within a population.To develop effective and efficient breeding and selection strategies, one must conduct an analysis that reveals the degree of diversity and similarity.This study aimed to investigate the phenotypic diversity of the third backcross of F1 generation (BC3F1) tomatoes resulting from crosses between commercial and Sletr1-2 mutant tomatoes and to gain the advanced backcross genotype with high similarity to their commercial parents.The ultimate objective was to produce tomatoes with the mutant allele Sletr1-2 and similar desirable traits to their commercial parents.
This study did not use a research design.Planting was carried out in experimental plots without replication.The characteristics observed were plant height (cm), number of flowers and fruit per bunch, fruit weight per plant (g), the average weight per fruit (g), fruit length (mm) and diameter (mm), the pericarp thickness (mm), and the number of locules.Furthermore, mutant alleles were observed from the plant's phenotype, seen in the flower crowns that did not wither even though the fruit had formed.
Determination of the criteria for the phenotypic variability of the backcross population is done by comparing the phenotypic variance with the standard deviation.Observational characters have a high variability value if the variance value is equal to or greater than twice the standard deviation of the variance (σ 2 f > 2Sd σ 2 f).The following is the formula for estimating the phenotypic variance [8]: The similarities in the tested tomato genotypes will show the genetic relationship proximity between these genotypes.Cluster analysis between genotypes will be carried out with the application SPSS 23.A T-test was used to statistically compare the BC3F1 genotype's performance to the parent plant.The calculations were as follow:

Result and discussion
Based on the observations of quantitative traits in the 'Intan' BC3F1 and 'Mutiara' BC3F1 populations (Table 1), the results showed high variance values for plant height, fruit weight per plant, fruit weight per fruit, diameter, and fruit length, following [8].In contrast, variance estimates for other traits were narrow, except for the flower per cluster trait in the 'Mutiara' BC3F1 population.The high variance in the backcross population indicates significant genetic variation among the backcrossed individuals, even though they come from the same population or have the same parents.Variability is a critical factor in the successful development of superior plant varieties [9].Genetic heterogeneity of the parents in their cross combinations is essential for generating variability [10] and provides a wide range of opportunities for trait improvement [11].Environmental elements are also believed to have contributed to this study's high diversity of traits [12].This high diversity is because most of the genome of the recurring parent, the commercial variety, has not yet been passed on to the offspring.Characters with high variability values offer more excellent opportunities for selection to obtain the desired traits [13].Selection is a crucial step in developing superior cultivars [14], and understanding the variability of characters in a population is crucial for effective selection.Therefore, selecting individuals with desirable traits and backcrossing in a more uniform environment should be conducted to obtain offspring that carry the donor genes and have most of the genes of the recurrent parent.Conversely, Characters with narrow variability indicate that the phenotype of these characters is relatively uniform, so selecting these characters may not be effective [15].
Based on the cluster analysis results shown in Figure 1, some backcross genotypes are similar to the 'Intan' variety.At the 85% similarity level, five genotypes are similar to 'Intan'.Furthermore, at the 90% similarity level, two of the six backcross populations are similar to 'Intan', namely IBC3F1.37.2 and IBC3F1.45.3.One of the expectations in this research is obtaining a genotype with characteristics like 'Intan' but carrying the Sletr1-2 gene from the donor parent that extended shelf-life.Therefore, both genotypes above can be selected to be continued in the following breeding activities.Cluster analysis is intended to classify data (observations) into several classes (groups).Grouping based on plant phenotypes and estimating genetic kinship is beneficial to determine the similarity or closeness of these lines.Grouping can identify whether the lines have similar or almost identical characteristics and have close kinship relationships [16].Grouping at 90% similarity for the BC3F1 population with the genetic background 'Mutiara' resulted in three clusters.In this grouping, two populations have similarities with the 'Mutiara' variety, namely MBC3F1.34, and MBC3F1.34.2.At the same time, the grouping at a similarity level of more than 90% for both the 'Intan' and 'Mutiara' genetic backgrounds showed that none of the BC3F1 population genotypes had similarities with their commercial parents.The characteristics used in this grouping were plant height, flowers per bunch, fruit per bunch, fruit weight per plant, weight per fruit, fruit length, fruit diameter, number of locules, and fruit flesh thickness.The genetic similarity based on the vegetative characters and components of the yield cannot accurately show the kinship relationship between populations because these characters are generally still influenced by environmental factors.However, the information obtained is helpful in quickly knowing the similarity (genetic distance) between genotypes.The similarity of characters in the tested tomato genotypes shows the proximity of the genetic relationship between these genotypes.The selection of BC3F1 genotypes that differ from 'Intan' and 'Mutiara' and have a high variability is still adequate.
The analysis of the difference in mean values using the t-test showed that the 'Intan' variety and the IBC3F1.45.3 genotype had similarities in all the observed characters, as presented in Table 2.In comparison, the IBC3F1.37.2 genotype still has one characteristic that differs from Intan: the number of flowers per cluster.Based on the cluster analysis results, these two genotypes are more similar to 'Intan' than the other four.These results reveal that in terms of vegetative growth and productivity, indicated by yield per plant, these two genotypes have the same advantages as 'Intan.'In addition, these two genotypes have their advantages compared to 'Intan,' namely carrying the Sletr1-2 gene that controls the shelf life of the fruit.Therefore, the two genotypes can be observed further to see their feasibility of being released as superior varieties with a long fruit shelf life.A t-test analysis on 'Mutiara' and its backcross population revealed that one genotype, namely MBC3F1 34.2, had no difference with 'Mutiara' in all the observed characters.The backcrossing has returned almost all the genome proportions of 'Mutiara' in that genotype.Meanwhile, in the population of MBC3F1.34,there are still several characteristics that are different from Mutiara, namely plant height and fruit diameter.The plant height of the MBC3F1.34genotype was still shorter, and the fruit diameter was less than 'Mutiara.'The lower performance of plants and fruit in the backcross population is caused by the genetic background of the Sletr1-2 mutant, namely Micro-Tom, which has dwarf plants and small fruit sizes [6].
Plants with the Sletr1-2 allele display a distinct phenotype in their flowers compared to other plants [17].Specifically, the corolla of their flowers undergoes delayed senescence, as shown in Figure 3.As a result, even one week after pollination, when the fruit has already developed, the corolla maintains its fresh appearance.This phenomenon is likely due to the inhibiting activity of ethylene, a hormone that typically promotes flower aging [18].Six plants were selected from the four backcross populations that had high similarity with their parents and showed the presence of the Sletr1-2 allele.Therefore, these six plants can be considered carriers of the Sletr1-2 allele, which possesses the same advantages as its parent and can be used as breeding material to produce high-quality, long-shelf-life tomatoes.The selection of tomato commodities focuses on plants that yield high-quality fruits and high production.Following the backcrossing and selection of the desired parent phenotype, the resulting genotype will be identical to the parent, except for the introgressed genes from the donor, which can be reduced in the offspring due to repeated backcrossing.

Conclusions
The BC3F1 populations of "Intan" and "Mutiara" exhibit high variance values for multiple traits, indicating significant genetic variation among the backcrossed individuals.Cluster analysis and a t-test suggest that the genotypes IBC3F1.37.2 and IBC3F1.45.3 are similar to "Intan," while MBC3F1.34 and MBC3F1.34.2 display strikingly identical characteristics to "Mutiara."Furthermore, six individual plants carrying the Sletr1-2 mutant allele in these four genotypes can produce longer-lasting tomato fruit and high-yielding varieties.
phenotype variance   = the individual value in the data set. = the total number of data in the set = the t-value or test statistic ̅ = the sample means. 0 = the assumed population means in the null hypothesis. = the sample standard deviation  = the sample size.

Figure 3 .
Figure 3.The visual appearance of the corolla in plants carrying the Sletr1-2 allele.