Determination of Genome Size Differentiation and Ploidy Levels in Some Citrus Rootstock Populations

In plants, knowing the ploidy level of plant material used in breeding studies, and especially for biotechnology applications, carries great importance. The presence of a rapid variety of dynamics in citrus fruits allows their use as rootstock and varieties ensuring adaptability to various climate and soil conditions with different breeding methods. A variety of appropriate rootstocks are used for commercial citrus species. This study investigated the genome sizes and ploidy levels in citrus rootstocks commonly used around the world with ow cytometry in seedling populations. The study used Gou-Tou, C-35, Troyer Citrange, Taiwanica, Citremon, Yuzu, Sunki mandarin, Flying Dragon, Yuma Citrange, Macrophylla and Chinese orange rootstocks. Fresh leaf tissues were mixed with the triploid Tahiti lime leaf tissue, used as standard species, and cell nuclei were isolated. Cells stained with propidium iodide were read with ow cytometry and histograms and cytograms were obtained. According to the obtained results, all seedlings of species had diploid genome volumes. In terms of genome volume, there were differences found between species. Yuzu seedlings were determined to be the species with largest genome volume (0.808 pg/2C), while Flying Dragon trifoliate had smallest genome volume (0.700 pg/2C).


Introduction
Citrus, originating in southeast Asia, are commonly cultivated in Turkey as in the rest of the world.
According to the FAO Statistical Database [9], 4,769,726 tons of citrus fruits from different species were produced in Turkey in 2017 contributing to 3.25% of the total citrus production in the world. Citrus fruits have a very signi cant share of global fruit production. Citrus species have been cultivated in subtropical and tropical regions for years. Rootstock selection is very important for citrus cultivation. Rootstock has very large effects on tolerance to cold and diseases, adaptation to different climate and soil conditions, fruit yield and quality. As a result, the use of rootstocks for citrus species has become mandatory. An ideal citrus rootstock has high ability to adapt to different soil conditions, is compatible with citrus species and varieties, resistant to biotic and abiotic stress conditions and has high polyembryony rates. Gou-Tou, C-35, Troyer Citrange, Taiwanica, Citremon, Yuzu, Sunki mandarin, Flying Dragon, Yuma Citrange, Macrophylla and Chinese citrus rootstocks are among the rootstocks commonly used in citrus cultivation [6].
As a signi cant proportion of citrus rootstocks have a tendency toward nucellar embryony, seeds display the same characteristics as main plants and they display performance like clonal proliferation.
Genome volumes are very important for identi cation of signi cant citrus genotypes. With ow cytometry analyses, genome volumes and different ploidy structures may be determined in citrus species and varieties. For calculation of the nuclear DNA content of a species, it is necessary to determine ploidy level, chromosome counts and how big chromosomes are. Before beginning breeding studies, differences in ploidy level between individuals to be used as genotype and identi cation of these differences is very important in terms of breeding studies being successful. Flow cytometry is currently the most sensitive, rapid and reliable method used for determination of nuclear DNA content, and use with this aim has become more common in recent years [34]. Determination of ploidy level in plants with traditional methods includes counting chromosomes during mitosis division in preparations made from root tip tissues with light microscopy; however, this method is very demanding, slow and performing ploidy analysis of many plant species takes time. Additionally, it is not useful for determination of ploidy levels in plant species with small chromosomes and high ploidy levels, and may cause misclassi cation of species. As the plant sample for ploidy level determination increases, the light microscopy method may not be su cient. As studies may not be su cient for determination of ploidy level using root tip tissues, the ow cytometry method has become a method chosen for ploidy analyses in recent years due to being convenient, rapid, sensitive and reliable [3,13,35]. As chromosomes are found in the nuclei of cells in plants, there is a close correlation between the nuclear DNA amount and ploidy level. As a result, due to the convenience, speed and reliability of the ow cytometry method, in recent times ploidy analysis has been performed by determining the nuclear DNA content in plant cells with the ow cytometry method.
Flow cytometry analysis has many advantages compared to the chromosome count method in traditional methods such as preparation of samples being easy, it is rapid and there is no need for a root tip cell in mitosis to perform analyses, just a small leaf tissue is su cient [7]. Flow cytometry was rst developed as a method to identify the nuclear DNA content of organisms and it is a very important and easily applied method in these studies [30]. The ow cytometry method is a method based on analysis of light after being absorbed by cells as they pass singly through uorescence detectors generally. However, in order to ensure light absorption by cells, the cell nuclei from enzymatically degraded leaf samples are freed and cell nuclei stained with uorescent stains like DAPI or PI are used to determine nuclear DNA amounts. With the ow cytometry method, one person can analyze over 100 plants per day, it performs chromosome counts very rapidly, provides accurate and reliable results, and increases signi cance and this situation has led to ow cytometry currently being the most chosen method for chromosome counts.
However, the disadvantage of the method is that it must be set to the plant species to be analyzed [8]. Applications where ow cytometry is commonly used in plants include determination of ploidy level, nuclear DNA content and estimation of genome volume [25].
In this study, the nuclear DNA amounts in seedling populations of citrus genotypes were examined with the ow cytometry method and ploidy levels and genome volumes were determined. Thus, the ploidy levels between genotypes were investigated for presence of signi cance from a statistical viewpoint.

Plant Materials
In this research seeds and seedlings were used during the experiment.
Two hundred seeds from the following citrus rootstocks were sown and germination rates were precisely determined during four weeks. Seedling populations comprising 100 plants from each rootstock were obtained.
Gou Tou Sour Orange (Citrus aurantium L. var. Gou Tou): A rootstock of Chinese origin, apart from being resistant to tristeza disease, there is no information about soil wants or resistance to other diseases. In Florida plants grafted onto Gou Tou rootstock have larger crown structure compared to those grafted on other bitter oranges [31]. Gou Tou sour orange is reported to reduce yield in grapefruits [16]. Gou Tou sour orange is tolerant of Phytophthora citrophthora and Phytophthora parasitical diseases [17].
C-35 citrange (Poncirus trifoliata (L.) Raf. X Citrus sinensis. Osb. 'Ruby'): A rootstock obtained by hybridization of Ruby Blood orange and trifoliate orange. It is tolerant of gummosis (Pytophthora citrophthora (Smith and Smith) Leon.) and Tristeza diseases and resistant to nematodes. Resistance to cold is equivalent or slightly better than Carrizo citrange. Trees have moderate size and those grafted on Troyer have 25% smaller crown. Good compatibility with sandy, sandy-clayey and clayey soils; however, is more susceptible to limey soils than Carrizo citrange [31].
Troyer citrange (Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osb.): A sweet orange and trifoliate orange hybrid. Generally, it has trifoliate properties, with more compliant characteristics in terms of environmental conditions and compatibility with varieties so it is used more often in recent years and in most cases is chosen as alternative to sour orange rootstock. Proliferation with seed and grafting is easy, growth is moderate, yield is high, maturation and fruit setting are early, effects on fruit quality are high and economic lifespan is at moderate levels [23].
Alemow (macrophylla) (Citrus macrophylla Wester): Important features are resistance to salinity and boron. Generally, it has good compatibility with all varieties. However, it is used as rootstock for lemon and lime mainly due to susceptibility to Tristeza and Xyloporosis diseases. It appears to be tolerant of Exocortis and Psorosis diseases. Varieties grafted on it grow rapidly and set early fruit. However, quality of fruit is negatively affected. It is susceptible to cold.
Flying Dragon (Poncirus trifoliata var. Monstrosa): The trifoliate clone Flying Dragon was found in America in 1915. This rootstock ensures tight crown formation for lime, grapefruit and tangelo, and has the effect of dwar ng mandarin and orange. It is very sensitive to calcium and chlorosis. It develops excessively slowly on mild sandy soils. It is resistant to tristeza (CTV) virus and Phytophthora spp. (root neck rot). It is sensitive to Exocortis. The Eureka group showed incompatibility with lemons. Due to showing dwar ng effect on all citrus types and varieties, it is appropriate for dense planting, ensuring convenient harvesting of grafted varieties. It has positive effects on fruit quality, like the trifoliate rootstock. The body having zigzag form and many thorns makes grafting di cult [2]. The Flying Dragon rootstock which is resistant to cold and humid conditions and sensitive to high-lime soils, does not have a tendency to form nucellar plants at high rates [1,10].
Sunki mandarin (Citrus sunki (Hayata) hort ex. Tanaka): It is very widely used as rootstock in China. It is tolerant of Tristeza and Xyloporosis, but sensitive to Exocortis. Studies have reported Sunki is susceptible to Phytophthora brown rot. This rootstock is tolerant of salt, has moderate resistance to low temperatures, and can withstand chlorosis in limey soils. It is a polyembryonic rootstock.
[16] reported adaptation to limey soils was good, and that it was tolerant to iron chlorosis. Fruit yield, fruit juice amounts and sugar content in fruit juice was equivalent or superior to fruit obtained from trees grafted on bitter orange [31].
Yuzu (Citrus junos Sieb. ex Ten.): It is a common rootstock in the southern regions of China. From China, production spread to Japan and it forms an important commercial rootstock in Japan. Proliferation from seeds is easy, with slow growing features. It is a rootstock with high fruit quality and yield. It is resistant to phytophthora, fungus and nematodes. It is tolerant of tristeza, dwar ng and spalling diseases. It has moderate levels of resistance to limey and salty soils. It has high resistance to low temperatures and polyembryony tendency.
Taiwanica (Citrus taiwanica Tan. and Shim.): It is a rootstock that is easily proliferated from seeds, and has moderate levels of tree growth, fruit quality and yield. It is resistant to phytophthora disease, very susceptible to fungal disease and susceptible to nematode damage. It is tolerant of tristeza, dwar ng and spalling diseases. It has moderate levels of resistance to limey soils, with weak resistance of saline soil conditions. It has moderate resistance to low temperatures and is a rootstock with very high tendency for polyembryony.
Yuma Citrange (P. trifoliata × C. sinensis): A trifoliate orange hybrid. It matures in the months of October-November. It is a rootstock susceptible to iron de ciency. It has smaller fruits than Citrumelo. It forms trees of moderate size, with trifoliate leaves, and crown volume in global structure. It has low tendency toward polyembryony, and forms zygotic plants at high rates [12]. It is a very suitable rootstock for grapefruit. In terms of features like susceptibility to disease and nematodes, fruit quality and adaptation to soil types, Yuma citrange is similar to Carrizo and Troyer citranges.
Chinese bitter orange (C. myrtifolia Ra nesque): Chinese bitter orange, susceptible to CTV, matures from January-March. Leaves are small and don't have pointed tips. Fruit are small and rounded, with variability in seed numbers from low to high, and it forms small tress. The peel of the fruit has moderate roughness, with color ranging from orange to dark orange. It originated in China.

Isolation And Staining Of Nuclei
To release cell nuclei, approximately 50 mg of fresh tissue form each seedling leaf was mixed with Tahiti lime (Citrus latifolia Tan.) leaf pieces, which were used as a control, and chopped into small pieces with a sharp razor in a sterile Petri dish containing 300 µl of nuclei buffer (pH7.4) of the following composition: 0.14 M NaCl, 0.003 M KCl, 0.012 M NaH 2 PO 4 , 0.002 M KH 2 PO 4 , 0.1% Triton 100, 50 µg of RNAse and 100 µl of dithiothreitol. For measurements of absolute DNA values, Tahiti lime leaf tissues were included as an internal standard, as previously described by [19,20]. Tahiti lime was described as triploid and nuclear DNA content was found to be 1.17 pg/2C by [19,20]. The suspension was ltered through a 50 µm pore nylon lter into microcentrifuge tubes. After ltration, 100 ll (1 mg/ml) of propidium iodide was added for staining of the DNA. Then the suspensions were incubated for approximately 5 min at room temperature.
After incubation, each sample was run on a ow cytometer [33].
For estimation of DNA content of nuclei, the relative uorescence of nuclei was measured by using a CA-III Flow Cytometer (Partec GmbH, Münster, Germany) with an Argon laser light source operating at a wavelength of 488 nm. Histograms and cytograms were evaluated on DPAC Software (Partec GmbH, Münster, Germany). From 2000 to 5000 nuclei were counted per ow cytometry measurement. The nuclear DNA contents of different seedlings were calculated by comparison of relative positions for G 0−1 peaks corresponding to the sample nuclei and the nuclei isolated from Tahiti lime or mungbean, respectively. This permits accurate determination of the unknown DNA content [33].
Data analysis and estimation of nuclear genome size: The nuclear DNA contents of the different rootstock seedlings were calculated by comparison of the relative positions for the G 0−1 peaks corresponding to the sample nuclei and the nuclei isolated from Tahiti lime, respectively. This permits accurate determination of the unknown DNA content. Calculation was made according to the formula: where Q = unknown DNA content (pg/2C), R = standard 2C DNA content (1.17 pg), E = sample G 0−1 peak mean, and S = standard G 0−1 peak mean.

Statistical Analysis
Statistical analyses were carried out with the genome results obtained from each seedling. Signi cance of genome size variation among the seedlings was determined from analysis of variance by using SAS statistical analysis software. Analysis of variance was computed using the General Linear Model (GLM).

Results
Germination rates the seeds of different citrus rootstocks were given in (Table 1) and compared in (Fig. 2). According to the obtained results germination rates were differed statistically important among the rootstocks. The highest germination rate was found on Troyer citrange (16,3%) at the end of rst week. The lowest rates were found in Flying Dragon (5,0%), Taiwanica (5,3%) and Citremon (6,2%). Troyer citrange seeds can be considered that having earliest tendency for germination.
To summarize, 1921 seedlings were obtained at the end of seed germination studies from 2200 seeds. The number of seedlings per genotype was recorded maximum in C35 citrange (196 seedlings) whereas the minimum in Yuzu (148 seedlings) followed by Flying Dragon trifoliate orange (163 seedlings). The difference for germination rates could be due to genotypic difference. A study conducted by [5] C35 citrange had the lowest germination rate under in vitro germination conditions. Contrarily, C35 citrange had the highest germination rate in our research. The reason for the high difference between two researches could be origin of seed source and seed conservation conditions. Some of the citrus genotypes like C-35 citrange and Troyer citrange produced two or more seedlings from one single seed due to nucellar embryony.
[18] stated that Troyer citrange has the highest graft success if two weeks old seedlings used for shoot tip grafting. The seedlings obtained from each rootstock were uniform for further evaluation. Signi cance ** ** ** ** Flow cytometry analyses are rapid, preparation of nuclear suspensions is easy and statistical distribution of DNA content of large populations is completed rapidly. Cutting fresh leaf tissues from young seedlings with a sharp razor ensures mixing of large numbers of cell nuclei with the nuclear buffer solution. Leaves from young citrus seedlings and Tahiti lime were mixed and prepared for analysis and ow cytometry formed two large peaks showing G 0-1 peaks for both species. Calculation of total genome in G 0-1 peak ratio was successfully completed as published by many researchers. Results obtained from ow cytometry analyses show seedlings of the species used in this study had diploid genome. The G 0-1 peak ratios were smaller than the Tahiti lime genome used as control species. The mean genome volumes for seedling species analyzed in this study are given in (Table 2) and genome size variations among species are given in (Fig. 1). According to the obtained results, Yuzu (0.808 pg) has the largest genome while Flying Dragon trifoliate had the smallest genome (0.700 pg). There were statistical differences determined between the seedling populations of the eleven different rootstocks. The Yuzu rootstock is easily proliferated from seeds, has slow growth characteristic, and is a rootstock with high fruit quality and yield. Yuzu has high resistance to low temperatures and polyembryony tendencies. Though it is resistant to phytophthora, fungus and nematodes and tolerant of tristeza, having dwar ng effect, it has moderate resistance to lime and salty soils so commercial use in the world has not become widespread. With nucellar embryony several embryos occur from one seed and these plants carry all the characteristics of the mother plant. As a result, seedlings obtained from species and varieties with excess polyembryony tendency have diminished genetic expansion [27,37]. Seedlings occurring from nucellar embryos may be used as rootstock due to having similar features to the mother. Tetraploid plants can be found in zygotic citrus seedling populations and polyploidy level could be reach up 2.5% in Rutaceae according to the previous articles [4,29,33].
A study by [33], determined that trifoliate seedlings had the smallest genome among citrus species. As a result, both common trifoliate orange and Flying Dragon trifoliate orange were revealed to have small genomes. The Poncirus genus was determined to have smaller genome volume than the citrus genus.
Again, Sunki mandarin was found to have smaller genome volume. The study carried out by [33] determined Cleopatra mandarin has smaller genome than other citrus species.
Mandarins having small genome is considered to be effective on both species having small canopy volume, small leaves and small fruit. As a result, Sunki mandarin and Cleopatra mandarin are two valuable rootstocks especially for mandarin species and varieties. C-35, Carrizo and Troyer citrange species have larger genome compared to trifoliates. The statistical differences between them are signi cant. The reason for this is associated with citranges having larger genome volume than the other parent of orange. [5] demonstrated C-35 citrange seedlings were diploid with 0.794 pg/2c relative genome sizes. This result con rmed our ndings in C-35 seedlings.

Conclusions
Genome size refers to the amount of DNA in an organism's non-replicated haploid set of chromosomes [32]. In diploid (2n=2x) organisms, genome size refers to the amount of DNA contained in haploid (n number of chromosomes) chromosomes. Genome size is expressed as the C value, which is the amount of DNA content in the genome in picograms. The 2C value is the amount of DNA in the nucleus of a somatic cell, regardless of its ploidy level [14,35]. Signi cant (about 1000-fold) differences are observed between species in terms of genome size (C value). On the other hand, the genome size remains constant among different individuals of a species and therefore becomes species speci c. Therefore, the C values of species are extremely important for biology, genetics, taxonomy and evolution studies [21,22,24,26,28]. Flow cytometry is the newest, fast, sensitive and economical method used to determine genome size today. Since there is a very close relationship between the C values determined by ow cytometry and the chromosome numbers of the species, this parameter is also used to determine the ploidy levels of the species [15,36]. This study showed that nucellar seedling populations of Gou-Tou sour orange, C-35 citrange, Troyer citrange, Taiwanica, Citremon, Yuzu, Sunki mandarin, Flying Dragon trifoliate orange, Yuma Citrange, Macrophylla and Chinese orange rootstocks had only diploid genomes. In a study conducted by [11], similar to our study results, they reported that the Gou-Tou sour orange rootstocks they used as study material was diploid. There were no polyploids in the surveyed seedling population due to high tendency to nucellar embriyony. Polyploidy may have great potential for citrus rootstock breeding.
Autotetraploid and allotetrapoid rootstocks may have potential especially for dwar ng of citrus trees.
Declarations Figure 1 Genome size variations among citrus rootstocks (bar indicates the standard error)