Selection of Tolerant of Some Citrus Hybrids F1 to Calcareous Stress and Identification Sexuall Individuals by SSR Marker

The research was conducted during two seasons 2018-2019 and 2019-2020, at the Scientific Research Center in Lattakia-Syria, where the cross between Sour orange, Cleopatra mandarin and Trifoliate orange. The first season was (Sour orange ♂ × Cleopatra mandarin ♀) and (Cleopatra mandarin ♂ × sour orange ♀) and (sour orange ♂ × trifoliate orange ♀), and the crossing in the second season was (Trifoliate orange ♂ × Sour orange ♀), and (Trifoliate orange ♂ × Cleopatra mandarin ♀). The plants of the first generation F1 were exposed to calcareous stress according to two methods. The first one for a long term (two years) method was grown in plots containing 15% and 35% Calcium Carbonate and the control free of Calcium Carbonate for the first cross, and the second one short term (two months) by watering method was with a solution of 1 mol and 2 mol of Calcium Carbonate for the second cross. The indicators of proline and chlorophyll A, B and T were measured in the first-generation individuals, once at the beginning of the experiment and a second time at the end of study. Seven SSR primers were used to identify sexual individuals, five primers were able to identify sexual F1 individuals are CSM13, CSM17, CSM147, CSM18 and TAA27, F1 individuals in the first season (mixture experiment) gave high values of chlorophyll and were tolerant to calcareous stress from crossing Cleopatra mandarin and Sour orange, where they did not show a deterioration in chlorophyll, but rather increased chlorophyll values, and the Trifoliate orange hybrids were sensitive to this stress, and in the watering experiment the F1 showed significant deterioration in Chlorophyll in all seedlings, and symptoms of Lime-induced chlorosis appeared on the more sensitive seedlings.


Introduction
In Syria, Citrus is important fruit tree, it's 42% of total fruit production ( Figure   1) [1]. It is mainly cultivated in Lattakia, but suffers from Lime-induced chlorosis in calcareous soils that contain high concentrations of HCO 3 -bicarbonate in the soil solution, which causes a high pH (7.5 -8.5), and affects the metabolic processes in roots and leaves of citrus [2]. The genus Citrus belongs to the family of Rutaceae and subfamily of Aurantioideae [3].
Rootstocks have a major role in the success of citrus cultivation because they play an important role in the growth, development and production [4]. More than 20 horticultural characteristics are influenced by the rootstock including adaptation to certain soil conditions such as high pH [5]. Sour orange is one of the most major rootstocks in Citrus fruits, but it began to be dispensed with after CTV (Citrus Tristeza Virus). However, in Syria, it is still the only rootstock in citrus nurseries.
Hybridization in citrus aims to obtain tolerant rootstocks to biotic and abiotic stresses [6]. The problem of high calcium carbonate in the soil is also one of the most important nutrition problems [7]. Cultivation of economic citrus cultivars on suitable rootstock tolerant of environmental conditions is one of the environmentally friendly techniques [8] [9].
The aim of this research is to evaluate the F1 individuals, then select the more tolerant to Lime-induced chlorosis, by chemical indicators. Hybridization carried out three parents namely: Sour orange, Cleopatra mandarin and Trifoliate orange, then, identification the nucellar (asexual) and zygotic (sexual) seedlings of F1 by PCR based SSR markers.
Proline is one of the most abundant amino acids in the tissues of Citrus. It contains a non-free amino group (NH2). It is an important soluble nitrogen solution in citrus leaves [10]. Proline accumulation is important primary indicator in determining the degree of tolerance of plants to abiotic stresses and has a role as protective osmolyte compound at the cellular level in the internal osmotic adjustment of plants exposed to stress [11] [12]. It usually accumulates in plant tissues in response to its adaptation or sensitivity to certain stresses such as low temperatures, salinity or lack of water, and this can be known early during the plant cycle. Increasing the level of Proline content protects the plant from stress but also acts as part of a cascade of signals to alter plant development [13]. It is a protective factor against swelling and tolerant rootstocks can accumulate larger amounts of Proline [14]. Tolerant genotypes released more Proline than sensitive ones, there is a relationship between the resistance of the genotype to a specific stress state with the amount of accumulated amino acids, Proline accumulated in the root can be used as an early sign of the degree of stress tolerance, as the plant may not accumulate proline in the leaves to a large extent if it is not stressed enough to accumulate it [15].
Chlorophyll is the basic pigment that performs photosynthesis by absorbing light and converting it into chemical energy, so life on Earth depends largely on chlorophyll [16].
The ratio of chlorophyll A to chlorophyll B in plants has been used as an indicator of response to light shade conditions [17] [18], The low value of chlorophyll A/B refers to sensitive genotype to environmental stress [19]. The color of Citrus leaves varies greatly from dark green to light green, due to the amount of chlorophyll in the leaves, and even in the same plant, it varies according to the growing season, the maturity of the leaf, light intensity, nutrition, stress and rootstock. However, after exposure to various stresses, chlorophyll begins to break down, and in general, is affected by the stresses [20] [21]. Chlorophyll shows damage at a high level of stress [22]. Lime-induced chlorosis in the Mediterranean region is one of the main abiotic stresses affecting fruit trees [23]. [24] studied the chlorosis of 16 rootstocks in high pH conditions in growth chambers and found that Sour orange, Cleopatra mandarin was the most tolerant, Pomeroy trifoliate (Pon) trifoliata var. Pomeroy), was sensitive and the local Trifoliate orange was most sensitive to calcareous soil and high pH. The researchers note that the Trifoliate suffers more severely from chlorosis than the other rootstocks, while Cleopatra mandarin orange was showed little or no symptoms of chlorosis [24] [25] [26]. The values of total chlorophyll were significantly higher in resistant rootstock than in sensitive ones [24].
Microsatellites or simple sequence repeats are short sequence composed of repeat units one to seven basepairs [27]. These sequences are highly polymorphic and used in population genetics [28], and genome mapping [29]. SSR markers are becoming popular due to high polymorphism, co-dominance, simplicity of analysis and repeatability [30].  [31]. In most of the crossing combinations, aimed at citrus improvement, it would not be possible to determine the genetic origin of seedlings from morphology. Identification of young seedlings is very essential for the plant breeders to avoid 5 -10 years' cost of growing and maintaining unwanted nucellar seedlings. Thus, it is mandatory to identify the zygotic seedlings while discarding the nucellar ones for smooth running of citrus breeding programmes. Genetic studies using microsatellite markers have increased rapidly because these are highly polymorphic, heterozygous conserved sequences that can be used as co-dominant markers [32].
[33] evaluated primeres CSM17, CSM147, CSM13, CSM18 in distinguishing hybrids from nucelling seedlings caused by Citrus. reticulata Blanco. and they gave high efficacy. [34] also used SSR indicators to identify the sexual from asexual of Mexican lemons seedlings from previous cross-breeding programs for disease resistance, such as the CTV and Huanglongbing (greening) that were threatened in citrus cultivation.

Plant Material
Hybridization has been made between three Citrus rootstocks: Citrus aurantium L. (Sour orange), it is good compatibility with commercial cultivars, and tolerant of calcareous soils [35].
Poncirus trifoliata (L.) Raf. (Trifoliate orange) It is deciduous rootstock, becoming very dormant and extremely freeze-hardy, used as a rootstock in cold regions [37]. It is an important genetic source for Citrus rootstocks genetic improvement programs, for stress tolerance and diseases resistance [38]. Important rootstocks were obtained by cross-breeding between Citrus and Trifoliate orange [39].

Hybridization
The crosses were carried out for two successive seasons 2018-2019 and 2019-2020 during March and April of each season, more than 1500 flowers for each seed parent, and the crosses were as Table 1.
The hybrid seeds of Sour orange and Cleopatra mandarin were planted in  Hybrids were transferred to 2 kg planting pots containing an earthen mixture consisting of turb, sand and soil in equal proportions.
1) Season 1, 2018-2019: The hybrid seedlings for the first season of crossing were transferred in April 2019 to previously prepared soil mixtures according to Table 2.
2) Season 2, 2019-2020: As for the f1 individuals of the second season, were transferred in September 2019 to 2 kg agricultural pots and soil containing traces of calcium carbonate to start the experiment of the calcareous stress watering at the beginning of October. The treatments were as Table 3.

Proline
The proline content of leaves was measured before stress and after stress according to [40].
extention volum of extraction solution concentration ppm proline mg g plant tissue sample solution volum weight extract sample 1000 Chlorophyll will be extracted according to [41]. Chlorophyll A, chlorophyll B and total chlorophyll were calculated according to equations:  Electrophoresis PCR products were analyzed using 2 percent agarose gel% (1% agarose and 1% PeqGold MoSieve Agarose) containing ethidium bromide. Fragment sizes of PCR products were estimated from the gel by comparison with 50-base pair DNA ladder marker. The bands were recorded as present (1) or absent (0) and complied into a two-way matrix (accession × marker), were estimated from the gel by comparison with 50-base pair DNA ladder marker.

Statistical Analysis
The significant differences between the values of the studied indicators (Proline, Chlorophyll) before and after the stress were calculated using the T-student for the linked samples.
Criticale Difference was calculated to determine the significant difference of the studied indicator in the special coping ability of the individuales after exposure to stress.
V. E. Hasan et al. The molecular seedlings were compared with the profile of the mother for ascertaining the nature of the seedling. Proline before and after stress is significant (Figure 3).

2) Season 2
Calcareous stress 1 mol The highest value of Proline is (2.43) mg/g in the hybrid S102 (sour orange ♀) before exposure to stress (Figure 4).
V. E. Hasan et al.   Critical Difference indicates that superiority of hybrid K78 (seed parent Cleopatra mandarin) over the rest of the crosses, and the superiority of hybrid K71 (seed parents Cleopatra mandarin) over all hybrids except K80 and K79, there are no significant differences between the latter. in the rest of the crosses. There is no significant difference in the amount of chlorophyll A in individuals exposed to stress 15% ( Figure 6).

Chlorophyll B
There is a significant difference in the concentration of Chl. B at 15% stress, the highest concentration of it is 0.55 mg/g for hybrid B17 (Figure 7), because tolerant of its parent seed (Cleopatra mandarin ♀), and 0.54, 0.53 for hybrids A14 (sour orange ♂) and B15 respectively. The lowest value is 0.25 for hybrid B4 and 0.32 for hybrid B16, and there are many hybrids that have deteriorated and exited from the experiment such as A27 and C15.

Clorophyll T
There are no significant differences in the Chl. T in the date 2 compared with date 1 (Figure 8), the highest concentration is 1.61 and 1.48 for hybrid A11 and B15, respectively, and the lowest value was 0.78 for hybrid B4.

Calcareous stress 35%
Chlorophyll A Figure 5. Accumulation of proline at the individuals exposed calcareous stress 2 mol.    stress, but it is not significant (Figure 11).

2) Season 2
Calcareous stress 1 mol Chlorophyll A The Chl. A decreased after stress with a high statistical significance ( Figure 12).

Chlorophyll B
Chl. B in all individuals destructs after calcareous stress with a high statistical significance. The highest value of Chl. B is (0.761, 0.813 and 0.663) mg/g of (S89, K50 and S85) respectively. The lowest concentration of Chl. B is (0.015 and 0.029) mg/g of (K49 and K46) respectively. According Critical Difference, that hybrids S89, S85, and K50 are significantly superior to all hybrids, no significant difference between S89, K50, and between S89, S85 ( Figure 13).
Chlorophyll T Figure 9. Chlorophyll A concentration in two dates, the first after six months of the experiment and the second at the end of the experiment after two years at 35% calcium stress.
V. E. Hasan et al. Figure 10. Chlorophyll B concentration in two dates, the first after six months of the experiment and the second at the end of the experiment after two years at 35% calcium stress. Figure 11. Chlorophyll T concentration in two dates, the first after six months of the experiment and the second at the end of the experiment after two years at 35% calcium stress.  . Chlorophyll B index in individuals exposed to calcareous stress 1 mol. Figure 14. Chlorophyll T index in individuals exposed to calcareous stress 1 mol.

Calcareous stress 2 mol
Chlorophyll A The value of Chl. A decreased in all individuals (seed parent Cleopatra mandarin and Sour orange) after calcareous stress, decrease is significant. The highest value is 1.584 mg/g for S99, (0.801) for S97 and (0.51) mg/g for S98 (the seed parent is Sour orange), the hybrids K69 and K76 are valued (0.264, 0.263) respectively which the seed parent is Cleopatra mandarin. According to Critical Difference, the hybrid S99 is significantly superior to all crosses followed by the hybrid S97 which is morally superior to the rest of the hybrids, the S98 hybrid is morally superior to the rest of the hybrids (Figure 15).

Chlorophyll B
The value of Chl. B decreased after stress with a high statistical significance ( Figure 16 Figure 17).
When plotting the graph of the total chlorophyll and Proline values at the end of the experiment, the higher values of both total chlorophyll and Proline is for the most tolerant rootstocks compared to the sensitive ones, and the higher the sensitivity of the rootstocks is the lower of Proline, so total chlorophyll in their leaves ( Figure 18 and Figure 19). Figure 15. Chlorophyll A index in individuals exposed to calcareous stress 2 mol.

Molecular Marker
Seven SSR primers were tested for discrimination of hybrids from the nucellar seedlings of F1 ( Figure 20). Tow SSR primers selected from TTA series TTA15, were not able to discriminate seedlings and generated single amplicon after PCR reaction, but the primers selected from CCSM series (CCSM13, CCSM17, CCSM18 and CCSM147) and TTA27 were able to differentiate sexual seedlings ( Figure 18). [43] were able to identify hybrid with primers from TTA series. TTA15 proved useful for hybrid detection, but in our experiment only a Figure 20. Visual symptoms of calcareous-induced chlorosis in individuales F1 exposed to calcareous stress 1 mol and 2 mol. There is a similarity between individuals and parents of the absence or presence of the amplicon (Figure 21), therefore the individuals which have difference with the seed parent, those were regarded a hybrid. In our study, all the studied individuals recorded a difference with the seed parent and she was a hybrid.
According to Figure 22, the studied individuals were divided into two groups, a group that included individual S97 independently, and a group Z that included several clusters, including group Z1 individuals B, C, and S86 within one cluster, and individual A3 independently, and group Z2 that included individuals S107 and C11 within the binary and the other cluster which contained individual B12 separately, A and B21 that formed a binary As for the genetic similarity values between individuals tolerant of calcareous stress, Table 5 shows the highest value was between individuals B21 and B12, reaching 0.875, and the lowest value was 0.25 between individuals (S97 and B21) and individuals (S97 and A3).

Discussion
According to variation in the behavior of the individuals of F1 exposed to calcareous stress, some of the hybrids were not affected by the stress 15%, but rather the concentration of total chlorophyll increased as the hybrids B7, B17 and B15 (Sour orange ♂ × Cleopatra mandarin ♀), Which parents are known tolerant to calcareous stress and A14 (Sour orange ♂ × Trifoliate orange ♀) its pollen parent is also tolerant, this applied stress is low for them, so chlorophyll was not affected at this stress, the [22] found that chlorophyll was damaged at high level of stress.
In 35%, total chlorophyll increased in F1 individuals of the same parents as B21, B3, B2 (Sour orange ♂ × Cleopatra mandarin ♀), and C3 (Cleopatra mandarin ♂ × Sour orange ♀). On the other hand, we observed the accumulation of proline in all hybrids in all stress treatments, which protects the plant cells from damage, as a protective osmolyte compound in response to stress, it has an important role at the cellular level in the internal osmotic of plants exposed to stress [10] [12] [40], we obtained tolerant hybrids by crossing (Sour orange × Cleopatra mandarin).

V. E. Hasan et al. American Journal of Plant Sciences
While watering method (1 mol and 2 mol) calcareous solution, caused clear symptoms of Lime induced chlorosis ( Figure 19)associated with a significant deterioration of chlorophyll, the reason may be that pollen seed is Trifoliate orange (sensitive), the trifoliate hybrids suffer severely from chlorosis in calcareous soil, while no symptoms on Cleopatra mandarin and Sour orange hybrids [24] [25] [26].
Long term (two years) made the individuals able to adapt to conditions of calcareous stress, and tolerant ones kept the Chlorophyll T in 15%, 35% treatments, according to [23] the values of total chlorophyll were significantly higher in resistant rootstocks than in sensitive ones. In contrast, the short term (two months) of application stress show clear symptoms of Lime-induced chlorosis are noted in Figure 20, these symptoms are due to deterioration of chlorophyll, and the chlorosis varies between plants depending on several factors, including genetic and the surrounding conditions [19] [20].
The careful analysis of different groups of parents is essential for the progress of citrus breeding programs and this made the high accuracy of the SSR primers effective in identifying the vegetative plantings by matching them to the parents, the mother.