Grey Correlative Degree Analysis on the Cold-Resistant Traits of Parthenocarpic Eggplant

In order to breed new eggplant variety with parthenocarpic ability and strong cold-resistance, the six cold-resistant traits of fifteen eggplant resources with parthenocarpic ability were evaluated with grey correlative degree analysis. One non-parthenocarpic resource ‘Shenggao No. 2’ was used as control. The results indicated that ‘29’, ‘32’, ‘30’, ‘XBL’, ‘31-2-1’, ‘TXQ’ and ‘31-2-2’ had strong cold-resistance; ‘HLMQ’, ‘HQ’, ‘ZHQ’, control and ‘HXZ’ had weak cold-resistance; the other four resources had moderate cold-resistance. When suffered cold stress, the resources with strong cold-resistance had a larger increase of SOD, POD, CAT activities and a least increase of MDA content and EL. Among the strong cold-resistant resources, ‘29’, ‘30’, ‘31-2-1’, ‘TXQ’ and ‘31-2-2’ had purplish red fruits and significantly stronger cold-resistance than control, which were suitable for cultivation in south China. ‘32’ and ‘XBL’ with white fruits could be used as breeding materials.


Introduction
As an agronomically important solanaceous crop, eggplant (Solanum melongena L.) is a kind of thermophile vegetable and is widely grown in both north and south China.It often encounters chilling injury in winter and early spring cultivation, which results in delaying the growth, declining of yield and quality.The optimum growth temperature for eggplant is 22-30 o C. When temperature is lower than 17 o C, the eggplant growth will be slowed down; when lower than 10 o C, the plant will suffer metabolic disturbance (Song, 2011).
Under cold stress, eggplant resources with parthenocarpic ability can bear large number of fruits without pollination while the non-parthenocarpic resources can't fruit.Parthenocarpy can overcome loosing flowers and fruits, improve yield and reduce cost.Meanwhile, parthenocarpic fruits have no seed, which improves quality (Gao et al., 2012).To mitigate these defects in fruit set under low temperature, the breeding of parthenocarpic variety may be a cost-effective alternative.
Parthenocarpic round eggplant with strong cold-resistance was screened out from local varieties (Liu et al., 2005).Pan et al. (2005) found parthenocarpic material from inbred lines of round eggplant.Some parthenocarpic lines suitable for protected cultivation were selected from abroad eggplants (Zhang et al., 2008;Yang et al., 2009).However, the parthenocarpic lines selected already are suitable for cultivation in north China, the purplish red and long eggplant with parthenocarpic ability and suitable for cultivation in south China has not been selected yet.
The antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) have been extensively studied for their roles in plant defense (Aniszewski et al., 2008;Kong et al., 2015;Shi et al., 2015;Wei et al., 2016).Malondialdehyde (MDA) content, electrolyte leakage (EL) and chilling injury (CI) index are closely related to cold-resistance (Shi et al., 2015;Wei et al., 2016).Grey correlative degree analysis is one of the multi-parameter diagnostic methods, which was conducted to make a comprehensively quantitative evaluation on sixteen eggplant resources according to their six main cold-resistant traits.It would help to know the relationship between parthenocarpy and cold-resistance, which would also lay foundation for breeding new variety with strong cold-resistance and suitable for cultivation in south China.

Plant Materials
Sixteen eggplant resources were used as materials, the characters of which were showed in Table 1.'Shenggao No. 2' without parthenocarpic ability was used as control.The seedlings with four leaves of sixteen eggplant resources were cultivated in incubators.), the leaves were taken for analyzing the activities of antioxidase and MDA content.The weight of each sample was 0.5 g.After weighing, leaves were immediately frozen in liquid nitrogen, then stored at -80 o C until use.At the above four stages, fresh leaves were taken respectively and immediately used for EL analysis.The experiment was repeated three times (three independent samples).

CI Index
On the sixth and eighth day, external CI symptoms were visually analyzed.CI was determined according to the following scale: 0 = no damage, 1 = the edge of the leaf wilting, 2 = one leaf wilting, 3 = two leaves wilting, 4 = over two leaves wilting.Observations were made on 20 plants for each resource.The CI index was calculated according to the following equation: The highest level × total number of plants (1)

The Activity of SOD, POD and CAT
For sample preparation, 0.5 g leaves were ground in a mortar with 5 ml 0.2 mol/L phosphate buffer solution (pH = 7.0).The suspension was transferred to a tube, then mixed and centrifuged at 12,000 rpm for 15 min at 4 o C.
The supernatant was collected.Three extracts were done for each resource.
The SOD activity was measured using Total Superoxide Dismutase assay kit (Hydroxylamine method) (Nanjing jiancheng bioengineering institute) following the manufacturer's instructions.SOD activity was calculated as the Equation (2): Where, Ack: the absorbance of control; Ae: the absorbance of sample; Vt: the volume of the reaction liquid (ml); W: the fresh weight (FW) of leaves (g); V1: the volume of the enzyme solution in reaction liquid (ml); V2: the total volume of the enzyme extraction (ml).
The POD activity was measured by guaiacol method (Wu et al., 2006), which was calculated as the following equation: Where, ∆A 470 : the variation of the absorbance during the reaction time; V2: the total volume of the enzyme extraction (ml); W: the FW of leaves (g); V1: the volume of the enzyme solution in reaction liquid (ml); t: the reaction time (min).
The CAT activity was measured by ultraviolet absorption method (Li, 2005), which was calculated as the Equation ( 4): Where, ∆A 240 : the variation of the absorbance during the reaction time; V2: the total volume of the enzyme extraction (ml); W: the FW of leaves (g); V1: the volume of the enzyme solution in reaction liquid (ml); t: the reaction time (min).

MDA Content
MDA content was measured using thiobarbituric acid (TBA) method (Ma, 2009).0.5 g leaves were ground in a mortar with 5 ml 5% trichloroacetic acid (TCA).The suspension was transferred to a tube and centrifuged at 3,000 rpm for 10 min at room temperature.3 ml supernatant and 3 ml TBA were mixed and boiling for 30 min.
The absorbance of supernatant at 450 nm, 532 nm and 600 nm was measured.Blank control: 3 ml TCA + 3 ml TBA.Three extracts were done for each resource.MDA content was calculated as Equation ( 5): Where, Vt: the volume of the reaction liquid (ml); V2: the total volume of the enzyme extraction (ml); W: the FW of leaves (g); V1: the volume of the enzyme solution in reaction liquid (ml).

Electrolyte Leakage
EL was analyzed as described in the experimental guide (Chen & Wang, 2006).Ten discs (1 cm × 1 cm) from the leaves were obtained with a hole-punch and rinsed with deionized water for three times.When dried out with filter paper, the leaves were soaked in 10 ml deionized water for 2.5 h.The conductivity of the bathing solution was measured with a conductimeter (R1).Afterwards, the bathing solution was boiled for 15 min, when cooled to room temperature, the conductivity of the bathing solution was measured (R2).EL was calculated as: EL = R1/R2 × 100%.Measurements were done in triplicate.

Data Analysis
Microsoft Excel 2010 and DPS v7.05 were used for data collecting and analyzing.
Grey correlative degree analysis: Sixteen eggplant resources could be considered as a grey system, in which each resource was a contributing factor.Now suppose there were i eggplant resources and j main cold-resistant traits which would be analyzed.'Reference resource' had the best cold-resistant traits, which consisted of the reference data array X 0 , and each cold-resistant trait of test resources consisted of Xi accordingly.Kj represented each cold-resistant trait.The averages of cold-resistant traits on the sixth day were showed in Table 2. MDA content, EL and CI index were negative traits, and the data would be converted according to '1-actual data'.For contrast, all the data of cold-resistant traits would be initialized.Equal-weighted correlative degree would be calculated according to the equation below: In the formula, the letter 'n' stands for the number of cold-resistant traits,' i (k)' stands for the correlative coefficient of X i to X 0 , '' represents identification coefficient (artificial setting 0.5), and 'r i ' represents the equal-weighted correlative degree of test resources (Table 2).

Chilling Injury Index
On the sixth day, the CI indexes of '30','32','29','TXQ','HL','XBL', were significantly lower than that of control, which indicated they had stronger cold-resistance than control (Figure 1).However, the CI indexes of 'HQ', 'ZHQ' and 'HLMQ' were significantly higher than that of control, and visible symptoms of CI were detected in 'HQ'.When recovered for 2 days, only the CI index of 'HL' kept increasing to 0.74, which suggested the recovery capability of 'HL' was relatively weak; the CI index of other resources decreased rapidly.

The SOD, POD and CAT Activities
Under cold stress, SOD, POD and CAT activities of all resources increased (Appendix A-C).However, the SOD, POD and CAT activities of some resources reached the peak on the fourth day, some on the sixth day.The resources with stronger cold-resistance had a larger increase of SOD, POD and CAT activities than those with weaker cold-resistance.

MDA Content and EL
At the first three stages, the MDA content and EL of all resources increased (Appendix D-E), when recovered for 2 days, all MDA content and EL decreased.The resources with stronger cold-resistance had a least increase of MDA content and EL, which suggested they had the least damage.

Grey Correlative Degree Analysis
According to the equal-weighted correlative degree, the test resources were ranked (Table 3).The results showed '29' had the largest correlative degree (r = 0.899) with the reference resource, which revealed '29' had the strongest cold-resistance, followed by '32' and '30', with the value of 0.877 and 0.820 respectively.'HXZ' had the smallest correlative degree of 0.665, which was smaller than that of control.

Discussion
Visible symptom of chilling injury is wilting of leaves.CI index had significant negative correlation with cold-resistance, which was also an important index to evaluate the cold-resistance in seedling stage (Wang et al., 2015b).The result of CI index was highly consistent with that of grey correlative degree.One main difference was 'HL' had strong cold-resistance according to CI index and moderate cold-resistance according to the latter.So combined with other cold-resistant traits, CI index could evaluate more objectively.
In order to clear away the harmful materials caused by cold stress, the activity of antioxidase such as SOD, POD and CAT will increase rapidly (Fu et al., 2010;Hao et al., 2014;Yan et al., 2011).The variety with strong cold-resistance had a larger increase of SOD and POD activities (Zhang & Yan, 2013).Wang et al. (2013) reported that rice with extremely strong chilling-tolerance had a bigger jump of SOD and POD activities than chilling-sensitive rice.Under cold water stress, CAT activity of chilling-sensitive rice and chilling-tolerant rice increased significantly (Wang, 2015).Jia et al. (2011) reported that CAT activity of wild Lily species with strong cold-resistance increased and maintained a higher level when treated at 11 o C/5 o C (day/night).The results in this work were in accordance with these reports.
Low temperature is harmful to the cell membrane system which causes the selectivity of cell membrane lost and MDA produced.Electrolyte and small molecule organic compounds will permeate out, which lead to the increase of relative conductivity.Under cold stress, materials with strong cold-resistance had a smaller change in MDA content and EL (Zhang et al., 2012(Zhang et al., , 2013;;Wang et al., 2013).
Grey correlative degree analysis is one of the multi-parameter diagnostic methods, which was applied in many crops (Yang et al., 2012;Wang, 2013;Long, 2013;Xing, 2015;Lu et al., 2016;Wang et al., 2015a;Chen et al., 2016).Through grey correlative degree analysis, Chen et al. (2015) selected two varieties which were better than control in agronomic traits and disease-resistance.In this work, cold-resistance of sixteen resources was analyzed.Among the fifteen resources with parthenocarpic ability, only one resource had weaker cold-resistance than control, so most of the resources with parthenocarpic ability had strong cold-resistance.
According to the equal-weighted correlative degree, the resources were divided into three grades.Seven resources had strong cold-resistance and five resources including control had weak cold-resistance.'29', '30', '31-2-1', 'TXQ' and '31-2-2' had purplish red fruits and significantly stronger cold-resistance than control, which were suitable for cultivation in south China.'32' and 'XBL' with white fruits could be used as breeding materials. Appendix

Figure 1 .
Figure 1.Chilling injury index of sixteen resources on the sixth and eighth day Note.The capital letters show significantly different (P  0.01).Percent error line (5%).

Table 1 .
The characters of sixteen eggplant resources

Table 2 .
Main cold-resistant traits of reference resource and test resources

Table 3 .
The equal-weighted correlative degree of test resources B. The mean values of POD activity