To screen out Camellia oleifera cultivars with strong heat tolerance and provide a theoretical basis for breeding new heat-tolerant varieties.

A pot experiment was conducted using the method of high-temperature stress in an intelligent greenhouse on seedlings of two C. oleifera cultivars, ‘Dabieshan 1’ and ‘Changlin 53’. Nine time gradients were set: 0 (CK), 4, 7, 14, 21, 28, 35, 42 and 49 d. Ten physiological and biochemical indicators were measured, including relative water content, relative electrical conductivity, malondialdehyde content, hydrogen peroxide content, superoxide dismutase activity, peroxidase activity, catalase activity, soluble sugar content, soluble protein content, and free proline content. The heat tolerance of the two C. oleifera cultivars was comprehensively evaluated using the average membership function value of each physiological indicator.

1) The leaf relative water content of both cultivars decreased continuously with the extension of high-temperature treatment, and the decline accelerated after 7 days of stress. After 49 days of high-temperature treatment, the leaf relative water content of both cultivars reached its minimum, with ‘Dabieshan 1’ at 71.40% and ‘Changlin 53’ at 63.32%. The relative electrical conductivity, malondialdehyde content, and hydrogen peroxide content increased continuously with the extension of high-temperature treatment and reached their maximum at the 49th day; 2) The activities of superoxide dismutase and peroxidase in the leaves of both cultivars first increased and then decreased. The catalase activity in ‘Dabieshan 1’ leaves showed a continuous upward trend, while that in ‘Changlin 53’ increased in the early stage of stress and began to decrease after 42 d of stress; 3) The proline, soluble sugar, and soluble protein contents in ‘Dabieshan 1’ leaves continued to increase. In ‘Changlin 53’ leaves, proline and soluble protein contents continued to increase in the early stage of stress, reached their maximum at the 42th day, and then decreased; soluble sugar content continued to increase in the early stage of stress, reached its maximum at the 35th day, and then began to decrease; 4) The average membership function value of ‘Dabieshan 1’ was 0.559, and that of ‘Changlin 53’ was 0.449, indicating that the heat tolerance of ‘Dabieshan 1’ was superior to that of ‘Changlin 53’.

Under high-temperature stress, the physiological response patterns of the two C. oleifera cultivars seedlings were different. The comprehensive membership function evaluation showed that the average membership function value of ‘Dabieshan 1’ (0.559) was higher than that of ‘Changlin 53’ (0.449). The heat tolerance of ‘Dabieshan 1’ was superior to that of ‘Changlin 53’.

In order to optimize the SSR-PCR reaction system of Carya illinoinensis, we conducted a screening of highly polymorphic SSR primers specific to C. illinoinensis. This screening process has provided a valuable tool for various subsequent research activities, including fingerprint construction, genetic relationship analysis, and variety identification of C. illinoinensis.

A combination of univariate experiments and L₁₆ (4⁵) orthogonal experimental designs was used. The DNA of C. illinoinensis was used as a template. Firstly, the effects of six factors (DNA, Mg²⁺, 10×PCR Buffer, primer, Taq enzyme, dNTPs) on the SSR-PCR reaction system of C. illinoinensis were tested individually. Based on the results of the single factor tests, the appropriate dosage range for each factor was determined, and an orthogonal test was designed accordingly.

Based on the results amplified by orthogonal test, the optimal reaction system of C. illinoinensis was determined as follows: Taq enzyme 0.15 U, Mg²⁺ 2.0 mmol/L, dNTPs 0.1 mmol/L, primer 1.0 μmol/L, 50 ng DNA template 1.0 μL, 10×PCR Buffer 1.0 μL, and ddH₂O 5.8 μL. The extremum difference analysis of orthogonal test revealed that the five factors affecting the amplification effect of the SSR-PCR reaction system of C. illinoinensis ranked in the following order: Taq enzyme > primer = DNA > Mg²⁺ > dNTPs. To validate the optimized SSR-PCR reaction system of C. illinoinensis, four pairs of C. illinoinensis primers were used with the DNA of eight species of C. illinoinensis as a template. The results showed the successful amplification of bright and clear bands, confirming the stability and reliability of the optimized reaction system. Out of the 283 pairs of walnut and C. cathayensis primers, 12 pairs of highly polymorphic primers were selected using the optimized reaction system. The number of polymorphic sites exceeded 0.5.

The optimized reaction system and 12 pairs of primers are screened and can be directly utilized for subsequent SSR molecular marker experiments. This lays a solid foundation for the identification of genetic relationships and mating system analysis of C. illinoinensis.