International Journal of Molecular Evolution and Biodiversity 2024, Vol.14, No.2, 62-70 http://ecoevopublisher.com/index.php/ijmeb 68 The calculation of Wright’s FST value indicated a genetic differentiation of 39.09% between the Sanya and Qionghai populations of common wild rice. This value exceeds the average for wind-pollinated plants (GST=0.099) and the genetic differentiation average for perennial herbaceous plants (GST=0.227) (Gamba and Muchhala, 2020). However, this value is lower than that found in two other wild rice species in China—Oryza granulata and Oryza officinalis, with FST values of 0.8700 and 0.788, respectively (Lan et al., 2006). This difference in differentiation levels may be due to geographical isolation and different environmental pressures, or it may reflect the impact of human activities on population genetic structure. The genetic research of the Sanya and Qionghai populations of common wild rice not only revealed their rich genetic diversity but also emphasized the importance of conserving these genetic resources. Genetic diversity is crucial for species to adapt to environmental changes, especially in the context of current global climate change. Maintaining genetic diversity is essential for the long-term survival of species (Lubis and Iksan, 2023). Moreover, since common wild rice contains many important genes that can enhance the disease resistance and adaptability of cultivated rice, conserving these wild resources is invaluable for maintaining agricultural biodiversity and crop improvement. In the future, we should strengthen the protection of the natural habitats of common wild rice to maintain its high genetic variation. Additionally, future research should more extensively use high-throughput genetic marker technologies, such as single nucleotide polymorphism (SNP) markers, to obtain more detailed genetic information (Morales et al., 2020). Furthermore, research should be expanded to more geographical locations and larger populations to comprehensively assess the genetic diversity and structure of common wild rice globally. By thoroughly analyzing the genetic resources of common wild rice, we can not only enhance our understanding of its genetic structure but also better utilize these resources to promote sustainable agricultural development and biodiversity conservation. 3 Materials and Methods 3.1 Study area and sample collection This study selected Qionghai and Sanya regions on Hainan Island as sample collection sites for common wild rice. In the Qionghai region, samples were collected from 11 points centered around Fenglou Village. In the Sanya region, samples were collected around the Hainan Institute of Tropical Agricultural Resources Development and Utilization. Eleven samples were collected from each region using a random sampling method to ensure the representativeness and reliability of the data. During sample collection, fresh leaves were collected using sterile tools, immediately frozen in liquid nitrogen, and then transferred to a -80°C freezer to ensure the integrity and viability of the DNA. 3.2 DNA extraction DNA extraction from common wild rice was performed using a modified CTAB method (Aboul-Maaty and Oraby, 2019). The specific steps are as follows: First, 2~3 g of fresh leaf samples were weighed, frozen with liquid nitrogen, and ground into a powder. The powder was transferred to a preheated 15 mL centrifuge tube containing 7 mL of preheated 1.5× CTAB extraction buffer. The mixture was incubated in a 65°C water bath for 30 minutes. It was then cooled to room temperature, and 4 mL of chloroform/isoamyl alcohol (24:1) was added. After mixing, the solution was centrifuged at 4 000 rpm at room temperature for 20 minutes. The supernatant was transferred to a new centrifuge tube, 1/10 volume of 10% CTAB and an equal volume of chloroform/isoamyl alcohol were added, mixed, and centrifuged again at 4000 rpm for 20 minutes. The supernatant was transferred to a new centrifuge tube, and an equal volume of 1% CTAB precipitation buffer was added, gently shaken until a flocculent DNA precipitate formed, and centrifuged at 3000 rpm for 10 minutes to pellet the DNA. The pellet was resuspended in 1.5~2 mL of 1N NaCl and 5 μL of RNase A, and incubated overnight at 56°C. Once the DNA was fully dissolved, 2~3 mL of 95% ice-cold ethanol (pre-cooled to 4°C) was added to precipitate the DNA, which was then collected and washed with 70% ethanol for 30 minutes, followed by a 5-minute wash with 95% ethanol, and air-dried. The air-dried DNA was dissolved in 1 mL of TE buffer, stored at 4°C for immediate use, or dried for long-term storage.
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