www.nature.com/scientificreports OPEN Conservation recommendations for Oryza rufpogon Grif. in China based on genetic diversity analysis Junrui Wang1,6, Jinxia Shi2,6, Sha Liu1, Xiping Sun3, Juan Huang1,4, Weihua Qiao1,5, Yunlian Cheng1, Lifang Zhang1, Xiaoming Zheng1,5* & Qingwen Yang1,5* Over the past 30 years, human disturbance and habitat fragmentation have severely endangered the survival of common wild rice (Oryza rufpogon Grif.) in China. A better understanding of the genetic structure of O. rufpogon populations will therefore be useful for the development of conservation strategies. We examined the diversity and genetic structure of natural O. rufpogon populations at the national, provincial, and local levels using simple sequence repeat (SSR) markers. Twenty representative populations from sites across China showed high levels of genetic variability, and approximately 44% of the total genetic variation was among populations. At the local level, we studied fourteen populations in Guangxi Province and four populations in Jiangxi Province. Populations from similar ecosystems showed less genetic diferentiation, and local environmental conditions rather than geographic distance appeared to have infuenced gene fow during population genetic evolution. We identifed a triangular area, including northern Hainan, southern Guangdong, and southwestern Guangxi, as the genetic diversity center of O. rufpogon in China, and we proposed that this area should be given priority during the development of ex situ and in situ conservation strategies. Populations from less common ecosystem types should also be given priority for in situ conservation. Common wild rice (Oryza rufpogon Grif.) is the putative progenitor of Asian cultivated rice, one of the most important food crops in the world. It is also an important source of germplasm for rice improvement 1–3. Ding Ying found wild rice (O. rufpogon) in Guangzhou in 1926, and the wild × cultivated cross Zhong Shan No. 1 was widely planted in South China for more than 50 years. In 1970, Yuan Longping and his assistant discovered wild rice with male sterility in Hainan and used it to breed high yielding “three-line” hybrid rice varieties4,5. Te increased yield of this hybrid rice saved thousands of lives in China and around the world. In recent years, wild rice has been used to introduce genes that confer agriculturally benefcial traits into cultivated species, and it holds great potential for future rice breeding eforts. Before the 1970s, O. rufpogon was found in 113 counties of eight provinces in southern China, including Guangdong, Guangxi, Hainan, Yunnan, Hunan, Jiangxi, Fujian, and Taiwan, although the populations in Taiwan disappeared in 19786,7. Since the 1980s, many wild rice habitats have been converted to agricultural or industrial use because of the rapid development of the rural economy and the population expansion in rural China. Conse- quently, areas of wild rice cultivation have dramatically decreased. Our recent work indicates that approximately 70% of the O. rufpogon populations have disappeared, and all large populations (growth area > 33 hm2) have either disappeared or decreased dramatically (unpublished data). Te threatened status of wild rice has attracted increasing attention in China, and there is a desire to col- lect samples for ex situ conservation and to develop in situ conservation programs. Te Chinese government began to invest in the construction of in situ conservation sites in 2001, and 15 such sites were established by the end of 2014. Te government also established eight other in situ conservation sites using a mainstreaming approach, a new conservation strategy in which farmers are encouraged to participate in conservation activities that span physical boundaries such as hills and rivers. Most in situ conservation sites were selected based on 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. 2Shanghai Normal University, Shanghai, China. 3Shanxi Agricultural University, Jinzhong, China. 4Institute of Rice Research, Guangxi Academy of Agricultural Sciences, Nanning, China. 5Agricultural Science and Technology Innovation Program/Crop Germplasm Resources Preservation and Sharing Innovation Team, Beijing, China. 6These authors contributed equally: Junrui Wang and Jinxia Shi. *email: [email protected]; [email protected] SCIENTIFIC REPORTS | (2020) 10:14375 | https://doi.org/10.1038/s41598-020-70989-w 1 Vol.:(0123456789) www.nature.com/scientificreports/ Population code Population locality Sample size A Ae He Ho I Private alleles N_HN1 Sanya County, Hainan 7 1.57 1.54 0.27 0.53 0.38 0 N_HN2 Qionghai County, Hainan 38 2.29 1.8 0.39 0.7 0.57 1 N_HN3 Wenchang County, Hainan 43 1.89 1.67 0.34 0.66 0.48 1 N_HN4 Chengmai County, Hainan 24 2.54 1.91 0.44 0.81 0.66 0 N_HN5 Qiongshan County, Hainan 52 5.71 2.84 0.58 0.75 1.14 11 N_GD1 Leizhou County,Guangdong 6 2.57 1.8 0.38 0.42 0.64 1 N_GD2 Suixi County, Guangdong 31 4.61 2.75 0.59 0.65 1.1 4 N_GD3 Enping County, Guangdong 21 4.82 2.91 0.6 0.6 1.15 2 N_GD4 Huiyang County, Guangdong 19 3.39 1.83 0.4 0.42 0.72 2 N_GD5 Gaozhou County, Guangdong 30 2.5 1.78 0.39 0.69 0.59 1 N_GD6 Zengcheng County, Guangdong 11 1.82 1.59 0.3 0.53 0.44 1 N_GD7 Huilai County, Guangdong 32 4.86 2.31 0.5 0.53 0.97 1 N_GX1 Fusui County, Guangxi 33 4.04 2.48 0.56 0.67 1.01 0 N_GX2 Fangchenggang County, Guangxi 46 4.96 3.01 0.63 0.72 1.19 6 N_GX3 Wuxuan County, Guangxi 63 6.4 2.66 0.54 0.51 1.12 12 N_GX4 Hezhou County, Guangxi 45 4.46 2.58 0.5 0.56 0.95 1 N_GX5 Zhongshan County, Guangxi 25 3.61 2.18 0.45 0.33 0.83 5 Zhangpu County, Fujian (conserva- N_FJ1 11 3.04 1.89 0.4 0.43 0.7 1 tion site) Chaling County, Hunan (conserva- N_HuN1 35 4.93 2.58 0.53 0.36 1.04 0 tion site) Dongxiang County, Jiangxi (conserva- N_JX1 56 4 2.25 0.49 0.44 0.88 1 tion site) Populations from the diversity center 4.72** 2.63** 0.56** 0.66** 1.04** without N_GD1 Means Populations from the diversity center 4.45* 2.53** 0.53** 0.63** 0.99* Populations from the whole country 3.7 2.22 0.46 0.57 0.83 Table 1. Population codes, geographical localities, sample sizes and genetic diversity parameters of all O. rufpogon populations. A mean number of alleles per locus, Ae efective number of alleles, He expected heterozygosity, Ho observed heterozygosity, I Shannon–Weaver information index. *P < 0.05; **P < 0.01. scientifc expertise or local government recommendations, although it was still necessary to justify their value and rationale. Detailed information on the population genetic structure of O. rufpogon is therefore useful to guide the selection of future sites. Information generated using molecular methods has direct and indirect consequences for the practical man- agement and conservation of germplasm. Genetic diversity data can be useful for understanding the taxonomy and evolution of crop species, and this basic knowledge supports their conservation 8. More directly, genetic diversity studies can help us to adjust our strategies for collection, evaluation, and breeding. Although some studies have recently documented the population genetic structure of O. rufpogon in China 9–13, few have focused on the development of conservation strategies based on this population genetic structure. Our study used SSR markers to examine the genetic diversity and population genetic structure of natural populations at three diferent levels: national (China), provincial (Guangxi Province), and local (Dongxiang population in Jiangxi Province). Results Genetic diversity of O. rufpogon populations. Twenty-four SSR primer pairs from previous studies14 with polymorphisms and a uniform distribution among chromosomes were selected for use in the analysis of population genetic diversity and genetic structure (Supplementary Table S1). All loci were found to be in Hardy– Weinberg equilibrium. High levels of genetic variability at 24 loci were detected in 628 individuals from 20 populations sampled across China (Table 1; Fig. 1a). A total of 340 alleles were detected across the loci, ranging from 23 alleles at RM253 to seven alleles at RM244 and RM345 (Supplementary Table S2). Te average number of alleles was 14.17. Te overall means of AE (the efective number of alleles), HO (the observed heterozygo- sity), HE (the expected heterozygosity) and I (the Shannon–Weaver information index) across all loci were 6.97, 0.58, 0.83, and 2.08, respectively. Te values varied widely among loci: AE ranged from 2.80 (RM244) to 15.41 (RM336); HO ranged from 0.15 (RM244) to 0.88 (RM336); HE ranged from 0.64 (RM244) to 0.93 (RM253); and I ranged from 1.13 (RM244) to 2.85 (RM336). Te genetic structure of populations across China was analyzed. Te mean value of FST was 0.44, and it varied from 0.25 to 0.59, indicating that there was substantial genetic variation among populations (Supplementary Table S2). Genetic population diferentiation was also measured by AMOVA analysis (Table 2) and pairwise pop- ulation diferentiation (Supplementary Table S3). AMOVA analysis showed that 41.2% of the variation occurred among populations. Te signifcant diferentiation (the P value of FST < 0.001) among populations also refected larger diferences between all populations from the whole country (Table 2). FIS ranged from − 0.49 to 0.44 with SCIENTIFIC REPORTS | (2020) 10:14375 | https://doi.org/10.1038/s41598-020-70989-w 2 Vol:.(1234567890) www.nature.com/scientificreports/ a b R_GX13 Jiuwan Mountain Shanzhao Ling Liu River R_GX10 Yu River Diaozha Mountain R_GX11 R_GX7 R_GX9 R_GX14 Xun River Dayao Mountain R_GX8 R_GX12 Darong Mountain You River Liuwan Mountain R_GX2 Gutong Mountain R_GX1 Nanliu River R_GX6 R_GX4 R_GX5 R_GX3 50 km 200 km c concrete wall 500 m Figure 1.