Introduction
Artificial selection is a selection process by which human instead of nature select animals or plants with desirable traits to reproduce. After thousands of years, domesticated animals and plants often have distinct characteristics comparing with their wild progenitors. To study the molecular mechanism of artificial selection, it is important to map genes affected by artificial selection during domestication because these genes are commonly related to economic traits or biological processes associated with development and reproduction (Gepts, Reference Gepts2014). To date, there are different methods and software available to detect artificial selection (Li and Stephan, Reference Li and Stephan2006; Sabeti et al., Reference Sabeti, Schaffner, Fry, Lohmueller, Varilly, Shamovsky, Palma, Mikkelsen, Altshuler and Lander2006; Li Reference Li2011; Lin et al., Reference Lin, Li, Schlotterer and Futschik2011; Fu and Akey, Reference Fu and Akey2013; Waldmann et al., Reference Waldmann, Pfeiffer and Meszaros2020). When artificial selection occurs in one of populations, it increases the fixation index (FST) on the selected locus. FST is a measurement of population differentiation (Hartl and Clark, Reference Hartl and Clark1997), and a locus with high FST indicates that two populations are highly diverged on the locus. Therefore, high FST has been frequently used as one of the signatures of selection in rice and many other species (Gao, Reference Gao2004; Yu et al., Reference Yu, Tang, Qian, Wang, Yan, Zeng, Han, Wu, Shi and Li2008; Fabian et al., Reference Fabian, Kapun, Nolte, Kofler, Schmidt, Schlotterer and Flatt2012; Huang et al., Reference Huang, Kurata, Wei, Wang, Wang, Zhao, Zhao, Liu, Lu, Li, Guo, Lu, Zhou, Fan, Weng, Zhu, Huang, Zhang, Wang, Feng, Furuumi, Kubo, Miyabayashi, Yuan, Xu, Dong, Zhan, Li, Fujiyama, Toyoda, Lu, Feng, Qian, Li and Han2012; Daub et al., Reference Daub, Hofer, Cutivet, Dupanloup, Quintana-Murci, Robinson-Rechavi and Excoffier2013; Shen et al., Reference Shen, Bo, Xu and Wu2014; Kumagai et al., Reference Kumagai, Kanehara, Shoda, Fujita, Onuki, Ueda and Wang2016; Islam et al., Reference Islam, Khalequzzaman, Prince, Siddique, Rashid, Ahmed, Pittendrigh and Ali2018; Cheng et al., Reference Cheng, Kim and Park2019).
It has been well-known that demographic factors, such as population structure and varying population size, may generate selection-like signatures in the genetic variation of domesticated animals or plants (Catriona and Emma, Reference Catriona and Emma2006; Li and Stephan, Reference Li and Stephan2006). Thus, it may hinder the efforts to detect artificial selection. To examine the confounding effects of demography, we investigated whether the polymorphism on highly diverged loci (i.e. high FST loci) could be accounted by neutral demographic scenarios alone, without invoking the hypothesis of artificial selection.
We used Asian rice to examine the confounding effects of demography, including population structure and varying population size. Asian rice (Oryza sativa L.) has two subspecies, indica and japonica, and it domesticated from its wild rice ancestor O. rufipogon Griff. (He et al., Reference He, Zhai, Wen, Tang, Wang, Lu, Greenberg, Hudson, Wu and Shi2011; House et al., Reference House, Griswold and Lukens2014; Zhang et al., Reference Zhang, Xu, Mao, Yan, Chen, Wu, Chen, Luo, Xie and Gao2016). An entangled history of rice domestication has been revealed (Huang et al., Reference Huang, Kurata, Wei, Wang, Wang, Zhao, Zhao, Liu, Lu, Li, Guo, Lu, Zhou, Fan, Weng, Zhu, Huang, Zhang, Wang, Feng, Furuumi, Kubo, Miyabayashi, Yuan, Xu, Dong, Zhan, Li, Fujiyama, Toyoda, Lu, Feng, Qian, Li and Han2012). As common variants in rice (Shen et al., Reference Shen, Jiang, Jin, Zhang, Xi, He, Wang, Wang, Qian, Li, Yu, Liu, Chen, Gao, Huang, Shi and Yang2004), InDels (insertion/deletion) may be good neutral markers and are easy to be genotyped. Polymorphisms of InDels have gained long-term interests in the rice research community (Shen et al., Reference Shen, Jiang, Jin, Zhang, Xi, He, Wang, Wang, Qian, Li, Yu, Liu, Chen, Gao, Huang, Shi and Yang2004; Liu et al., Reference Liu, Li, Qu and Yan2015a; Lu et al., Reference Lu, Cui, Li, Huang, Zong, Yao, Li, Zhang and Yuan2015; Moonsap et al., Reference Moonsap, Laksanavilat, Sinumporn, Tasanasuwan, Kate-Ngam and Jantasuriyarat2019), and these markers were used to conduct QTL mapping (Kim et al., Reference Kim, Ramos, Ashikari, Virk, Torres, Nissila, Hechanova, Mauleon and Jena2016) and study genetic differentiation in rice (Liu et al., Reference Liu, Cai and Lu2012; Sahu et al., Reference Sahu, Mondal, Sharma, Vishwakarma, Kumar and Das2017). In this study, we selected the highly diverged InDels between the rice cultivars (japonica or indica) and their wild ancestors, O. rufipogon and O. nivara (also recognized as the annual type of O. rufipogon) (Yamanaka et al., Reference Yamanaka, Nakamura, Nakai and Sato2003; Zheng and Ge Reference Zheng and Ge2010; Liu et al., Reference Liu, Zheng, Zhou, Zhou and Ge2015b) and genotyped these loci in 172 accessions of rice collected through the East, South and Southeast Asia. Then we conducted large-scale coalescent-based simulations on neutral demographic scenarios to examine whether the polymorphic profiles on the highly diverged loci can be explained by demography alone. This study would shed lights on how genes affected by artificial selection could be detected in Asian cultivated rice and other domesticated animals and plants.
Materials and methods
Sampling and InDels genotyping
In this study, total 172 accessions of japonica (n = 63), indica (n = 66), O. rufipogon (n = 25) and O. nivara (n = 18) were collected through the East, South and Southeast Asia (Table S1). These samples were provided by the International Rice Research Institute, the Shanghai Center for Agricultural Biological Genetics, Zhejiang University and Yunnan Agricultural University, respectively, and most of their genomes have not been sequenced. The 45 InDels were genotyped in the collected accessions (Table S2). Those InDels were initially discovered by comparing the genomic sequences of indica (93–11) and japonica rice (Nipponbare) (Shen et al., Reference Shen, Jiang, Jin, Zhang, Xi, He, Wang, Wang, Qian, Li, Yu, Liu, Chen, Gao, Huang, Shi and Yang2004; Lu et al., Reference Lu, Cai and Jin2009). They are randomly distributed along the rice genome.
Genomic DNA extraction and InDels genotyping was conducted according to our previous published methods (Lu et al., Reference Lu, Cai and Jin2009). Seedlings of rice samples were germinated in an incubator at 37 °C and then transferred into a glasshouse at around 25 °C. About 0.5 g of fresh leaf samples were collected from seedlings at about the 3–4-leaf stage and then placed in a plastic bag containing silica gel for fast drying. Genomic DNA was extracted using the hot CTAB procedure (Murray and Thompson, Reference Murray and Thompson1980). PCR analysis and electrophoresis were performed. The primers for the 45 InDels were obtained by the previous research (Shen et al., Reference Shen, Jiang, Jin, Zhang, Xi, He, Wang, Wang, Qian, Li, Yu, Liu, Chen, Gao, Huang, Shi and Yang2004) and listed in Supplemental Table S3. PCR products were resolved on a 4% denaturing polyacrylamide gel. After electrophoresis, bands were revealed using the silver-staining procedure. The electrophoretic banding patterns were used for genotyping and subsequent calculation of allele frequency.
Heterozygosity and FST calculation follows the textbook (Hartl and Clark, Reference Hartl and Clark1997). Heterozygosity of wild rice, japonica and indica at each InDel was calculated from allele frequency. To calculate FST, we used the following formula: $F_{ST} = {{{\rm H}_{\rm T}-{\rm H}_{\rm S}} \over {{\rm H}_{\rm T}}}$
, where HT is expected heterozygosity for total population and HS is average expected heterozygosity in subpopulations.
Model selection and simulated neutral demographic scenarios
Four demographic models were considered here (Fig. 1). Two of them represent the single evolutionary origin of rice, and the other two models stand for the multiple origins. Here, the single origin means that O. sativa was domesticated once and diverged to japonica and indica (Sang and Ge, Reference Sang and Ge2007; Gross and Zhao, Reference Gross and Zhao2014). The multiple origins indicate that japonica and indica have independent origins from distinct wild rice subpopulations (Oka, Reference Oka1988).
Four demographic scenarios of rice populations considered in this study. (a) The single origin of rice: the model I: japonica rice was first domesticated; the model II: indica rice was first domesticated. (b) The two origins of rice: the model III: japonica rice was first domesticated; the model IV: indica rice was first domesticated.
By using a software ms (Hudson, Reference Hudson2002) we simulated the neutral data under the demographic scenarios. The parameters of the four demographic models were obtained from the previous study (Molina et al., Reference Molina, Sikora, Garud, Flowers, Rubinstein, Reynolds, Huang, Jackson, Schaal, Bustamante, Boyko and Purugganan2011). Thus, to simulate the data, the ms command lines are as following:
Model I (single origin, japonica domesticated first):
Model II (single origin, indica domesticated first):
Model III (multiple origins, japonica domesticated first):
Model IV (multiple origins, indica domesticated first):
Neutrality test
The null hypothesis is one of the neutral demographic scenarios described above. The alternative hypothesis is that, a locus evolved neutrally in the progenitor population, but was subject to artificial selection in one of the cultivated rice populations. As expected, artificial selection could cause a high FST between progenitor-japonica, progenitor-indica or japonica-indica. We pooled simulated results as a sample by rejection-sampling algorithm (Tavare et al., Reference Tavare, Balding, Griffiths and Donnelly1997) that has been commonly used to compare simulated and observed data (Li et al., Reference Li, Xiang-Yu, Dai, Gu, Ming, Yang, Ryder, Li, Fu and Zhang2016). The neutrality test was conducted as $P( f_j > f_{j, obs}{\rm \vert }\;\vert {f_p-f_{p, obs}} \vert < \varepsilon )$
and $P( f_i > f_{i, obs}{\rm \vert }\;\vert {f_p-f_{p, obs}} \vert < \varepsilon )$
, where f p, f j and f i are the allele frequency of mutant in the progenitor, japonica and indica population, and $\varepsilon$
is a fixed tolerance.
When $\varepsilon$
is very small, the computational load would be very large to estimate the probability, whereas the precision of the probability will be poor when $\varepsilon$
is large. Our experience suggests that $\varepsilon = 0.02$
works well.
Results
In this study, we surveyed the genetic diversity of 129 accessions of Asian cultivated rice (O. sativa ssp. japonica and O. sativa ssp. indica) and 43 accessions of wild rice (O. rufipogon and O. nivara). FST at 45 InDels loci was calculated among wild rice and Asian cultivated rice (Table S2). FST was widely used to measure the level of population differentiation and detect artificial selection in rice populations. Twelve of the 45 loci between wild rice and japonica (Table 1), 5 of the 45 loci between wild rice and indica (Table 2), and 35 of the 45 loci between japonica and indica showed a FST value greater than 0.4. We defined highly diverged locus between japonica (or indica) and wild rice with FST > 0.4 as previous study (He et al., Reference He, Zhai, Wen, Tang, Wang, Lu, Greenberg, Hudson, Wu and Shi2011). So 17 loci are highly diverged between japonica (or indica) and wild rice. These loci are R1M7, R1M37, R1M47, R2M10, R2M24, R2M26, R2M50, M3M10, R3M23, R3M30, R4M17, R4M43, R4M50, R6M44, R7M37, R8M23 and R9M42. Because neutral demographic scenarios could affect FST, we tested whether the polymorphism pattern at these highly diverged loci can be explained by neutral demographic scenarios alone.
Probability value for the test of neutrality in the 12 highly diverged InDel loci between japonica and wild rice
a Model I (single origin, japonica domesticated first).
b Model II (single origin, indica domesticated first).
c Model III (multiple origins, japonica domesticated first).
d Model IV (multiple origins, indica domesticated first).
Probability value for the test of neutrality in the five highly diverged InDel loci between indica and wild rice
a Model I (single origin, japonica domesticated first).
b Model II (single origin, indica domesticated first).
c Model III (multiple origins, japonica domesticated first).
d Model IV (multiple origins, indica domesticated first).
The rice domestication process has been studied well (Gross and Zhao, Reference Gross and Zhao2014; Choi et al., Reference Choi, Platts, Fuller, Hsing, Wing and Purugganan2017; Fornasiero et al., Reference Fornasiero, Wing and Ronald2022; Izawa, Reference Izawa2022; Shang et al., Reference Shang, Li, He, Yuan, Song, Wei, Lin, Hu, Zhao, Zhang, Li, Gao, Wang, Liu, Zhang, Zhang, Cao, Yu, Zhang, Zhang, Tan, Qin, Ai, Yang, Zhang, Hu, Wang, Lv, Wang, Ma, Wang, Lu, Wu, Liu, Sun, Zhang, Guo, Li, Zhou, Li, Zhu, Xiong, Ruan and Qian2022). It was suggested that the first domestication of Asian cultivated rice occurred in the Yangtze River basin at about 9000 years ago (Fornasiero et al., Reference Fornasiero, Wing and Ronald2022), but the two subspecies (japonica and indica) originated independently (Choi et al., Reference Choi, Platts, Fuller, Hsing, Wing and Purugganan2017), indicating that different loci may have different evolutionary histories due to migration and introgression. Therefore, we simulated four different neutral demographic scenarios (the models I, II, II and IV) of the single and the multiple origins (Fig. 1), which represent the possible neutral evolutionary histories of genes in two cultivated rice subspecies.
We then compared the simulated and observed data using the proposed neutrality test (see Materials and Methods). At 17 highly diverged loci in the four models, we only observed R6M44 in the model I (single origin, japonica being domesticated first) with a marginally significant difference (p = 0.042) (Tables 1 and 2). The value becomes insignificantly different when the multiple testing is considered. Other p values are between 0.101 and 1.000. Therefore, the high FST at 17 loci may have resulted from demography other than selection during japonica and indica domestication.
Discussion
In this study, we genotyped 45 InDels in the privately collected 172 accessions and identified 17 highly diverged InDels between the rice cultivars and their wild ancestors. Large-scale coalescent-based simulations were conducted on four neutral demographic scenarios to test the neutral evolutionary hypothesis of the 17 InDels. The results of neutrality test suggested that demography alone could explain the polymorphic profiles on the highly diverged InDels, irrespective of which domestication hypothesis was used and which subspecies was first domesticated. Therefore, FST is informative for detecting artificial selection in rice, but high FST should not be used as conclusive evidence that candidate loci are affected by artificial selection.
To examine these 17 highly diverged loci, the new statistical test was developed. It has a number of advantages. First, the test was conducted conditional on a mutation, thus evolutionary rate heterogeneity in the rice genome (Zhao et al., Reference Zhao, Feng, Lu, Li, Wang, Tian, Zhan, Lu, Huang, Wang, Fan, Zhao, Wang, Zhou, Chen, Zhu, Li, Weng, Xu, Wang, Wei, Han and Huang2018) does not affect our analyses. Second, the test can be used to analyse a single-point mutation because we did not limit the mutation type as InDels (Yu et al., Reference Shang, Li, He, Yuan, Song, Wei, Lin, Hu, Zhao, Zhang, Li, Gao, Wang, Liu, Zhang, Zhang, Cao, Yu, Zhang, Zhang, Tan, Qin, Ai, Yang, Zhang, Hu, Wang, Lv, Wang, Ma, Wang, Lu, Wu, Liu, Sun, Zhang, Guo, Li, Zhou, Li, Zhu, Xiong, Ruan and Qian2022). Third, the test is based on a single locus assuming no recombination occurred within the locus (Yang et al., Reference Yang, Li, Wiehe and Li2018). Therefore, the test is ready to be applied to other sexual and asexual species.
Though demography alone could explain the polymorphic profiles on the 17 loci with high FST, our results do not indicate that there is no artificial selection during the domestication of rice. The number of loci examined in this study is small, comparing with those in genome-wide surveys. Our results indicate that it is extremely important to consider the demography as a confounding factor when detecting artificial selection in rice (Gao and Innan, Reference Gao and Innan2008; He et al., Reference He, Zhai, Wen, Tang, Wang, Lu, Greenberg, Hudson, Wu and Shi2011; Molina et al., Reference Molina, Sikora, Garud, Flowers, Rubinstein, Reynolds, Huang, Jackson, Schaal, Bustamante, Boyko and Purugganan2011). When genome-wide scans are conducted for searching the signal of artificial selection, the number of windows examined increases and it is very likely to observe a number of loci with high FST due to random demographic effects, such as demography.
Because the detection of selection may be hindered by the demographic effects, it is further suggested that the combination with different signatures of selection (Lin et al., Reference Lin, Li, Schlotterer and Futschik2011; Horscroft et al., Reference Horscroft, Ennis, Pengelly, Sluckin and Collins2019) could be considered when detecting artificial selection. Moreover, the confounding effects of demography can be addressed by controlling its false-positive rate, which is generally composed by two-step analysis (Li and Stephan, Reference Li and Stephan2006; Koropoulis et al., Reference Koropoulis, Alachiotis and Pavlidis2020). Genome-wide DNA polymorphism is used to infer the demography (Speidel et al., Reference Speidel, Forest, Shi and Myers2019; Hu et al., Reference Hu, Hao, Du, Di Vincenzo, Manzi, Cui, Fu, Pan and Li2023) and the detection of positive selection is followed conditional on the inferred demography. The alternative approaches could be to use neutrality tests that are insensitive to demographic events (Li, Reference Li2011; Hunter-Zinck and Clark, Reference Hunter-Zinck and Clark2015). Overall, our results suggest that it should be more cautious about the explanation of highly diverged loci when studying domestication of indica and japonica rice varieties.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262123001132.
Author contributions
J. X.-Y., H. L. and B.-R. L. developed the concepts. J. X.-Y. and Z. G. performed the analyses. B.-R. L. provided the resources. J. X.-Y., Z. G. and H. L. wrote the original manuscript. H. L. and B.-R. L. reviewed and edited the manuscript. All authors read and approved the manuscript.
Funding statement
This work was supported by grants from the National Natural Science Foundation of China (nos. 32270674 and 91131010), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDPB17).
Competing interests
None.
Ethics approval
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
The datasets generated and analysed during the current study are available from the corresponding authors on reasonable request.
