Conservation and Utilization of Genetic Resources of Wild Rice in China

YANG Ziyi,XU Zhijian,YANG Qingwen,QIAO Weihua

(1Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China;2Rice Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,China)

Abstract:The abundant genetic resources of wild rice in China represent a key gene pool for modern rice breeding,contributing to food production and agricultural development in China and worldwide.Between the 1970s and the 2010s,two national wild rice surveys were carried out in China.More than 20 000 accessions of three species Oryza rufipogon,O.officinalis and O.meyeriana have been conserved ex situ.An in situ conservation system has also been set up to protect notable and endangered populations.This review summarized the geographical distribution of wild rice in China,the current status of conservation,the discovery of elite genes,and the application of research into the origin and domestication of rice.

Key words:wild rice;conservation;valuable gene;origin and domestication

The importance of rice as the major food supporting half of the world’s population makes it a critical target of research.The genusOryzais pantropical in distribution,and there are 2 domesticated and 24 wild species that have considerable geographical,morphological and physiological diversity (Lu,1999).Cultivated rice is domesticated in Asia asO.sativaand in Africa asO.glaberrima(Oka,1988).WildOryzaspecies,with 2n=24 or 48 chromosomes and genome constitutions of AA,BB,CC,BBCC,CCDD,EE,FF,GG or HHJJ,are important reservoirs of genes with potential for use in rice breeding (Aggarwal et al,1997),biological research,as well as being a foundation for food production and agricultural development (Tanksley and McCouch,1997).

China is one of the original regions ofO.sativa.The domestication of rice started during the Neolithic period and a rice farming culture has persisted in China for thousands of years.Archaeological data from Shangshan ruins in Zhejiang Province of China indicate that ancient humans learned to cultivate rice as early as 11 400 years ago.The first cultivated rice variety Yaston 1 was bred using wild rice in the 1920s by Professor DING Ying.Since then,varieties derived from Yaston 1 flourished for more than half a century.The Chinese three-line hybrid rice,which solves the problem of food shortages in China and perhaps in other parts of the world,was also developed by using a sterility gene from wild rice in Hainan Province,China.Therefore,the Chinese government has paid considerable attention to wild rice research and utilization since the 1970s,and in the last 50 years,there has been a series of notable achievements.More than 20 000 accessions of wild rice have been collected and conserved in the National Crop Genebank and National Field Genebank for Wild rice.A technology system combingin situandex situconservation was established in the 2000s,protecting most endangered wild rice populations.Since then,more than 100 new elite rice varieties have been developed by using wild rice resources or innovative germplasm with wild rice genes,with accumulatively popularized 15 million hectares.Considerable great achievements have also been made in studies of the function and evolution of important genes in wild rice.In this review,we provided a general discussion of the conservation and use of wild rice in China,including its history and current status,ex situandin situconservation,and wild rice genes or resources that have been used in breeding programs.We also considered prospects for the future study of wild rice in China.

History of field survey of wild rice in China

The first report was by Roschev and Merrillv in 1917 following their discovery of common wild rice (O.rufipogon) between Luofu Mountain and Shilong Plain in Guangdong Province,China.In 1926,DING Ying discovered common wild rice in a swamp of Xiniuwei area near Guangzhou City,Guangdong Province (Zhang and Yang,2003).From that time,Chinese scientists gradually increased their studies on wild rice.In the following 40 years,species were continuously discovered in southern China.For example,O.meyerianawas found in Cheli county,Yunnan Province,andO.officinaliswas discovered in Yulin county,Guangxi Province and in Luoding and Yingde counties in Guangdong Province.In addition,O.rfipogonrice was found in Hsinchu and Taoyuan counties in Taiwan,China in 1935 (Chang,1987;Xu et al,2020).By the end of the 1970s,however,all surveys were still being carried out by local scientists and there were no surveys of wild rice resources at a national level.

Wild rice species and distributions in China

To obtain basic information on wild rice species and distribution in China,Chinese government organized the first nationwide field survey of wild rice in 1978-1982.The results indicated that three species of wild rice grow in China:O.rufipogon,O.officinalisandO.meyeriana.It was found in seven provinces in southern China:Guangdong,Guangxi,Hainan,Yunnan,Fujian,Hunan and Jiangxi,from 18°09′ N to 28°14′ N,and from 121°15′ E to 97°56′ E.O.rufipogonis the most widely distributed species,found in all the seven provinces,whileO.officinalisis distributed in Guangdong,Guangxi,Yunnan and Hainan provinces,andO.meyerianais only found in Yunnan and Hainan provinces (Fig.1) (Zhang and Yang,2003).

However,since the 1980s,with the rapid development of economy,natural populations of wild rice have suffered serious damage.Many populations are declining sharply and facing the risk of extinction in their original habitats.For example,of 26O.rufipogonsites found in Yunnan Province in the early 1980s,only one was found in the 1990s.The largest wild rice habitat,an area of more than 14 hm2,was found in Guigang City,Guangxi Province during the first national field survey in 1978-1982,but 80% of it disappeared around 2000.In Yunnan and Hainan provinces,O.meyerianaandO.officinalisalso faced devastation owing to the cultivation of rubber trees.Prompted by these reports,Chinese Academy of Agricultural Science (CAAS) organized the second nationwide field survey of wild rice from 2002 to 2013.A comparison of the first and second national surveys is shown in Fig.2-A.The surveyors collected basic information including distribution,ecological system,original habitat,physiological characters and genetic diversity.The results of the second national survey showed that 76% wild rice populations had disappeared (Fig.2-B).Of the 2 147 populations ofO.rufipogondocumented in the first national survey,only 461 were found to have survived (Fig.2-C) in the second national survey.Additionly,58 new wild rice sites were found in the second national survey,including in Heyuan county in Guangdong Province and Pu’er county in Yunnan Province.Pu’er population in Yunnan Province and Dongxiang population in Jiangxi Province grow at the highest altitude location (1 068 m) ofO.meyerianaand the northernmost location (28°14′ N) ofO.rufipogonin the world,respectively (Xu et al,2020).

ex situ conservation

Theex situconservation of wild rice resources in China can be divided into seed genebank and field genebank.China has successfully established a large-scale,long-term national seed genebank and a medium-term genebank at CAAS.Given the perennial and strong heterogeneous nature of wild rice,it is preferable to conserve individual plants to maintain their genetic integrity.For this reason,two national wild rice field genebanks were constructed in Guangzhou,Guangdong Province,and Nanning,Guangxi Province.By the end of 2020,a total of 24 752 accessions of wild rice had been conserved in field genebanks,including about 3 000 accessions collected during the first national field survey,more than 20 000 accessions collected during the second national field survey,and a few accessions exchanged with other countries.China has more conserved wild rice accessions than any other countries or the International Rice Research Institute (IRRI) (Fig.2-D).Of these,7 003 accessions have also been conserved in the China National Genebank as seeds.Different numbers of the three wild rice species in China have been conservedex situ.With respect toO.rufipogon,6 697 accessions are kept in the China National Genebank as seeds,and 5 158 and 12 000 accessions are kept in the Guangzhou Field Genebank and Nanning Field Genebank,respectively (Xu et al,2020).

in situ conservation

Site selection criteria

Valuable traits in wild populations,such as resistance to biotic stress and tolerance to abiotic stress,are well conserved under natural selection.For this reason,anin situconservation system has also been developed to protect wild populations.Before 2001,there was no research onin situwild rice conservation in China or globally.But,after 20 years of experimentation,Chinese scientists have constructed a system that is easy to use.First,an overall assessment of potentialin situconserved populations is performed,and the population size,distribution,ecological environment,mating system,genetic erosion,invasive species,and human and animal activities are considered.Wild rice is distributed widely in China,and so the selection ofin situconservation sites of wild rice is based on the following principles:(1) the populations must be large and of high genetic diversity;(2) the distribution area is special or represents an unique ecosystem;(3) the populations are seriously endangered;(4) local governments and farmers understand the importance of biodiversity conservation;and (5) thein situconservation sites will continue to play a positive role in biodiversity protection in the area that is native to the species (Yang et al,2003,2013).

Site selection also depends on studies of genetic diversity that examine populations throughout China.Research into genetic structure and the characteristics of the ecological environment found that the genetic structure of each wild rice population is closely related to geographical isolation:it is restricted by mountains and has typical watershed characteristics.Southern Guangdong,Guangxi and northern Hainan provinces are identified as the genetic diversity centers ofO.rufipogonandO.officinalisin China;Hainan Province and southwestern Yunnan Province as the genetic diversity centers ofO.meyeriana(Huang et al,2012;Wei et al,2012).

Conservation approaches

Oncein situconservation sites/areas have been established,the populations within the protected areas or those outside the protected areas must be managed and monitored.All conservation areas should have management plans,and their main purpose is to ensure the stable management of the reserve areas and the sustainability ofin situconservation.Chinese scientists developed two approaches for management of wild ricein situconservation sites:(1) closed management using fencing around plants or physical barriers to isolate thein situconservation sites from the outside,and (2) the mainstreaming approach,which mainly focuses on the participation of local farmer (Yang et al,2013).The government made a series of incentive policy,according to the local conditions,the farmers aroundin situconservation sites get benefits from reduced land taxes,subsides from property rights,creation of jobs and markets.The center government also provided persistent investment once thein situconservation site was construed.Popularizing the significance of wild rice conservation and enhancing the awareness of local residents can effectively avoid the destruction of wild rice environments by human activities.

in situ conservation results

A series of national technical standards have been devised and issued by the Ministry of Agriculture and Rural Affairs of China (MARA).So far,a total of 65 wild ricein situconservation sites (30 at the national level and 35 at the provincial level) have been established.Some unique populations,such as the Dongxiang population (O.rufipogon) in Jiangxi Province (the northernmost wild rice in the world,able to survive under extreme low temperatures),and the Pu’er population (O.meyeriana) in Yunnan Province (grows at the highest recorded altitude)are well protected (Fig.1).in situconservation provides abundant genetic resources for gene discovery,germplasm innovation and sustainable use of wild rice.

Assessment of in situ conservation effectiveness

From the 30in situconservation sites at the national level,23 critically endangered wild rice populations have been identified through the endangered status survey and included in MARA’s biological resources protection plan.A monitoring and early warning technology system forin situconservation sites has been established,and a significant reduction is found in the main threat factors toin situconservation sites.For example,surveys from Dongxiang wild ricein situconservation sites indicate that the average observed heterozygosity (Ho) and the genetic diversity index (Shannon’s information index,I) remain basically stable (Fig.1-I).In 2003,2010 and 2015,Iwas 0.35,0.37 and 0.42,respectively,andHowas 0.47,0.50 and 0.51,respectively,indicating that the protection of the original wild rice habitat is effective and that the monitoring index is reasonable (Liu et al,2017).Wang et al (2009) found that the genetic diversity of the conserved populations might be representative of theO.rufipogonpopulations in China.They also found that the number of specific alleles of the conserved populations is higher than that of the non-conserved populations,indicating that the conserved populations harbor more unique genes.

Evaluation and gene discovery

The most successfully used wild rice in China isO.rufipogon,which has the same AA genome as cultivated rice.It can be crossed directly with cultivated rice although F1hybrids have some pollen sterility.A key moment is the capture of the cytoplasmic male sterility genes fromO.rufipogon,which allows the development of hybrid rice,a notable achievement.Potentially useful traits focusing on high resistance to biotic and abiotic stresses have also been identified inO.officinalisandO.meyeriana.O.meyerianaexhibits high level of resistance or immunity to bacterial blight (Wu et al,2018;A et al,2021),however,the genes responsible for these traits have not been characterized to date because of the complexity of its genomes.

Chinese wild rice confers broad-spectrum resistance to rice pathogen disease,such as rice blast,bacterial blight,bacterial stripe disease,and brown and white-backed planthoppers (Table 1).It also has strong abiotic tolerance to stress,such as cold,heat,salt,and other excellent traits (Wu et al,2020).These characteristics play a vital role in breeding high-yielding rice that has resistance to biotic and abiotic stresses.Broad-spectrum rice blast resistance genes were identified fromO.rufipogonin Southern China.Xa23fromO.rufipogonis an executor R protein and confers broad-spectrum disease resistance in rice (Wang et al,2015).At least 34 loci for resistance to brown planthopper have been found in wild rice,and 28 major resistance genes have been located,among which the dominant genesBph3,Bph14andBph27have been successfully cloned (Li R B et al,2006;Huang et al,2013).However,resistant germplasm has not been found for some rice diseases,such as southern black stripe dwarf disease.

Table 1.Status of excavation of elite genes in Chinese wild rice.

Quan et al (2018) reported that genes/quantitative trait loci (QTLs) from Dongxiang wild rice can markedly improve the salt tolerance of cultivated rice.Liu et al (2018,2019) reported that a transcription factor,bZIP73 fromO.rufipogonplays an important role in rice cold tolerance at the seedling and reproductive stages.A G-protein signaling regulating geneCOLD1fromO.rufipogonenhances rice cold tolerance (Ma et al,2015),and over-expressing this gene injaponicarice significantly enhances cold tolerance.The wild rice-derived haplotype of CTB4a is retained by artificial selection duringtemperatejaponicadomestication in cold habitats (Zhang et al,2017).There are few reports of cloned heat-resistance genes from wild rice in recent years,but many QTLs relating to heat tolerance at the flowering stage have been discovered in wild rice fromO.rufipogonof Yuanjiang county,Yunnan Province (Chen et al,2008;Kui et al,2008).

Although the overall yield traits of wild rice are inferior to those of cultivated rice,potential genes hidden in wild rice are essential for yield-related traitimprovement by modern molecular biological studies (Huang et al,2012;Qi et al,2018).NOG1identified from Dongxiang wild rice regulates the number of grains (Huo et al,2017),andGAD1fromO.rufipogonregulates grain length,grain number and awn development (Jin et al,2016).These genes have profound significance for molecular breeding of improved rice yield.Closed panicle and short awn are traits selected during rice domestication.The spread panicle architecture of wild rice is controlled by a dominant geneLG1,which regulates the closed panicle trait and plays an important role in the control of inflorescence architecture during rice domestication (Ishii et al,2013;Zhu et al,2013).Luo et al (2013) found thatAn-1controls the awn length,grain size and grain number inO.rufipogon.Hua et al (2015) found a gene,LABA1,which controls awn growth in common wild rice in Yuanjiang.These are likely to be important in explaining the closed panicle and shortened awns of cultivated rice,as well as increased grain yield.

Although many beneficial genes have been identified and cloned from wild rice,few are used directly in rice breeding,because most of them have genetic links with disadvantageous traits.An excellent population of genetic materials is important for comprehensive understanding of wild rice relatives.Some particular wild rice populations,like the Yuanjiang population in Yunnan Province and the Dongxiang population in Jiangxi Province,have been used for the construction of genetic populations with a cultivated rice background (Tian et al,2006;Tan et al,2007;Qiao et al,2016).Many useful QTLs/genes related to biotic and abiotic tolerances,yield and some important domestication traits have been identified using Chinese wild rice resources and these genetic materials (Table 1).Recently,due to the advancements in sequencing technologies,chromosome-level genome assembly of the wild rice facilitates tracing the origins of cultivated rice,and provides new insights for excavating the favorable genes in wild rice (Li et al,2020;Xie et al,2020).

Genomics research into origin and domestication

Common wild rice is considered to be the direct ancestor of modern cultivated rice.However,O.rufipogonhas diverged into several groups that may represent the different origins of modern cultivated rice.The origin and domestication of cultivated rice and the differentiation of the two subspecies,indica(Xian) andjaponica(Geng),remain controversial.There are two main hypotheses for the origin of distinct rice subspecies such asindica,japonicaandaus:single origin and multiple origin (Choi and Purugganan,2017).Molina et al (2011) reported that the earliest cultivated rice appeared in China’s Yangtze River Basin between 8 200 and 13 500 years ago,which supports the single origin,and confirms that rice is originated in China for the first time.Civáň et al (2015) examined selected footprints on the genomes of different types of cultivated rice,and concluded thatjaponicarice originates in southern China and the Yangtze River Basin;indicarice originates in Indochina and the Yarlung Zangbo valley;andausoriginates in central India or Bangladesh.These results indicate that rice has a multi-origin domestication.

Huang et al (2012) re-sequenced 446 geographically distinctO.rufipogonand 1 083 cultivated rice samples.They believed thatjaponicarice has been domesticated from a particular population ofO.rufipogon,which originates in the central part of the Pearl River in southern China,and thatindicarice is created through a cross betweenjaponicaand a wild ancestor and then spreads to southern and southeastern Asia.This result also supports the idea of a single origin.However,some studies of the genomic data of domesticated rice and its wild ancestors support an Asian rice multiple-origin theory,in which cultivated rice originates from multiple wild rice ancestral subgroups (Choi and Purugganan,2017).

Artificial selection of genes possibly provides clues for understanding the evolutionary origin of cultivated rice.Some well-documented rice genes are involved in domestication,such assh4for reducing grain shattering (Li C B et al,2006) andprog1for erect growth (Tan et al,2008),supporting the single-origin hypothesis.Some rice genes,likeOsC1for leaf color,support the multiple-origin hypothesis (Zheng et al,2019).

Because common wild rice populations are widely affected by the gene flow of cultivated rice,results of research into the history of rice domestication differ owing to the complex population structure of rice and its wild ancestors (Wang et al,2017).Asian wild rice is a hybrid swarm with extensive gene flow and feralization from domesticated rice.

Future prospects

Till now,there are no published reports of wild ricein situconservation in other countries,and therefore,thein situconservation technology developed by Chinese scientists is of reference value to other countries.Collecting,researching and using wild rice resources is a long-term and arduous process,and requires continuous effort and perseverance from one generation of researchers to the next.Although some achievements have been made in the protection of wild rice,there is still considerable concern about its habitats,and more advanced measures are urgently needed to protect them and to improve conservation technology.We recommend,based on the ongoing genetic diversity studies of Chinese rice germplasm resources,that many morein situconservation sites should be established,particularly in the southern part of Guangxi and Guangdong provinces.

There should also be further improvements in combiningin situandex situconservation systems.The current very limitedex situcollections in seed banks need to be supplemented by much more extensive collections.Genetic resource exchange with other countries should be strengthened.According to the Nagoya Protocol on Access to Genetic Resources and Benefit Sharing,wild rice resources in other countries are indispensable supplements to China’s rice breeding program as well as to food security.Global climate change increases the risk of extinction of some wild rice populations,and thusin situconservation needs a long-term dynamic monitoring.

Identification and evaluation of wild rice should focus on new rice diseases and pests generated by climate change,and major demands of modern rice breeding,such as low-oxygen tolerance for direct-seeding rice and suitability for mechanical harvesting.The use of favorable genes in wild rice to cultivate environmental-friendly germplasm resources can promote characteristics such as salt,flood and drought tolerances,and regeneration.This will drive in high-yielding breeding and green agricultural production.We believe that the combination of modern biotechnology and increasingly comprehensive genomics information will enable the beneficial properties of wild rice to be used more quickly with greater benefit.

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (Grant No.31471471),and the Project of Sanya Yazhou Bay Science and Technology City,China (Grant No.SKJC-2020-02-001).We especially thank Prof.Robert HENRY (The University of Queensland,Australia) for critical reading of the manuscript,and Prof.LIANG Yuntao (Rice Research Institute,Guangxi Academy of Agricultural Sciences,China) for wild rice collection and investigation.