Genetic diversity among perennial wild rice Oryza rufipogon Griff., in the Mekong Delta

Abstract Oryza rufipogon Griff. is a perennial species of wild rice widely distributed along the channels and rivers of the Mekong Delta, Vietnam. This study attempted to find centers of diversity among wild rice populations in this area and their inter‐relationships. The highest genetic diversity was found in the Dong Thap population and the lowest in the Can Tho population. Maternal diversity evaluated using chloroplast INDELs detected ten plastid types, five of which were novel relative to other Asian countries. The mitochondrial genome suggested two unique deletions. One 699‐bp deletion via short tandem repeats was accompanied by another deletion including orf153. All accessions carrying the mitochondrial type were found in a particular plastid type. This unique maternal lineage was confined to specific channels where it showed vigorous vegetative growth in comparison to upstream areas where various maternal lineages and maximum genetic diversity occurred. This area along the Mekong Delta is a center of not only nuclear but also maternal diversity.


| INTRODUC TI ON
The wild species of genus Oryzais regarded as valuable resource for rice improvement because of its high genetic diversity (Brar, 2003;Sun, Wang, Li, Yoshimura, & Iwata, 2001). Application of wild rice to breeding programs can facilitate adaptation to climate change and meet the demand for food security in the face of rapid world population growth (Henry, 2016;Henry et al., 2010;Mickelbart, Hasegawa, & Bailey-Serres, 2015;Moner et al., 2018).In this context, Oryza rufipogon and its relatives can provide a rich repository of genes and alleles for potential utilization in rice improvement with the help of genomics-assisted breeding. Such studies can provide specific insight into natural genetic resources that can be preserved and utilized efficiently.
The wild rice species Oryza rufipogon Griff. is a common perennial known to be the progenitor of the Asian cultivated rice species, O. sativa L. (Oka, 1988;Vaughan, 1994). Many valuable genes conferring resistance to major biotic and abiotic stresses are being introduced into improved varieties (Brar & Khush, 1997;Ram, Majumder, Mishra, Ansari, & Padmavathi, 2007;Xiao et al., 1996;Yuan, Virmani, & Mao, 1989), for example, resistance to bacterial leaf blight (BB), brown plant hopper (BPH) and tungro virus, tolerance to aluminum toxicity, sulfate soil, and so on. Despite these advantages of wild rice, it is under serious threat and facing extinction due to ecological changes and human disturbance. Hence, effective conservation of this wild rice has become an urgent priority in many countries (Akimoto, Shimamoto, & Morishima, 1999;Gao, Zhang, Zhou, Gre, & Hong, 1996;Zhou, Chen, Wang, & Zhong, 1992).
The Mekong Delta, Vietnam, where the Mekong River flows out into the East Sea, has been considered a "biological treasure trove." The delta shows high biodiversity of fauna and flora with 1,068 new species having been discovered (Fantz, 2008). According to the FAO database (FAOSTAT: http://www.fao.org/faostat/en/#home), the regional yield of paddy rice ranks 23rd in the world, but 6th in terms of production quantity, in view of the multiple cropping system made possible by the rich soil and abundant constantly available water resources. The area is also rich in wild rice species such as O. rufipogon, O. nivara, and O. officinalis. The delta is also the biggest rice granary in the country, playing a pivotal role in food security and accounting for more than 50% of total production, making Vietnam the second largest rice exporter in the world (Buu & Lang, 2007;Gephart, Blate, McQuistan, & Thompson, 2010;Ti et al., 2003).
Because it is a rich source of not only wild rice species but also rice landraces, the Mekong Delta is considered to be one of the most important rice gene pools in the country (Buu, 1994(Buu, , 1996Xuan, 1975). The wild species of rice are widely distributed from upstream to downstream in the delta and their perennial nature makes them different from those in neighboring Cambodia (based on our field observation). Annual type is predominated around Phnom Penh area, whereas a few perennial populations were reported (Orn et al., 2015). Perennial type could be observed but became extinct because of the size and drastic infrastructure development. These wild rice are widespread along the river and channel systems in Mekong Delta, as well as occasionally in rice fields or marshes; particularly  (Buu & Lang, 1997. As a result, many useful accessions have been exploited for rice improvement over the last few decades, mostly to improve resistance to the brown plant hopper and blast, and tolerance to phosphorus deficiency, aluminum toxicity and acid sulfate soil (Buu & Lang, 2003;Nguyen et al., 2003).
Although genetic variation of O. rufipogon in Vietnam has been studied, nucleus in genetic and maternal diversity has not yet been elucidated adequately (Cai, Wang, & Morishima, 2004;Ishii et al., 2011).
The aims of the present study were to clarify (a) the genetic diversity of O. rufipogon in the Mekong Delta by using nuclear and cytoplasmic markers, and (b) their distribution along the delta based on maternal lineage. It was anticipated that the results would provide insight into the natural wild rice resources in this area that could be useful for biological conservation as well as exploitation in rice breeding programs.  of 55 accessions ( Figure 1). Subpopulations were subsequently identified comprising several individuals corresponding to different collection sites. A maximum of eight individuals for each subpopulation were collected (Appendix Table A1).

| Field collection and plant materials
A core collection derived from the National Bio-Resource (NBR) Project in Japan (Nonomura et al., 2010) and 85 Thai wild rice accessions were also applied for verification (Kaewcheenchai et al., 2018). Additionally, one hundred wild stocks preserved at Cuu Long Rice Research Institute (CLRRI) were used to trace mitochondrial deletions in order to analyze mitochondrial variations from the past (Appendix Table A2).

| Mitochondrial genome markers
A wild rice accession from the Can Tho population was subjected to next-generation sequencing to obtain resequencing data against the mitochondrial genome. Details of the NGS protocol have been reported previously (Waters et al., 2012).

| Molecular markers
Eight chloroplast INDELs (cpINDELs) developed in our previous study (Kaewcheenchai et al., 2018) were used to trace maternal TA B L E 2 Frequency distribution of plastid types found in Vietnames wild rice, Thai wild rice, and NBR  Kaewcheenchai et al. (in press).
F I G U R E 2 Compositions of maternal lineages among the Dong Thap, My Tho, and Intermediate populations lineages. Twenty nuclear SSR markers were applied to evaluate genetic diversity (Table 1). PCR products were amplified using a basic cycle of preheating at 94ºC for 3 min, followed by 30 rounds of 95ºC for 10 s, 55ºC for 30 s, and 72ºC for 30 s, and postheating at 72ºC for 5 min with Thermopol Taq polymerase (NEB Ltd., Japan). The amplified DNA fragments of both chloroplast and nuclear were mixed with a loading dye for electrophoresis on 6% denaturing polyacrylamide gel at 1,500 V for 2 hr in 0.5 × TBE buffer. The gels were then visualized by silver staining (Promega Co., Japan).

| Data analysis
The data were subjected to principal component analysis using

| Chloroplast genome variations (maternal lineage)
In order to trace maternal lineages of wild rice along the Mekong river, eight chloroplast (cp) INDELs were genotyped. Only cpINDEL5 was monomorphic among all of the accessions collected in Vietnam.
Remaining cpINDELs represented alternative genotypes except for cpINDEL3 carrying multiple alleles. These allelic combinations were used to identify different chloroplast types as plastid types (Appendix Table A3).  Tho populations comprised six plastid types. Type 15 was found in all of the populations, but characterized canals at Can Tho, and also predominated in the Intermediate population ( Figure 2).
Although wild rice populations in Vietnam have been cataloged (Buu & Lang, 2011), maternal lineages have not yet been traced.
Therefore, this result shows that perennial wild rice species in Vietnam have identical maternal lineages, thus, contributing to our understanding of the origin of maternal genetics.

| Tracing maternal lineages with unique deletions in the mitochondrial genome
Resequencing of the mitochondrial genome yielded two absence/ deletion markers, which were confirmed by PCR amplification ( Figure 3).One of these presumed deletions, termed the 699-bp deletion, extending from bp 328, 592 to bp 329, 291 in the Nipponbare mitochondrial genome, was amplified and sequenced. It was flanked by tandem duplications of TTGCTA in Nipponbare. Using PCR amplificon, we also tried to confirm another presumed deletion that included orf153. However, this region was not amplified in a particular Vietnamese wild rice accession. The PCR products of orf153 and its upstream region were used as probes to confirm the deletion by Southern hybridization. Specific Vietnamese accessions, P75-1 and P75-2, in the P75 subpopulation at Can Tho did not yield any signals.
In order to clarify the mitochondrial rearrangement, a flanking probe was used. This showed that two accessions in the P75 subpopulation exhibited polymorphism relativeto Nipponbare and W0106, suggesting that a highly complex rearrangement may have deleted orf153 in the P75 subpopulation.
All wild accessions from the Mekong Delta were screened for both mitochondrial INDEL markers, and this revealed that the two deletions were present in a single maternal lineage (Figure 4)  Can Tho population. In addition, only two accessions at Dong Thap shared these deletions but not in others in the CLRRI collection (Table 3). All accessions carrying the deletions corresponded to a particular plastid type, Type 15.

| Genetic diversity and phylogenetic relationships evaluated using nuclear SSR markers
Genetic diversity was estimated using 20 SSR markers (Table 4). The

| D ISCUSS I ON
Previous studies have attempted to characterize and exploit wild rice species in the Mekong Delta without investigating their origin, or clarifying genetic variations among them (Buu, 1996;Buu & Lang, 2007;Lang et al., 2012). The present study focused on genetic variation in O. rufipogon specimen collection along the Mekong River and attempted to know how they distribute along the river system by In contrast to the unique population in Can Tho, high genetic variation was found in the upstream area, at Dong Thap. This higher variation allowed a breeding program that successfully created AS996, carrying higher acid sulfate tolerance (Can & Lang, 2007;Khush & Virk, 2005;Lang et al., 2012). This higher degree of diversity might be due to ecological factors that have a great influence on genetic differentiation among wild rice populations (Orn et al., 2015). In fact, the Dong Thap wild population is distributed widely in a government conservation area at Tram Chim National Park. Our assessment of the genetic diversity would be available to collect valuable resources efficiently before they would be extinct.

ACK N OWLED G M ENTS
This work was funded by a Grant-in-aid B (Overseas project, No.

16H05777) and partly by a Grant-in-Aid for Scientific Research on
Innovative Areas (15H05968). The valuable wild rice accessions used in this study were distributed by the National Institute of Genetics, supported by the National BioResource Project, MEXT, in Japan.
Sequencing was performed at the Gene Research Center, Hirosaki University.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
DTL and BCB managed core collection of perennial wild rice. DTL, BCB, NTL, IN, KT, and RI surveyed natural populations. DTL contributed to analyze with molecular markers. RI set up markers and genome analysis.

DATA ACCE SS I B I LIT Y
All data were included in this manuscript.