Joong Hyoun Chin. Yoo-Jin Lee. Wenzhu Jiang. Hee-Jong Koh. Michael J. Thomson

Transcription

1 Genet Resour Crop Evol (2017) 64: DOI /s RESEARCH ARTICLE Characterization of indica japonica subspecies-specific InDel loci in wild relatives of rice (Oryza sativa L. subsp. indica Kato and subsp. japonica Kato) Joong Hyoun Chin. Yoo-Jin Lee. Wenzhu Jiang. Hee-Jong Koh. Michael J. Thomson Received: 22 July 2015 / Accepted: 18 January 2016 / Published online: 3 March 2016 Springer Science+Business Media Dordrecht 2016 Abstract Insertion/deletion (InDel) polymorphisms are generally irreversible and, thus, are useful for evaluating the genetic relationships within the genus Oryza. Moreover, subspecies-specific (SS) InDel markers linked to conserved genomic regions specific to the indica and japonica subspecies of Oryza sativa can provide insight into the genetic relationships between cultivated and wild rice. The evolutionary relationship among Oryza species in respect to their indica and japonica alleles was investigated using 67 selected indica japonica InDel SS-STS primers across 290 accessions, including 61 Asian cultivated rice (O. sativa) cultivars, 27 African cultivated rice (O. glaberrima) accessions, and 202 accessions of wild Joong Hyoun Chin and Yoo-Jin Lee have contributed equally to this paper. Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. Y.-J. Lee M. J. Thomson (&) Plant Breeding, Genetics and Biotechnology, International Rice Research Institute, Los Baños, DAPO Box 7777, Manila, Laguna, Philippines m.thomson@tamu.edu J. H. Chin H.-J. Koh (&) Division of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul , Korea heejkoh@snu.ac.kr Orzya species. The average SS allele frequency of the various Oryza species, from AA-genome to BB * EE, and FF * HHKK showed an increased proportion of non-o. sativa and null alleles in the more distantly related wild species. Most of the wild species, except the more distant EE, GG, HHJJ, and HHKK genome accessions, consisted of relatively more indica than japonica alleles of SS markers. To validate the SS-STS study, PCR products of nine markers were sequenced across accessions. Sequencing results revealed that Oryza species share indica or japonica-like conserved InDel regions even across the different genomes. The presence of some japonica alleles beyond the AA genome at some SS InDel loci also suggests that japonica-specific alleles occurred early in the history of the Oryza genus. The O. sativa sub-species specific markers thus provide further insight into the evolutionary pathway in the genus Oryza and the process of differentiation between indica and japonica. W. Jiang Division of Agriculture, College of Plant Science, Jilin University, Changchun , China Present Address: M. J. Thomson Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843, USA

2 406 Genet Resour Crop Evol (2017) 64: Keywords indica japonica Insertion-deletion markers Oryza Subspecies-specific markers Wild rice Introduction The Oryza genus consists of eleven genomes: six diploid genomes (AA, BB, CC, EE, FF, and GG) and five allotetraploid genomes (BBCC, CCDD, HHJJ, KKLL, and HHKK), subdividing into 24 species (Vaughan et al. 2003; Marathi et al. 2015). Recent progress in structural and functional genomics has contributed to the understanding of evolutionary pathways in the Oryza genus which includes cultivated Asian rice (Oryza sativa L.). Although the domestication process of rice has been studied using a series of comparative methods, the domestication of Asian rice, in particular, remains a puzzle to be solved (Londo et al. 2006; Lu et al. 2009; Sang and Ge 2007, 2013; Vaughan et al. 2008b). Several ideas on the multiple pathways of domestication have been postulated on the basis of the archaeobotanical and geographic distribution of wild species, the hybridization and introgression between wild and cultivated populations, and the role of humans and animals in rice migration (Fuller 2007; Izawa 2008; Vaughan et al. 2008b; Purugganan 2010). One of the major breakthroughs of rice evolution is the emergence of the two O. sativa subspecies, O. sativa L. subsp. indica Kato and subsp. japonica Kato (hereafter referred to as indica and japonica for brevity), for which several pathways have been proposed (Izawa 2008; Kovach and McCouch 2008; Kovach et al. 2007; McCouch et al. 2007; Vaughan et al. 2008b). Sequence analyses have suggested an ancient divergence between the indica and japonica subspecies as early as 200, ,000 years ago that greatly predates rice domestication which is estimated to have occurred only about 10,000 years ago (Ma and Bennetzen 2004; Vitte et al. 2004). Moreover, the combination model of the multiple origin theory of rice genome evolution (Sang and Ge 2007) and the independent ancestors of aus- and aromatic-like O. rufipogon (McCouch et al. 2007; Sweeney and McCouch 2007) raised questions about the differentiation between indica and japonica. Even with large sequence data sets recent studies have not yet provided a conclusive answer which could provide support for multiple domestication events (Yang et al. 2012), for a single origin of domestication (Molina et al. 2011), and for a single origin leading to a domesticated japonica followed by subsequent hybridization and introgression with additional wild populations that will lead to indica (Huang et al. 2012; Sang and Ge 2013). It is generally recognized that future studies on the indica japonica diversification are needed which should include a large number of accessions of both subspecies and their wild ancestors to cover the diversity within each group (Garris et al. 2005; Kovach and McCouch 2008; Wang et al. 2008). Insertion/deletion (InDel) polymorphisms have been used for evaluating genetic variation in the Oryza genus. An important note in studying InDel polymorphisms is that the mutations are generally irreversible, having low levels of homoplasy (Hamdi et al. 1999; Rokas and Holland 2000). Other markers, especially simple sequence repeats (SSRs), have higher levels of homoplasy, which can lead to erroneous conclusions on true genetic relationships. Previous studies on the genetic variation between the subspecies indica and japonica employed in silico information between these sequences in one case examining miniature inverted repeat transposable elements and long terminal repeat retro-elements between the japonica variety Nipponbare and the indica variety GuangLuAi4 (Edwards et al. 2004), while in another case identifying 97 InDel markers between Nipponbare and the indica variety (Shen et al. 2004). Our previous work screened 765 sequence tagged site (STS) markers to identify a set of 67 InDel markers specific to each subspecies using 30 representative indica and japonica varieties (Chin et al. 2007), each of which clearly amplified only two alleles: one indica or one japonica allele (Supplementary Table 1). We propose that employing InDel markers that are specific for the indica and japonica subspecies will provide a novel view of the genetic relationships within the Oryza genus. These subspecies-specific STS (SS-STS) markers can track the genome-wide nature of rice accessions in relation to the indica and japonica subspecies and are therefore readily applicable to characterize germplasm for rice breeding programs. This was demonstrated after testing 320 O. sativa (133 japonica and 187 indica) accessions, with only a few varieties showing exceptions (Kim et al. 2009). Given that the indica japonica divergence predates domestication, the main objectives of this study were to explore the evolutionary relationships across wild

3 Genet Resour Crop Evol (2017) 64: accessions in the Oryza genus using indica japonica specific markers and to characterize the distribution of these InDel polymorphisms to better define the differentiation before and after domestication. Materials and methods Plant materials For the current study, 61 O. sativa varieties (O. sativa L. subsp. indica Kato and subsp. japonica Kato), 27 O. glaberrima accessions, and 202 accessions of 17 wild rice species were chosen to sample a broad range of genetic diversity across the cultivated and wild Orzya germplasm (Table 1). The Oryza species included: Oryza alta Swallen, Oryza australiensis Domin, Oryza barthii A. Chev., Oryza brachyantha A. Chev. et Roehr., Oryza coarctata Roxb., Oryza eichingeri Peter, Oryza glaberrima Steud., Oryza glumaepatula Steud., Oryza grandiglumis (Döll) Prodoehl, Oryza granulata Nees et Arn. ex Watt, Oryza latifolia Desv., Oryza longiglumis Jansen, Oryza longistaminata A. Chev. et Roehr., Oryza meridionalis Ng, Oryza minuta J. Presl, Oryza nivara Sharma et Shastry, Oryza officinalis Wall. ex Watt, Oryza punctata Kotschy ex Steud., Oryza ridleyi Hook. f., Oryza rhizomatis D. A. Vaughan, Oryza rufipogon Griff., and Oryza sativa L. (subsp. indica Kato and subsp. japonica Kato). The genome types and International Rice Germplasm Collection (IRGC) accession numbers at the International Rice Research Institute (IRRI) and accession numbers of Seoul National University (SNU) are listed in Supplementary Table 2. Accessions were grown and panicles on a single individual were bagged to harvest purified seed for each accession. DNA extraction and InDel marker genotyping Plants were grown on common paddy soil and DNA was extracted from young leaf blades of each accession according to the modified CTAB method (Pal et al. 2001). PCR amplification and detection for 67 SS-STS markers (Chin et al. 2007) was performed as described in Temnykh et al. (2000) with some modifications. Each 25 ll reaction mixture contained 50 ng DNA, 5 pmol of each primer, 2 ll PCR buffer [100 mm Tris (ph 8.3), 500 mm KCl, 15 mm MgCl 2 ], 250 lm of each dntps and unit Taq polymerase (SBS Genetech Co, Beijing, China). The MJ Research PCR system was used for DNA amplification. The thermocycler profile was: 5 min at 94 C, 35 cycles of 1 min at 94 C, 1 min at 55 C, 2 min at 72 C, and 5 min at 72 C for final extension. The amplified PCR products were resolved by electrophoresis on 8 % polyacrylamide gels. Each band was scored for the basic statistics of alleles as follows: 1 for indica-specific alleles, 2 for japonica-specific alleles, 3 for the heterozygote of indica and japonica alleles, 4 for alleles of another size, and 5 for missing or non-amplified alleles (null allele). For the PowerMarker and Structure analysis, the estimated size of each marker in base pairs (bp) was also recorded. Sequencing of InDel loci The sequencing of a subset of SS InDel loci was performed to validate the gel scoring. Nine InDel markers which had a single copy within each accession were chosen for sequencing based on three categories of gel scores: (1) three markers that amplified products only across the O. sativa complex (AA-genome), namely S01160, S09065, and S12030; (2) four markers that amplified bands across both the O. sativa and O. officinalis complex (AA to EE genomes) namely S02057B, S02081B, S02140, and S06001; and (3) two markers that amplified bands across all Oryza species (AA to HHKK genomes), namely S and S09093A. Representative accessions across all species were selected for DNA sequencing of these nine InDel markers, including 24 accessions of the O. sativa complex, 5 accessions of the O. officinalis complex, and 4 accessions of the O. ridleyi complex (Supplementary Table 3). PCR was performed using the TaKaRa Ex Taq polymerase (Takara Co., Japan). The amplified products were purified with MEGAquick-spin kits (intron Biotechnology Inc., Korea). Direct Sanger sequencing of the PCR amplicons using capillary electrophoresis (3730xl) was conducted by Macrogen (Seoul, Korea) using reads from both strands with trimming of low quality regions. SNP analysis For a genome-wide diversity analysis of the AA genome species, 96 accessions including IR64 and

4 408 Genet Resour Crop Evol (2017) 64: Table 1 The 290 Oryza spp. accessions and their origin in this study Complex a Species (genome) Abbrev. b Total no. of accessions Origin O. sativa complex O. officinalis complex O. barthii (AA) BAT 5 Chad (1), Guinea (1), Mali (1), Niger (2) O. glumaepatula (AA) GLU 6 Brazil (3), Colombia (1), French Guiana (1), Venezuela (1) O. meridionali (AA) MER 7 Australia (4), Indonesia (3) O. nivara (AA) NIV 32 Bangladesh (2), Cambodia (1), China (4), India (8), Laos (2), Malaysia (1), Myanmar (1), Nepal (7), Sri Lanka (2), Taiwan (1), Thailand (3) O. rufipogon (AA) RUF 84 Bangladesh (4), Cambodia (3), India (21), China (12), Indonesia (3), Laos (1), Malaysia (1), Myanmar (8), Nepal (10), Papua New Ginea (10), Philippines (1), Sri Lanka (1), Taiwan (1), Thailand (3), Vietnam (5) O. glaberrima (AA) GLA 27 Burkina Paso (2), Ivory Coast (1), Liberia (4), Mali (9), Nigeria (2), Senegal (3), Taiwan (1), Western Africa (5) O. longistaminata (AA) LON 5 Cameroon (1), Ethiopia (1), Madagascar (1), India (1), Unknown (1) O. sativa (AA) SAT 61 Afganistan (1), Bagladesh (3), Bhutan (1), China (3), India (16), Indonesia (3), Japan (2), Korea (13), Myanmar (2), Pakistan (1), Philippines/IRRI (14), USA (1), Western Africa (1) O. punctata (BB) PUN 6 Ghana (1), Kenya (1), Nigeria (1), Tanzania (1), Thailand (1), Unknown (1) O. minuta (BBCC) MIN 10 Philippines (10) O. officinalis (CC) OFF 9 Brunei (3), China (1), India (1), Papua New Guinea (1), Philippines (1), Thailand (1), Vietnam (1) O. rhizomatis (CC) RHI 3 Sri Lanka (3) O. eichingeri (CC) EIC 4 Sri Lanka (3), Uganda (1) O. alta (CCDD) ALT 4 Gyuana (1), India (1), Suriname (1), Unknown (1) O. latifolia (CCDD) LAT 9 Argentina (1), Costa Rica (5), Mexico (1), Paraguay (1), Unknown (1) O. grandiglumis GRAG 2 Brazil (2) (CCDD) O. australiensis (EE) AUS 5 Australia (3), Unknown (2) Unknown O. brachyantha (FF) BRA 1 Sierra Leone (1) complex O. ridleyi O. granulata (GG) GRAN 6 India (2), Nepal (1), Philippines (2), Unknown (1) complex O. longiglumis (HHJJ) LONG 1 Indonesia (1) O. ridleyi (HHJJ) RID 2 Thailand (2) O. coarctata (HHKK) c COA 1 Bangladesh (1) Total 290 a Oryza complex was defined and classified by Vaughan et al. (2005) b Abbrev is the abbreviation of each species in this study c O. coarctata (HHKK) is also designated as KKLL by Lu et al. (2009) Nipponbare were selected based on the SS-STS study and were assayed with a 384-plex SNP set using GoldenGate Assays with VeraCode on an Illumina BeadXpress Reader at the Genotyping Services Lab, IRRI (VC OPA; Thomson et al. 2012). Data analysis Genetic distance was calculated as the number of shared alleles. To identify the specific accessions which have a high proportion of either japonica or

5 Genet Resour Crop Evol (2017) 64: indica-specific alleles, the subspecies-prototype index (SPI) value (Chin et al. 2007) of each accession and its average across each Oryza species was calculated. The SPI value was estimated from the ratio of subspeciesspecific alleles in the model population: if the SPI of an accession is near either?1 or-1, its genome is considered to be close to japonica or indica, respectively. Phylogeny reconstruction was done by building a neighbor-joining tree using PowerMarker version 3.25 (Liu and Muse 2005). Bootstrap analyses were performed with 100 bootstraps for phylogenetic analysis within O. sativa and 1000 for the analysis including all accessions with AA genome by PowerMarker. The results of the bootstrap analysis were imported to Mega 5.05 (Tamura et al. 2011) to build combined phylogenetic trees. Sequence alignment and editing were carried out using ClustalW with BioEdit version (Tom Hall, North Carolina State University, North Carolina, USA) and all sequencing results were compared against (BGI, version of May 2014) and Nipponbare (MSU v.6) to confirm the expected sequences. Results Indica and japonica alleles across Oryza species A set of 67 SS InDel loci distributed over the 12 chromosomes was used to genotype 290 accessions of the Oryza genus, which included 61 O. sativa varieties, 27 O. glaberrima accessions, and 202 accessions of 17 wild rice species (Table 1). The 67 SS InDel markers differentially amplified DNA across the Oryza species, with an increased proportion of non-o. sativa and null alleles in the more distantly related wild species. A total of 11 out of the 67 SS markers amplified alleles in all wild species, while an additional 42 amplified across both the accessions of O. officinalis and O. sativa complex, and the remaining 14 SS markers were observed only in the O. sativa complex species (Fig. 1). Each O. sativa variety amplified only the two expected subspecies-specific alleles of indica and japonica, while additional alleles were identified in the other Oryza species at each SS InDel locus (Supplementary Table 4). For example, marker S03136 amplified five different alleles: (a) two O. sativa-specific alleles, 219-bp for indica and 200 bp for japonica in the AA, BB, BBCC, CC, CCDD, and EE genome Fig. 1 Number and distribution of amplified SS InDel markers across the three wild Oryza species complexes. The distribution of SS InDel markers is shown on the 12 chromosomes of rice, with 14 SS markers amplified in AA genome species only, 42 amplified across AA to EE, and 11 more amplified across all wild species accessions, (b) one allele at 160-bp only found in the wild species with the BB and BBCC genomes, but not in the CC genomes, (c) another wild species-specific allele of 180-bp in the four accessions of O. alta (CCDD) and O. australiensis (EE), and (d) a null allele in the HHJJ, GG, and KKLL (HHKK) genome accessions (Fig. 2). The indica-specific 219-bp allele was found in most of the wild rice species except in the FF * HHKK genome, while the japonica allele at this locus was observed only in the AA genome wild species. The overall allele constitution in each of the wild species as represented by the frequency ratio of amplified indica and japonica-specific alleles was calculated at 0.82 in the O. sativa complex and those of the O. officinalis complex and O. ridleyi complex fell to 0.32 and 0.09, respectively, due to the increased frequencies of non-o. sativa alleles or null alleles in the more distantly related wild species (Fig. 3). The frequency of indica alleles was higher than that of japonica in all the species in the AA * CCDD genome and in O. brachyantha (FF). However, the similar frequency of indica and japonica alleles was

6 410 Genet Resour Crop Evol (2017) 64: Fig. 2 Allele variation of 91 selected wild species accessions amplified by the subspecies-specific STS marker S Two japonica (CP-SLO and Nipponbare) and indica (IR64 and Dasanbyeo) varieties were used as controls to show japonica and indica-specific alleles, respectively. To check for contamination, water (W) was amplified instead of DNA in the last lane. An indica-specific allele (I, 219 bp) was also amplified in AA * EE wild rice accessions, while a japonica-specific allele (J, 200 bp) was not identified in the other accessions. One unique band (b 160 bp) was amplified in BB and BBCC genome and another band (c 180 bp) was identified in some CCDD and EE accessions, which implies the existence of duplication of SS loci in BB * EE species Non-O. sa va Ind/Jap Ind Jap JAP IND NIV RUF GLA GLU BAR LON MER PUN MIN OFF RHI EIC ALT LAT GRAG AUS BRA GRAN COA RID LONG O. sa va complex O. officinalis complex O. ridleyi complex Fig. 3 Genotype frequency of japonica, indica, heterozygous (indica and japonica), and non-o. sativa alleles of wild species. Three wild species complexes were described by Brar and Khush (2002) and Vaughan et al. (2005). See Materials and methods section for an explanation of the abbreviations of each species. The combined frequency of indica, japonica, and heterozygous (indica/japonica) alleles averaged across accessions was calculated for each complex (designated by the horizontal dashed lines). The higher indica allele frequency dominated that of the japonica alleles through all genome types except EE, GG, HHJJ, and HHKK

7 Genet Resour Crop Evol (2017) 64: found in the EE (O. australiensis) and the O. ridleyi complex species (O. brachyantha was not included in the O. ridleyi complex by Vaughan et al. 2005). Across the O. sativa complex, a higher frequency of japonica alleles was observed in O. glaberrima accessions compared to the other AA genome species, but these still had a greater proportion of indica alleles overall. Heterozygous alleles were also observed in the wild species of the O. sativa complex, O. punctata, and some species of tetraploids such as O. minuta (BBCC), O. latifolia (CCDD), O. alta (CCDD), and O. grandiglumis (CCDD). To identify the specific accessions which have a high proportion of either japonica or indica-specific alleles, the SPI value of each accession and its average across each Oryza species was calculated, with?1 indicating a japonica genome and -1 an indica genome. In the wild rice accessions, the SPI scores had mostly negative values, implying a trend in the indica type (Supplementary Figure 1). However, the SPI scores of O. granulata, O. longiglumis, and O. ridleyi were much larger positive values than the SPI of other wild species mainly due to the higher frequency of japonica than indica alleles generated by only a few SS InDel markers (Supplementary Table 2). Interestingly, most accessions in O. australiensis (EE) and O. granulata (GG) had positive SPI values, but they each had a single accession with a negative SPI. Very few accessions in other species showed a positive SPI value. It should be noted, however, that while the SPI value does not directly take into account null alleles, the SPI values for wild species distantly related to O. sativa must be considered with caution due to lower nucleotide homology leading to fewer markers being amplified overall. Two O. sativa varieties of mixed pedigree, IR68703-AC-24-1 and IR A, showed positive values even though they were originally classified as indica (Supplementary Figure 1a). In addition, three O. sativa aromatic accessions, Phudugey (O. sativa aus), JC 149 (O. sativa aromatic), and Basmati 370 (O. sativa aromatic), fell between indica and japonica with SPI values from to 0.13 (Supplementary Figure 1b). Phylogeny of Oryza species with SS InDel and SNP markers To confirm the ability of the SS InDel markers to differentiate between the indica and japonica accessions, a consensus phylogenetic tree of 100 bootstrap results using neighbor joining was first constructed for 61 O. sativa accessions. As expected, the 67 SS InDel markers clustered the two major indica and japonica groups, including the aus groups within indica (Supplementary Figure 2). The three O. sativa accessions were placed between indica and japonica groups, and there were two accessions of mixed ancestry, IR AC-24-1 and IR A, clustering with the japonica group. The SS InDel markers were then used to create a consensus phylogenetic tree of 1000 bootstrap results on 227 AA-genome accessions, not including the BB to HHKK accessions due to the high proportion of null alleles (Fig. 4). One distinct group contained the O. sativa japonica accessions with two O. nivara and two O. rufipogon accessions. The rest of the AA-genome accessions did not resolve into discrete clusters in the consensus tree due to the low bootstrap values, including the O. sativa indica and aus with scattered O. nivara, O. rufipogon, oneo. barthii, andtwoo. glumaepatula accessions, and the rest of the wild accessions from O. rufipogon, O. meridionalis, O. longistaminata, O. glumaepatula, O. barthii, O. glaberrima, and O. nivara. To compare the SS InDel results with other marker types, a set of 384 SNP markers was used to create a phylogenetic tree for the 96 AA-genome accessions (Supplementary Figure 3). The SNP markers showed several subgroups, with an indica cluster including IR64 and a number of O. rufipogon and O. nivara accessions, a separate cluster with O. barthii, O. glaberrima and more O. nivara and O. rufipogon accessions, with the remaining accessions outside those two clusters, including japonica (Nipponbare) and the other AA-genome species (Supplementary Figure 3). DNA sequencing of some subspecies-specific InDel loci To prove that the PCR amplicons in the wild Oryza accessions are corresponding to the O. sativa reference sequences and to confirm the InDel polymorphisms, nine SS InDel loci were selected for DNA sequencing across 33 representative accessions of 17 Oryza species (Supplementary Table 3). The amplicon sequences were compared by multiple alignment with indica (93-11) and japonica (Nipponbare) pseudomolecule sequences to visualize the SS InDel and flanking regions (Supplementary Figure 4).

8 412 Genet Resour Crop Evol (2017) 64: Fig. 4 Consensus tree of 227 accessions using 67 SS-STS markers across AA genome accessions. Major groups of Oryza accessions include the indica and aus accessions (shown in red) with some indica-like O. rufipogon and O. nivara accessions distributed between them, japonica and aromatic accessions The sequence data reflected the various patterns of indica and japonica allele amplification that was seen at the SS InDel marker loci. For example, locus S07103 had the predicted 12-bp insertion/deletion site and a predominantly japonica allele distribution, and most wild accessions from the AA to GG genome and even Kasalath and Black Gora also showing a japonica allele (Fig. 5a). Other loci had greater (shown in blue) with several japonica-like O. rufipogon and O. nivara accessions, and the other groups of wild accessions including O. meridionalis, O. longistaminata, O. glumarpatula, O. barthii, O. glaberrima and O. nivara from left to right. (Color figure online) variation than expected. For example, locus S09093A had the expected 27-bp indica deletion between the indica (IR64 and 93-11) versus the Nipponbare reference, but had a 10-bp deletion across many of the wild accessions along with a higher variation on the flanking region at nucleotide level among accessions (Fig. 5b). S09093A also included a second O. bathii- and O. glaberrima-specific InDel

9 Genet Resour Crop Evol (2017) 64: Fig. 5 Nucleotide variation at three SS InDel loci across AA genome to GG genome accessions. A S07103 locus: amplified well and aligned from AA to GG genome, with O. nivara and O. rufipogon showing indica or japonica alleles, although the japonica allele is more frequent across the accessions. B S09093A locus: showed higher variation on the flanking region at the nucleotide level among accessions even though most accessions showed similar size to the indica allele on the gel; moreover it became more diverse in the more distant genomes from AA to HHKK (black box). S09093A also includes O. bathii and O. glaberrima specific InDel (orange box) and an O. longistaminata specific InDel (yellow box). C S09065 locus: the AA genome accessions amplified well and aligned sequences showed most AA genome accessions had the indica allele across the accessions while O. meridionalis showed another species-specific InDel polymorphism. (Color figure online)

10 414 Genet Resour Crop Evol (2017) 64: and a third O. longistaminata-specific InDel in the flanking sequence (Fig. 5b). In another example, the locus S09065 amplified in the AA genome only and the sequence data showed that AA-genome species such as O. barthii, O. glumaepatula, O. rufipogon, O. nivara, O. glaberrima, and O. longistaminata have indica alleles without the 11-bp deletion seen in Nipponbare. However, the O. meridionalis species showed a species-specific InDel of 38-bp at the same location as the indica/japonica InDel (Fig. 5c). The other sequenced loci also confirmed many of the expected insertion/deletions but also revealed variation in the flanking sequences. The locus S02081B, where many AA-genome wild accessions showed a japonica allele, had several single basepair deletions in the flanking region (Supplementary Figure 4). The loci S02057B and S06001 showed that the O. sativa complex and O. officinalis complex were well aligned together, with a few accessions showing the japonica allele seen in Nipponbare, but most amplicons presented an indica allele with slight sequence variations among accessions (Supplementary Figure 4). Furthermore, the wild species also showed a number of single-nucleotide polymorphisms among accessions at the aligned InDel loci even though the amplicons were the same size. In a few instances, the sequences from the amplicons revealed InDel polymorphisms that differed between the expected insertion/deletion sizes from the reference sequences (93-11 and Nipponbare) and the actual SS InDel sequences observed. For instance, locus S01160 is supposed to have a 4-bp InDel polymorphism between the indica and japonica alleles, while the observed sequences had InDels varying from 4 to 12 bp due to the repeated 4-bp motif (Supplementary Figure 4). Likewise, locus S02057B had a slight variation in the insertion/deletion size likely due to the low sequence complexity at that locus (Supplementary Figure 4). But overall, the insertion/ deletions determining the indica and japonica alleles were shared across species in the Oryza genus. Discussion Usefulness of SS InDel markers Feltus et al. (2004) annotated whole-genome SNP and InDel polymorphisms between Nipponbare (japonica) and (indica) and predicted a total of 24,557 InDel polymorphisms between the two genomes. However, their specificity to indica and japonica varieties has not been tested. In another study, Chin et al. (2007) screened a total of 765 STS markers with 30 varieties and identified 67 subspecies-specific (SS) InDel makers. Selected indica japonica SS markers are unique in their application to evaluate the genomic similarity to indica and japonica prototypes in varieties derived through indica japonica hybridization and in unknown new landraces or germplasm (Chin et al. 2007; Degenkolbe et al. 2009; Kim et al. 2009). In cultivated O. sativa germplasm, these markers amplified only two unique and specific alleles of the indica and japonica subspecies (Chin et al. 2007; Kim et al. 2009). Moreover, with the distinct size of amplicons from the large differences in allele sizes, these markers are readily applicable to evaluate the indica japonica similarity in the genetic resource management of rice, even on the agarose gels (Chin et al. 2007). However, gel-based markers could have variations in the flanking sequence, requiring sequence analysis to decipher whether amplified sequences are corresponding to the target (Nishikawa et al. 2005). In the current study, SS InDel markers successfully provided information on the indica or japonica alleles across the Oryza genus because of the stretches of conserved flanking sequences at the InDel sites. This low level of nucleotide variation flanking SS InDel loci may also be explained by selective sweeps. For example, there were different levels of variation at the loci S01160 and S02057B which suggest that the S01160 locus underwent a stronger selective sweep than the S02057B locus (Xie et al. 2011; Zhao et al. 2010). Moreover, flanking conserved sequences may be due to the coding sequences of neighboring genes. To cite, S07103, which amplified across all wild species, is annotated as a PWWP domain containing protein related to chromatin remodeling (Stec et al. 2000). InDel mutations can be considered as excellent genetic sources of phylogenies to gain insight on rice evolution (Rokas and Holland 2000). Polymorphisms based on rare genomic changes (RGCs) have irreversible mutations such as Alu repeats (Hamdi et al. 1999) and SINEs (Cheng et al. 2003). Among various RGCs, including retrotransposons, cellular gene order changes, and gene duplications, InDels provide a wide-range taxonomic solution with low homoplasy

11 Genet Resour Crop Evol (2017) 64: and are applicable in the phylogenetic system of eukaryotes (Rokas and Holland 2000; Rogozin et al. 2008). The irreversible nature of the SS InDels used in this study is supported by the low numbers of alleles at each locus. Distribution of subspecies-specific alleles across Oryza species To date, several studies on rice domestication have focused mainly on the evolution of the AA genome, while the recent report on MOC1 using comparative genetics tools suggested that several kinds of variation have occurred in domestication-related genes across the AA to HHKK genomes to determine different genomes or species (Lu et al. 2009). The fact that more than 90 % of SS markers were amplified in the AA genome supports the previous reports on the genetic similarity of the wild species O. rufipogon and O. nivara, with japonica and indica (Li et al. 2006; Sweeney and McCouch 2007). African cultivated rice, O. glaberrima, showed the highest frequency of amplification compared with the wild Oryza species. In addition, although O. barthii and O. glaberrima shared some indica/japonica InDels, they have geographically specific polymorphisms as well. The domestication of African rice likely occurred later than in Asia and seemed to have diverged from Asian Oryza (Vaughan et al. 2008b). The genetic diversity of O. glaberrima in this study using 67 SS markers was very low as suggested by a previous report (Chang 1976). To better visualize the indica/japonica specific alleles across the wild rice species, a scatter plot displayed the number of japonica alleles on the x-axis and the number of indica alleles on the y-axis (Supplementary Figure 5). The japonica accessions were clustered at the bottom right aligning with the diagonal axis, whereas indica accessions were at the top left. Phudugey (aus), JC 149 (aromatic), and Basmati 370 (aromatic) were located in the middle of the indica and japonica groups, inferring a heterologous constitution of indica and japonica alleles at SS loci for these varieties. Almost all the O. rufipogon and O. nivara accessions were scattered near the indica group. Some accessions which have mostly equal indica and japonica alleles might be related to the domestication or emergence of O. sativa subspecies as previously reported (Garris et al. 2005; McCouch et al. 2007), but this should be further confirmed by examining specific genes and markers associated with domestication-related traits. The large gap between BB and AA accessions is explained by the larger proportion of indica alleles than japonica alleles that were amplified in the AA genome species. Heterozygous indica/japonica alleles in O. rufipogon accessions A number of O. rufipogon accessions from India and China showed a high heterozygous genotype frequency ranging from 14 to 25 %, including IRGC from India and IRGC , 99556, and from China (Supplementary Table 2). Most O. rufipogon and two O. nivara accessions are located between indica/japonica clusters, and a high frequency of indica/japonica heterozygote genotypes that have been conserved in O. rufipogon accessions from the Indo-China region shows that they still hold japonica alleles. Wang et al. (2008) analyzed 920 O. rufipogon accessions using 36 SSR markers in southern China. Interestingly, the significant difference in the frequency of japonica-specific and indica-specific alleles was found in Jiangxi and Hunan provinces near the Yangtze river where rice cultivation possibly started (Zong et al. 2007). On the other hand, the proportion of heterozygous genotypes in the populations in southern China was also reported to range from % in Yunnan province to % in Hainan province (Wang et al. 2008). Moreover, the area of the Pearl River was reported as the place of the first advent of cultivated rice since O. rufipogon accessions collected from southern China were closely clustered with cultivated rice as ancestral progenies (Huang et al. 2012). These heterozygous alleles from southern China may have been collected from the regions of admixture between indica and japonica populations (Kovach and McCouch 2008). Thus, the alleles only amplifying in the AA genome species and showing high heterozygous allele frequency in O. rufipogon accessions might be related with the allele evolution of SS InDel markers in the indica japonica differentiation in Asian rice. However, the question on the linkage between the region of genetic diversity with high heterozygous allele frequency as that in Hainan province and the region of high frequency of japonica-specific alleles (Jiangxi-Hunan province near Yangtze river) needs further study with respect

12 416 Genet Resour Crop Evol (2017) 64: to the population structure of germplasm from Southeast Asia. Unanswered questions on SS loci for rice evolution The 67 SS InDel markers are not evenly distributed in all chromosomes. Several clustered markers on the long arm of chromosome 9 showed an interesting distribution: only one marker, S09093A, amplified the DNA of almost all the accessions while the adjacent three markers S09062B, S09073, and S09075A appeared in the O. sativa complex and O. officinalis complex. Another five markers more widely distributed in this region amplified SS alleles of the O. sativa complex. Furthermore, sequencing results for the S09093A locus showed other significant speciesspecific InDel polymorphisms, including O. barthiiand O. glaberrima-specific and O. longistaminataspecific, aside from indica- and japonica-specific polymorphisms, and results for the S locus showed an O. meridionalis-specific InDel (Fig. 5 and Supplementary Figure 4). These complex patterns of InDel polymorphism will require further investigation to better understand the underlying forces that led to these results. A recent review on rice evolution and domestication suggested that the evolution of rice occurred more than once (Vaughan et al. 2008a). Since the SS InDel loci showed indica or japonica alleles beyond the AA genome, it suggests that these loci were present earlier than O. rufipogon or the AA genome divergence and that patterns of the SS InDel loci can be traced back into the O. sativa, O. officinalis, and O. ridleyi complexes. The major mechanisms involved could be hybridization-introgression (Vaughan et al. 2008a), admixture (Garris et al. 2005), leveraging natural diversity (Kovach and McCouch 2008), or domestication-superdomestication (Vaughan et al. 2007). A common theme throughout these studies was the effect of migration and adaptation, followed by introgression of specific chromosome segments after naturallyoccurring hybridization between divergent populations (Kovach et al. 2007). Even though we have the partial sequence data with nine SS InDel loci across the genome, it is insufficient to answer the deeper questions of the evolution of domesticated rice. Further investigation is required to assess whether the association among the SS InDel loci and domestication-related genes may provide more insight into the questions on bottleneck effects and the significance of selection as a result of evolutionary and domestication-related processes. Finally, comparative genetics using the sequence of SS loci of each Oryza species accessions might provide more information as the full genome sequences of the different wild Oryza species become available (Jacquemin et al. 2013). Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center, No. PJ ), Rural Development Administration, Republic of Korea and support from the Global Rice Science Partnership (GRiSP) to the International Rice Research Institute. The authors thank Christine Jade Dilla- Ermita, Joie Ramos and Eleazar Manalaysay for providing technical support. Compliance with ethical standards Conflict of interest conflict of interest. The authors declare that they have no Human and animal rights This research does not involve human participants or animals. References Brar DS, Khush GS (2002) Transferring genes from wild species into rice. In: Kang MS (ed) Quantitative genetics, genomics, and plant breeding. CAB International, Wallingford, pp Chang TT (1976) The origin, evolution, cultivation, dissemination, and diversification of Asian and African rices. Euphytica 25: Cheng C, Motohashi R, Tsuchimoto S, Fukuta Y, Ohtsubo H, Ohtsubo E (2003) Polyphyletic origin of cultivated rice: based on the interspersion pattern of SINEs. Mol Biol Evol 20:67 75 Chin JH, Kim HH, Jiang WZ, Chu SH, Woo MO, Han LZ, Brar DS, Koh HJ (2007) Identification of subspecies-specific STS markers and their association with segregation distortion in rice (Oryza sativa L.). J Crop Sci Biotech 10: Degenkolbe T, Do PT, Zuther E, Repsilber D, Walther D, Hincha DK, Köhl K (2009) Expression profiling of rice cultivars differing in their tolerance to long-term drought stress. Plant Mol Biol 69: Edwards JD, Lee VM, McCouch SR (2004) Sources and predictors of resolvable indel polymorphism assessed using rice as a model. Mol Gen Genomics 271: Feltus FA, Wan J, Schulze SR, Estill JC, Jiang N, Paterson AH (2004) An SNP resource for rice genetics and breeding based on subspecies Indica and Japonica genome alignments. Genome Res 14: Fuller DQ (2007) Contrasting patterns in crop domestication and domestication rates: recent archaeobotanical insights from the Old World. Ann Bot-Lond 100:

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