Breeding Science
Online ISSN : 1347-3735
Print ISSN : 1344-7610
ISSN-L : 1344-7610
Research Papers
Genetic diversity of the wx flanking region in rice landraces in northern Laos
Chiaki MutoRyuji IshikawaKenneth M. OlsenKazuaki KawanoChay BounphanousayToru MatohYo-Ichiro Sato
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Supplementary material

2016 Volume 66 Issue 4 Pages 580-590

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Abstract

A glutinous texture of endosperm is one of the important traits of rice (Oyza sativa L.). Northern Laos is known as a center of glutinous rice diversity. We genotyped INDEL, SSR and SNP markers in a sample of 297 rice landraces collected in northern Laos. These glutinous varieties were confirmed to share a loss-of-function mutation in Granule bound starch synthase I (Wx). INDEL markers revealed a high frequency of recombinant genotypes between indica and japonica. Principal component analysis using SSR genotypes of Wx flanking region revealed that glutinous indica landraces were scattered between non-glutinous indica and glutinous-japonica types. High ratios of heterozygosity were found especially in glutinous indica. Haplotype analysis using SNP markers around Wx locus revealed that glutinous indica landraces would have a few chromosome segments of glutinous japonica. Frequent recombinations were confirmed outside of this region in glutinous indica. This intricate genetic structure of landraces suggested that glutinous indica landraces in Laos were generated through repeated natural crossing with glutinous-japonica landraces and severe selection by local farmers.

Introduction

The endosperm of major seed crops such as rice, maize, and wheat is the major organ in which starch is stored. Many genes such as the starch biosynthesis and the color of the aleurone layer have been targeted for artificial selections (Olsen and Purugganan 2002, Palaisa et al. 2004, Sweeney et al. 2006, Wang et al. 1999, Whitt et al. 2002). One of the genes, Granule bound starch synthase I (GBSSI ; Wx) regulates amylose content of rice endosperm through three different functional alleles, Wxa, Wxb and wx (Sano 1984). Wxa, generally found in non-glutinous indica varieties and wild rice, confers relatively high amylose content (Umemoto and Terashima 1999) whereas Wxb carries a G-T exon 1 substitution resulting in an intron splicing mutation that confers a lower amylose content. The allele has tended to be found in non-glutinous japonica varieties (Hirano et al. 1998, Isshiki et al. 1998). The wx allele found in glutinous rice is defined by a complete loss of function of GBSSI which confers a lack of amylose synthesis (glutinous phenotype), generally caused by a 23-bp duplication in exon 2 (Inukai et al. 2000). Rice with lower amylose content and a glutinous phenotype predominates in hilly areas of Southeast Asia and also in northern Asia. There is molecular evidence of a strong selective sweep in a starch of about ~250 kb flanking the Wx locus among landraces cultivated in Asian countries (Olsen et al. 2006).

The Lao PDR (Laos) is characterized by consumption of glutinous cereals including rice, foxtail millet, and other millets, and for this reason it is known as the “glutinous zone” (Nakagahra 1986, Sakamoto 1987, Watabe 1967). The glutinous quality of the crops in this area probably reflects the preference of the local people, who constitute 49 independent ethnic groups (Sisouphanthong and Taillard 2000), most of whom consume predominantly glutinous rice as part of their culture (Schiller et al. 2006). The rice fields in northern Laos consist mostly of upland fields and partly paddy fields. Recently, some modern rice varieties have been introduced to paddy fields as cash crops, but not for private consumption (Ishikawa et al. 2002). However, the majority of the fields are cultivated for glutinous landraces mostly on 1.5–2 ha farms in hilly areas that are owned by single families. These landraces are sown as seeds harvested by the farmers themselves. Multiple landraces recognized from their morphological appearance are cultivated in single fields and later identified molecular bases (Ishikawa et al. 2002, Muto et al. 2010). Local farmers also recognized their phenotypic and agronomic differences created through unconscious selection. These are regarded as valuable genetic resources for improvement of modern varieties to supply stress resistance or novel phenotypes. Intensive efforts to collect these landraces have been made by local research organizations with international collaboration (Ishikawa et al. 2002, Muto et al. 2010). However, there are several factors that limit the accurate evaluation of these resources: 1) Landraces showing various phenotypes often share a single name, 2) genetically different individuals are cultivated as single landraces, 3) local people frequently exchange their seeds within or among villages, and 4) outcrossing between genetically different individuals is frequent (Ishikawa et al. 2002, 2006, Muto et al. 2010). Such factors resulted from natural outcrossing and management of heterogeneous seed stocks. These events are largely allowed under the traditional slash and burn system. It acted as genetic buffer to keep diversity. Thus, these landraces have huge potential for future rice breeding.

In the present study, we evaluated the genetic constitutions of these glutinous accessions at the cytoplasmic and nuclear levels and determined their fine structure around the Wx locus. These molecular data allowed us to grasp how glutinous landraces have been maintained through slash and burn system in Laos and how frequently outcrossing has occurred inside fields. In this study, we focus on Wx locus because the phenotype is a key trait for Lao farmers.

Materials and Methods

Plant materials

Two hundred ninety-seven rice (Oryza sativa L.) accessions comprising 276 landraces and 21 modern varieties were collected from 45 villages in six northern provinces of Laos—Luang Prabang, Luang Namtha, Oudom Xai, Phong Sali, Vientiane and Vientiane Municipality—between 2003 and 2005 (Fig. 1). The field survey was carried out under a material transfer agreement (MTA) between National Agriculture and Forestry Research Institute (NAFRI, Laos) and Research Institute for Humanity and Nature (RIHN, Japan). Dr. Chay Bouphanousay who was a researcher of NAFRI participated in the exploration. All of the collected materials were conserved in genebank of NAFRI. The duplications of these materials were used in this study. All of the materials had been grown by local farmers belonging to various ethnic groups. These landraces showed various appearances (Fig. 2). Differentiation between glutinous and non-glutinous endosperm was made on the basis of staining with 0.7% iodine-potassium-iodine (I/KI). After an overview of their genetic characteristics, 75 accessions were selected for further analysis (Table 1). DNA from all of these materials was extracted using the CTAB method from young leaves of single individual landrace plants.

Fig. 1

Sampling sites of local rice accessions in northern Laos. Rice accessions were collected from 45 local villages in six provinces, Luang Phrabang, Luang Namtha, Oudomxai, Phongsali, Vientiane, and Vientiane Municipality.

Fig. 2

Various appearances of seeds and hull in glutinous rice landraces collected from northern Laos. Accession No. and genotype of ORF100-INDEL (+; deletion type, −; non-deletion type) were shown in each photo.

Table 1 Information and indica-japonica classification of representative accessions judged by the four INDEL markers
Acc.No. Sample ID Site Name Ethnic Field condition Variety type J/I ORF100a Pgi 1b Cat 1c Acp 1d
LD23d Jwx105 LD ngo lamtan Khmu Up L J ND D ND INS
LL81b Jwx131 LL khao yuak Up L J ND D ND INS
LL81k Jwx139 LL no-record Low L J ND D ND INS
LL83f Jwx149 LL khao nang Khmu Up L J ND D ND INS
LL88e Jwx172 LL khao dong Khmu Up L J ND D ND INS
LL90d Jwx180 LL khao pat hin Khmu Up L J ND D ND INS
LL91d Jwx185 LL no-record Khmu Up L J ND D ND INS
LL93b Jwx192 LL khao vieng Khmu Up L J ND D ND INS
LL95c Jwx201 LL mak kheua khao Khmu Up L J ND D ND INS
LL96a Jwx204 LL no-record Khmu Up L J ND D ND INS
LL97a Jwx211 LL no-record Khmu Up L J ND D ND INS
LL101b Jwx222 LL fo hian Up L J ND D ND INS
LL104d Jwx240 LL khao deng du Tai Lue Up L J ND D ND INS
LL105f Jwx250 LL ngo tam Lao Up L J ND D ND INS
LD22d Jwx264 LL ngo muong Khmu Up L J ND D ND INS
KW1g Jwx307 LP mai platiyao Lanteng Up L J ND D ND INS
LD26a Jwx316 LD ngo yansuang Khmu Up L J ND D ND INS
LD32c Jwx380 LD ngo iinm Khmu Up L J ND D ND INS
LD25h Jnonwx56 LD ngo clock Khmu Up L J ND D ND INS
LD23e Jnonwx110 LD ngo salii Khmu Up L J ND D ND INS
LL87j Jnonwx167 LL khao chao Thai Dam Up L J ND D ND INS
LL90b Jnonwx178 LL khao deng Khmu Up L J ND D ND INS
LL92b Jnonwx189 LL khao ngo Lao Lum Up L J ND D ND INS
LL96c Jnonwx206 LL no-record Khmu Up L J ND D ND INS
LL105h Jnonwx252 LL ngo chao Khmu Up L J ND D ND INS
LD23a Jnonwx270 LD ngo chao Khmu Up L J ND D ND INS
KW2a Jnonwx308 LP manwa Lao ku Up L J ND D ND INS
KW3c Jnonwx315 LP hoche Akha Up L J ND D ND INS
KW4f Jnonwx327 LN cheju Akha Up L J ND D ND INS
KW4i Jnonwx330 LN lokuu Akha Up L J ND D ND INS
LD27g Jnonwx358 LD ngo chao clock Khmu Up L J ND D ND INS
LD31a Jnonwx370 LD ngo hiyan Khmu Up L J ND D ND INS
LL107b Jnonwx389 LL ngo prai Khmu Up L J ND D ND INS
LL107h Jnonwx395 LL hoichalyng Khmu Up L J ND D ND INS
ST22a Jnonwx451 LV khao niipun Low J ND D ND INS
LD24f Iwx1 LD khao kalong Tai Lue Low L I D ND D Non-INS
LD25g Iwx51 LD ngo ber Khmu Low L I D ND D Non-INS
LL82a Iwx142 LL khao pee pii Khmu Up L I D ND D Non-INS
LL94a Iwx196 LL khao leuan Up L I D ND D Non-INS
LL103f Iwx236 LL Khao Mea Tor Lao Low L I D ND D Non-INS
LD20e Iwx257 LD ngo pet Khmu Up L I D ND D Non-INS
LL103c Iwx233 LL khao Dor Dai Tai Lue Low L I D ND D Non-INS
ST02 Iwx400 LV khao kam Low L I D ND D Non-INS
ST5c Iwx405 LV khao angii Lao ND L I D ND D Non-INS
ST6c Iwx409 LV khao kii tom Lao Lum Low L I D ND D Non-INS
ST10b Iwx424 LV khao hoom neuang Lao Low L I D ND D Non-INS
ST16b Iwx434 LV khao Tomburi ND L I D ND D Non-INS
ST17b Iwx436 LV khao sai mai Lao Lum Low L I D ND D Non-INS
ST20b Iwx444 LV khao saam patone do Low L I D ND D Non-INS
LL81i Inonwx138 LL khao chao Up L I D ND D Non-INS
LL87i Inonwx166 LL khao chao pun Thai Dam Up L I D ND D Non-INS
LL93d Inonwx194 LL khao chao pun Khmu Up L I D ND D Non-INS
LL97i Inonwx218 LL no-record Khmu Up L I D ND D Non-INS
LL97j Inonwx219 LL no-record Khmu Up L I D ND D Non-INS
KW4c Inonwx324 LN khao lao soon Akha Low L I D ND D Non-INS
KW4e Inonwx326 LN teche Akha Low L I D ND D Non-INS
ST5a Inonwx403 LV RD8 Lao Low M I D ND D Non-INS
ST6b Inonwx408 LV khao chao hoom mali Lao Lum Low M I D ND D Non-INS
ST7a Inonwx413 LV khao ubong deeng Low M I D ND D Non-INS
ST8a Inonwx414 LV khao hoom mali Hmong Low M I D ND D Non-INS
ST9e Inonwx420 LV CR203 Low M I D ND D Non-INS
ST16a Inonwx433 LV khao chao hoom mali Dounhien Low M I D ND D Non-INS
ST20d Inonwx446 LV khao chaodo Low L I D ND D Non-INS
LL87a Jwx158-rec LL khao hin Thai dam Up L Rec ND ND ND INS
ST14a Jwx430-rec ST khao pee deng Lao Lum Up L Rec ND D D INS
LL97h Iwx217-rec LL no-record Up L Rec D D ND Non-INS
LL97k Iwx220-rec LL no-record (hybrid) Up M Rec D ND ND Non-INS
ST9c Iwx418-rec LV RD6 Low M Rec D D D Non-INS
ST9g Iwx422-rec LV no-record Low M Rec D D D Non-INS
ST11b Iwx426-rec LV khao pee deng Lao lum Up L Rec D D D Non-INS
ST19c Iwx441-rec LV khao phee dian Lao Lum Up L Rec D D D Non-INS
KW5d Jnonwx338-rec LN oyof Mushuda Up L Rec ND ND ND INS
ST4a Jnonwx402-rec LV khao chao laai Hmong Low L Rec ND ND ND INS
ST15a Jnonwx432-rec LV khao chao dam Hmong Up L Rec ND ND ND INS
KW6h Inonwx348-rec LN ngo suangungcho Khmu Khuen Low L Rec D D D Non-INS

Site: LL; Louang Phrabang, LN; Louang Namtha, LP; Phongsali, LD; Oudomxai, LV; Viengtiane.

Field condition: Up; Upland, Low; Lowland.

Variety type: L; Landrace, M; Modern variety.

J/I: J; japonica, I; indica, Rec; Recombinant. Judged by four INDEL genotypes. INDEL genotypes were classified by letters, D as deletion, ND as non-deletion, Non-INS as no insertion, INS as insertion.

a  ORF100: D; I type, ND; J type.

b  Pgi1: D; J type, ND; I type.

c  Cat1: D; I type ND; J type.

d  Acp2: INS; J type, Non-INS; I type.

INDEL markers for waxy and indica-japonica classification

Information of all primers used in this study is described in Supplemental Table 1. The causal mutation of glutinous rice, a 23-bp duplication in exon 2 of GBSSI, was confirmed using a primer pair: WaxyA1 and WaxyB1. PCR reactions were carried out as described in previous study (Muto et al. 2010). For genotyping, amplicons were electrophoresed on 1.5% agarose gels. Discrimination of accessions into indica-japonica types was based on the chloroplast INDEL marker (ORF100; Chen et al. 1993, 1994, Kanno et al. 1993) and nuclear INDEL markers (Pgi1-INDEL, Cat1-INDEL and Acp1-INDEL) were designed based on the isozymes that were flanked by indica-japonica diagnostic isozyme genes (Ishikawa et al. 1991, Sano and Morishima 1992). The INDEL patterns of these three nuclear INDEL markers were firstly confirmed by applying to a set of reference rice accessions provided as “Dr.Oka’s tester strain” by National Institute of Genetics that previously had been classified into indica or japonica based on its morphological and physiological characteristics (Oka 1958) and isozyme genotypes (Ishikawa et al. 1991). All PCR reactions were performed with 0.25 U rTaq (Takara Co., Japan) per single reaction. The conditions were; 95°C preheating for 5 min, followed by 35 cycles of 96°C for 30 s, 72°C for 1 min, and 75°C for 5 min. All INDEL markers were checked their performance with typical indica and japonica landraces, 40 accessions collected from Japan, China, and South-east Asian countries were compared for their genetic characteristics (Supplemental Table 2).

SSR analysis of the total genome and Wx flanking region

All 297 accessions were analyzed for genotypes at 12 SSR loci, which were randomly selected from 12 chromosomes in order to clarify the general genetic characteristics. In order to focus on polymorphism around the Wx locus, other set of 11 SSR markers dispersed at about 100-kb intervals around the Wx locus were genotyped for 75 representative accessions from northern Laos (Fig. 3). RM190 located in exon 1 of Wx is composed of multiple alleles (Temnykh et al. 2000). The multiple alleles were tightly linked with 23 bp tandem duplication which was causal mutation of glutinous rice (Bligh et al. 1995).

Fig. 3

Location of Wx flanked markers used in this study.

Haplotype analysis around the Wx locus

The Wxb allele was associated with low amylose resulting from a G to T substitution (hereafter, G-T substitution) at the splicing donor site between exon 1 and intron 1. Nucleotide diversity was determined with SNPs in 12 fragments dispersed at about 50 kb intervals around the Wx locus. Six fragments named KN5-0, KN5-2, KN5-4, KN5-6, KN5-8, and KN5-10 were located upstream of the Wx locus, and another six fragments named KN3-2, KN3-4, KN3-6, KN3-8, KN3-10, and KN3-12 were located downstream (Fig. 3). The PCR reaction consisted of preheating at 95°C for 5 min, followed by 30 cycles of 95°C for 10 s, 55°C for 30 s, 72°C for 30 s, and 75°C for 5 min. Amplicons were sequenced.

Data analysis

SSR data were analyzed using GenAlEx 6.1 (http://www.anu.edu.au/BoZo/GenAlEx/) to determine the number of alleles, allele frequencies, Nei’s genetic diversity, and for PCA (principal component analysis). Nucleotide diversity estimated as the Pai value was analyzed using Dna SP 5.10.1 (Librado and Rozas 2009).

Results

Overview of genetic structure of rice landraces in Laos

The landraces comprised 237 upland rice and 60 lowland rice accessions, and on the basis of endosperm traits, 231 were classified as glutinous and 66 as non-glutinous rice accessions. The origins of maternal lineages were identified with cpDNA INDEL (ORF100-INDEL) which was flanked by ORF100 (Chen et al. 1993, 1994, Kanno et al. 1993). Alternative plastid types, deletion or non-deletion type were defined as indicative of indica or japonica plastid type, respectively. Seventy-one percent of landraces, i.e. 223 out of 297 accessions, carried the non-deletion type whereas the remaining 74 carried the deletion type. Genomic diversity was surveyed by examining the genotypes of 12 randomly selected SSRs from 12 chromosomes. A general overview of the landraces was obtained using PCA based on the SSR genotypes and ORF100-INDEL (Fig. 4). The first and second components explained 41.0% and 17.4% of the total variation. The scatter plot showed that the population consisted of two distinct groups corresponding to indica and japonica group, respectively. Both groups contained glutinous and non-glutinous types. To perform sequence based analysis focused on the Wx flanking region, 75 accessions were chosen as representatives depending on endosperm phenotype (glutinous/non-glutinous) determined by I/KI staining and indica-japonica type of plastid determined by ORF100-INDEL type. These 75 accessions comprised 20 glutinous/non-deletion, 20 non-glutinous/non-deletion, 20 glutinous/deletion, and 15 non-glutinous/deletion accessions. In the last group, nine of the non-glutinous/deletion accessions were landraces, and the remaining six were modern varieties. Some of these included modern, recently introduced lowland varieties. For more exact indica-japonica classification, three INDELs for nuclear DNA—Acp1-INDEL, Cat1-INDEL, and Pgi1-INDEL were applied. These markers were designed tightly linked to each of the isozyme genes, Acp1, Cat1 and Pgi1, respectively. These three isozymes are locating different chromosomes respectively, and it was confirmed that there was no linkage disequilibrium among these three isozymes that have been widely used to know sub-species (Sano and Morishima 1992). Genotypes of these INDELs were first confirmed with indica-japonica reference accessions to follow the past result. Another chloroplast-based INDEL has been widely used to identify maternal linage (Garris et al. 2005). The results of ORF100-INDEL and three isozyme INDEL markers were consistent with the reference data (Ishikawa et al. 1991, Oka 1958, Supplemental Table 2). These genotypes were used to know rough classification of sub-species among landraces collected in Laos. Based on the genotypes of four INDEL markers (ORF100-INDEL, Acp1-INDEL, Cat1-INDEL, and Pgi1-INDEL), 18 accessions were classified as japonica/glutinous (J/wx), 17 as japonica/non-glutinous (J/Wx), 14 as indica/glutinous (I/wx), and 14 as indica/non-glutinous (I/Wx). Some of the accessions carried inconsistent combinations of nuclear INDELs and the ORF100-INDEL. These accessions were regarded as “recombinant type” (referred to as Rec-type hereafter). In total, eight recombinant/glutinous (Rec/wx) and four recombinant/non-glutinous (Rec/Wx) accessions were recognized (Table 1).

Fig. 4

Principal coordinates analysis by using SSR polymorphisms of 12 random SSR markers on each chromosome among 297 accessions. Horizontal and vertical axes show first and second principal components respectively. Non-deletion type of ORF100-INDEL and deletion type ORF100-INDEL were noted as circles and triangles. Glutinous type was noted as open symbols and non-glutinous type as solid symbols.

Wx locus is reported as a recombination hotspot in rice by previous studies (Inukai et al. 2000). As we had speculated about frequent recombination around the Wx locus, 11 SSR markers around the locus were chosen to monitor the compositions and genetic diversity of the chromosomal segments. The SSR polymorphism of 75 representatives were compared between two SSR sets, namely, 12 SSRs independent from Wx, and 11 SSRs flanked by Wx locus. The average heterozygosity (He) of the Wx independent SSRs was 0.654. PCA was performed using SSRs and INDEL markers. Two distinct groups corresponding to indica or japonica were recognized. However, the Rec-type accessions were involved in both groups (Fig. 5A). This trend of distribution is similar to that of 295 accessions shown in Fig. 4. From this result, it was confirmed that 75 accessions acted properly as representative. On the other hand, the average He over Wx flanked SSRs was 0.475 (Table 2). The RM588 showed lower He than its average in three groups, glutinous japonica, non-glutinous japonica and glutinous indica. But the other four SSRs (RM19291, RM597, RM586 and RM 19319) in upstream of Wx gene did not show effect of selective sweep because He was high in all groups (Table 2). PCA analysis revealed that glutinous indica accessions were scattered between the non-glutinous indica and japonica groups (Fig. 5B). Recombinant accessions were also scattered randomly. Number of heterozygotes obtained with Wx flanked SSRs indicated frequent heterozygote in Laos landraces (Table 3). The number of heterozygotes were eight out of 18 (44% of population size) in glutinous japonica, two out of 17 (12%) in non-glutinous japonica, 12 (86%) in glutinous indica, seven (50%) in non-glutinous indica, respectively. All of these value are higher than ever observed (Ishikawa et al. 2002, 2006), but especially glutinous indica showed quite a high value. The existence of heterozygous indicated that outcrossing recently occurred, because it should be replaced into homozygous during the selection. Observed heterozygosity (Ho) were calculated in two set of SSRs, Random-SSRs and Wx-flanked SSRs (Supplemental Table 3). Glutinous indica showed heterozygotes at nine SSR sites out of total 23, whereas glutinous japonica showed only three.

Fig. 5

Principal coordinates analysis by using SSR polymorphisms among 75 representative accessions. Horizontal and vertical axes show first and second principal components respectively. A: Result based on 12 Wx independent SSR markers. B: Result of 11 Wx flanking SSR markers. All accessions were distinguished additional diagnostic nuclear INDELs in order to distinguish accessions into indica or japonica. Recombinant type (Rec-type) carried combinations of the diagnostic INDELs.

Table 2 Average heterozygosity (He) at 11 Wx flanked SSRs
n RM19291 RM597 RM586 RM19319 RM588 RM190 RM19344 RM19350 RM6263 RM4923 RM587 Total
J/wx 18 0.691 0.424 0.796 0.695 0.000 0.685 0.623 0.364 0.346 0.391 0.648 0.515
J/Wx 17 0.791 0.570 0.828 0.742 0.117 0.422 0.758 0.305 0.000 0.648 0.555 0.521
I/wx 14 0.698 0.379 0.379 0.379 0.000 0.568 0.594 0.595 0.701 0.473 0.485 0.477
I/Wx 14 0.556 0.473 0.379 0.805 0.426 0.663 0.000 0.074 0.524 0.355 0.355 0.419
Rec/wx 8 0.694 0.541 0.653 0.667 0.337 0.245 0.571 0.653 0.663 0.245 0.735 0.546
Rec/Wx 4 0.444 0.444 0.444 0.444 0.000 0.444 0.000 0.667 0.278 0.444 0.444 0.369
Total 75 0.646* 0.472 0.580 0.622 0.147* 0.505 0.424 0.443 0.419 0.426 0.537 0.475
*  Significant difference (P = 0.05).

Table 3 Number of heterozygotes of random SSRs and Wx flanked SSRs among six groups of 75 represented accessions
Group n No. of heterozygotes
J/wx 18 8 (44%)
J/Wx 17 2 (12%)
I/wx 14 12 (86%)
I/Wx 14 7 (50%)
Rec/wx 8 7 (88%)
Rec/Wx 4 2 (50%)
Total 75 38

Molecular characterization of the Wx locus

All accessions were genotyped for a 23-bp duplication in exon 2 of the Wx gene and also for the G-T substitution at the exon1-intron1 junction of the gene. All glutinous accessions were found to carry the 23-bp duplication and G-T substitution at the splice junction. Ho was high in the glutinous indica group with Wx flanked SSRs (Table 3).

There were allelic variations at RM190 located inside exon 1. Eight alleles were found among 75 representatives. Glutinous japonica carried four alleles and glutinous indica shared two of them (Fig. 6). These multiple alleles were located quite close to the G-T substitution and the 23-bp duplication in the Wx locus, therefore, it may reflect Wx selection. Additionally, Japanese glutinous landraces shared a small proportion of these multiple alleles at RM190 (Supplemental Table 4). Glutinous landraces in Japan carried narrower ranges of multiple alleles at the locus. It was probably due to a bottle neck effect occurred when glutinous landraces was introduces into Japan.

Fig. 6

Haplotypes evaluated with SNPs of 12 DNA fragments Wx flanking region. N: not-determined.

In order to confirm how such glutinous landraces had arisen, DNA fragments flanking the Wx locus were sequenced. Nucleotide diversity (Pai) of silent SNPs was evaluated for 12 DNA fragments within 0.45 Mb of the Wx locus (Table 4). The indica group showed higher diversity (Pai = 0.028 for glutinous indica; 0.0039 for non-glutinous indica) than the japonica group (Pai = 0.002 for glutinous japonica; 0.008 for non-glutinous japonica) in terms of the Pai score throughout the regions. In KN5-10 and KN5-8, diversities were low in all groups irrespective of whether they were glutinous or non-glutinous suggesting that selection of this region was not for the glutinous phenotype. The Pai score for glutinous indica ranged from 0.0000 to 0.0005, and that of glutinous japonica was also low. The lower diversity of glutinous indica was not due to the small number of indica accessions examined because the Pai score for non-glutinous indica was higher. Comparative heterozygosity of Wx flanked SSRs also showed lower diversity between RM588 and RM19350. Relative to the 5′ region, higher diversity of Pai scores was found from KN3-4 to downstream in the 3′ region. Glutinous indica showed quite high diversity at KN3-4, KN3-6 and KN3-8. The major SNPs at each fragment were used to summarize the haplotypes (Fig. 6). The haplotypes were described according to their frequencies at each site, for example, most major haplotype was A and minor one was D. Haplotypes of KN3-2 were described with numbers, since there was a different tendency with other regions. Glutinous japonica shared single haplotypes except for RM190 and KN3-2 where multiple alleles were detected in all the groups. In the 5′ region, from KN5-6 to KN5-0, alternative haplotypes existed. All glutinous accessions except for one shared the single Haplotype A. Another Haplotype B was shared with non-glutinous indica and also Rec-groups. In contrast to the 5′ region, there were multiple haplotypes in the 3′ region. Glutinous japonica shared the single Haplotype A. Other groups showed a complex haplotype pattern in the 3′ region. Glutinous indica shared Haplotype A along KN3-4 to KN3-12 in 10 out of 14 accessions, but three of the remainder carried different haplotypes (B-B-B-B/C-B) which were typical of non-glutinous indica. Only one accession carried C-B-B-C-A. This haplotype could not be found in the other groups, but Haplotype C of KN3-4 appeared to be derived from Haplotype B, based on the compositions of the SNPs inside the haplotype. From these results, Haplotypes A and B were considered to be typical of either japonica or indica, respectively. Haplotypes C and D were considered to have resulted from recombination between indica and japonica (Supplemental Fig. 1). These complex recombination patterns in the 3′ region indicated that outcrossing had occurred frequently in these regions. Glutinous indica shared some haplotypes with glutinous japonica, but also with a proportion of non-glutinous indica. Lower diversity prevented any estimation of selective sweep in the 5′ region of the Wx locus. Higher polymorphism was maintained in the 3′ region in non-glutinous indica. Relatively frequent recombinations occurred at 3′ region when compared to its 5′ region. Higher number of haplotypes in glutinous indica revealed that multiple events involving to recombinations by outcrossing and under a process of self-fertilization happened after glutinous allele had been introduced into indica genetic background.

Table 4 Nucleotide diversity (Pai) of silent sites at 12 fragments Wx flanking region
Group site KN5-10
103 bp
KN5-8
300 bp
KN5-6* KN5-4
633 bp
KN5-2
370 bp
KN5-0
449 bp
KN3-2
152 bp
KN3-4
262 bp
KN3-6
308 bp
KN3-8
235 bp
KN3-10
471 bp
KN3-12
569 bp
Total
J/wx Pai 0.0000 0.0000 0.0004 0.0000 0.0000 0.0019 0.0000 0.0000 0.0000 0.0000 0.0004 0.0002
SD 0.0000 0.0000 0.0002 0.0000 0.0000 0.0008 0.0000 0.0000 0.0000 0.0000 0.0002 0.0001
J/Wx Pai 0.0000 0.0004 0.0005 0.0000 0.0000 0.0017 0.0000 0.0000 0.0103 0.0003 0.0000 0.0008
SD 0.0000 0.0003 0.0002 0.0000 0.0000 0.0010 0.0000 0.0000 0.0017 0.0002 0.0000 0.0001
I/wx Pai 0.0000 0.0000 0.0005 0.0000 0.0000 0.0000 0.0087 0.0045 0.0094 0.0025 0.0014 0.0028
SD 0.0000 0.0000 0.0004 0.0000 0.0000 0.0000 0.0024 0.0011 0.0024 0.0008 0.0005 0.0005
I/Wx Pai 0.0000 0.0000 0.0057 0.0018 0.0066 0.0000 0.0027 0.0016 0.0043 0.0029 0.0019 0.0039
SD 0.0000 0.0000 0.0008 0.0000 0.0006 0.0000 0.0023 0.0013 0.0027 0.0006 0.0002 0.0004
Total 0.0000 0.0001 0.0031 0.0007 0.0025 0.0010 0.0080 0.0038 0.0110 0.0027 0.0011 0.0030
*  No silent site in KN5-6 fragment.

KN3-2 showed multiple haplotypes, but two haplotypes, Haplotype 2 and Haplotype 3, predominated as was the case for multiple alleles in RM190 in glutinous indica. In glutinous japonica, although Wx flanking region were highly conserved, there are multiple alleles, (CT)12, (CT)16, (CT)17, and (CT)18 at RM190 which is located in exon 1 of Wx gene. On the other hand, glutinous indica also carried multiple alleles, (CT)17 and (CT)18, at RM190. To elucidate whether these two alleles of glutinous indica occurred after wx causal allele introduced into indica accessions, or introduced independently from glutinous japonica, combination patterns of alleles of RM190 and haplotype of KN3-2 fragment were compared between glutinous indica and glutinous japonica accession (Supplemental Table 5). Among glutinous japonica, accessions with (CT)17 carried Haplotype 1, 2, 3, 4, and 5 at KN3-2 fragment, and accessions with (CT)18 carried Haplotype 1, 3, and 5, respectively. All of haplotypes carried by (CT)18 were contained haplotypes carried by (CT)17. The (CT)17 is the dominant among multiple allele of RM190 locus. From these facts, (CT)18 allele was considered to be derived from (CT)17 allele in glutinous japonica population. On the other hand in glutinous indica, accessions with (CT)17 carried Haplotype 2 and 3, and accessions with (CT)18 carried Haplotype 2, 3, and 6, respectively. It is more likely that glutinous indica with (CT)18 was derived from (CT)17 of glutinous indica rather than (CT)18 of glutinous japonica.

Discussion

Introduction of wx causal allele into indica glutinous rice

According to previous studies, glutinous rice has a single origin derived from single causal mutation, 23-bp duplication in exon 2 of Wx gene (Wanchana et al. 2003), and it was occurred in japonica which have a G to T substitution at the splice junction if exon-intron 1 (Yamanaka et al. 2004). However, there is little reference focused on origin of indica glutinous rice. In this report, it was found that there was a minor glutinous landraces belonging to the indica type rice, although japonica was predominated. So we tried to discuss about the origin of indica wx allele in rice landraces in northern Laos where is known as the center of glutinous rice zone (Watabe 1967).

All glutinous accession in Laos carried both a 23-bp duplication inside exon 2 and G-T substitution at the splice junction in Wx gene. Thus, the glutinous origin was considered as a single. In glutinous japonica, although sequence compositions in Wx flanking region was highly conserved, there were four alleles at RM190 which is located in exon 1 of Wx gene. These alleles derived from a single wx allele. Glutinous japonica populations in Japan also carried the multiple alleles of RM190 but less than Laos. It implied that Laos has a long history as a center for japonica glutinous rice (Watabe 1967). On the other hand, glutinous indica also carried multiple alleles, (CT)17 and (CT)18, at RM190. Allelic frequency suggested that haplotype carrying (CT)17 repeat would be the origin (Supplemental Table 5). Haplotype data suggested that glutinous indica shared same haplotypes with glutinous japonica but not with non-glutinous indica. From these results, it was newly suggested that the wx causal mutation was occurred in japonica Wx gene which carried (CT)17 at RM190, then this wx allele was introduced into indica landraces through outcrossing.

Further recombination of Wx franking region of glutinous indica

The previous data indicated the selective sweep window spanning about ~250-kb of Wx flanking region. It corresponded with the region spanned from KN5-10 to KN3-2. The comparatively lower diversity around Wx locus in particular varietal groups suggested that there might be selection during cultivation. In our materials, two fragments, KN5-8 and KN5-10 in 5′ region of Wx gene showed quite low diversity through all groups (Table 4, Fig. 6). It was probably due to restricted materials collected inside Laos. Successive outcrossing among local landraces could be introduced much diversity not only flanking regions of Wx locus but also all genome regions. It was also proved by control sequence used by Olsen et al. (2006). The heterozygosity (He) of an SSR, RM588 which flanked the 3′ region of KN5-8 showed low diversity, whereas other SSRs such as RM19319 near KN5-10 showed diversity to some extent (Supplemental Table 6). It was considered that there could be any other reason to keep low genetic diversity not against for glutinous phenotype for KN5-8 to KN5-10. Therefore, this region should be excluded from sweep window for glutinous phenotype.

There was a gap between the preserved size of Wx flanking region of glutinous indica and glutinous japonica in landraces in Laos. In glutinous japonica, all fragments appeared quite low genetic diversity and single haplotype. The preserved region of glutinous japonica was estimated spanning about 404~-kb, from the 3′ terminal of KN5-8 (genomic position 1606959) to the 3′ terminal of KN3-12 (genomic position 2011364). It was wider than previously considered. The He of glutinous japonica was low in RM6263 and RM4923, which located downstream of KN3-12 (Supplemental Table 6). The sweep window of glutinous japonica possibly contained these regions. In addition to the existence of multiple alleles of RM190, it implies that glutinous japonica has been conserved quite long time in this area. On the other hand, many recombinant events were found at Wx flanking region in glutinous indica. The recombinant points were quite complicated in 5′ flanking region of Wx gene. Recombinant was also found in KN5-4 and KN5-6 where were regarded as inside of the selective sweep window in previous study. On the other hand, RM588 close to KN5-8 showed the lowest score in glutinous indica, but recover situation at RM19319. Therefore, sweep window of glutinous indica considered to be narrower than glutinous japonica but the still ~300-kb wide from RM19319 (genomic position 1550199) to KN3-4 (genomic position 1850705). The fragments derived from japonica genetic background was preserved to some extent among glutinous indica populations. According to Watabe (1967), glutinous japonica was cultivated primary in Laos and Thailand, and then non-glutinous indica expanded with replacing glutinous japonica gradually from Thailand after 10th century. A residual glutinous rise zone was formed after 18th century. It is considered that the glutinous indica occurred by outcrossing during this process, then expanded and stabilized with further recombination. Recombinants would result from high outcrossing rate in farms and on-going selection. In general, local farmers selected only from appearance of panicles. Thus, they allowed such recombinants to escape selection.

Genetic diversity conserved through the slash and burn farming system in northern Laos

In order to classify indica and japonica, we adopted a set of indicators including cytoplasmic (ORF100) and three nuclear markers (Acp1-indel Cat1-indel and Pgi1-indel). Generally, these four indicators show same genotype in a homozygote plant of indica or japonica. However, among rice in Laos, recombinant-type accessions were frequently found in glutinous indica diagnosed by deletion type ORF100. PCA clearly showed that the group contained scattered indica and japonica. Additional recombinant accessions were also found when Wx flanked SSRs were genotyped, although general indicators identified these accessions as indica. The introduction of wx allele and erosion of sweep window in glutinous indica could be also brought by frequent outcrossing events in slash and burn system.

All of these results indicated that frequent outcrossing and introgression had occurred frequently not only inside the population but also across the various populations. In general, outcrossing between different varietal types such as indica and japonica, or between cultivars and wild rice, has resulted in weedy types with a high rate of sterility and a tendency for seed shattering (Suh et al. 1997, Tang and Morishima 1996). However, no such weedy types with marked seed shattering or sterility were recognized in our field observations in northern Laos. Outcrossing and successive self-crossing may stabilize sterility in the progeny but still maintain diversity through introductions from other varietal groups. Such selection possesses may have resulted in plants with non-shattering seeds. The slash and burn farming system in Laos may have allowed such heterogeneity to exist in populations. From our field survey, with regard to rice seed propagation, local farmers tend to stock and re-use their harvested seeds for the following year, and scrupulous seed selection is performed once in a few years. Local farmers usually grow two or three landraces per a household. They tend to reserve various landraces. In some cases, there are more than 20 landraces in a village. They frequently exchange rice seed between neighbors, relatives and others beyond the ethnic or geographical communities. Thus, once gene flow occurs, heterogeneous gene pools would be inherited from generation to generation, and successive generations would have higher fertility and more stable traits. This system has allowed mixing of multiple crop species, various vegetables, and different rice landraces within a single field. Variation in genetic background may play an important role in the maintenance of stable cultivation in Laos to confer biotic and abiotic stress during upland cultivation. The heterozygous populations present in Laos may work to keep rice populations free of severe pandemic disease or insect damage. On the other hand, the preference of local farmers for glutinous rice may result in severe selection against the Wx allele. Only a single causal mutation at the Wx locus has been diversified and distributed intricately. This genetic structure of landraces may give us a chance to consider how such diversity can be maintained on farms or how efficiently genetic resources can collected from such farms before they are replaced by modern varieties introduced as cash crops.

Acknowledgements

This study was done as a research project of “A Trans-Disciplinary Study on the Regional Eco-History in Tropical Monsoon Asia: 1945–2005”. This study was also supported by the research project “Agriculture and Environment Interactions in Eurasia Past, Present and Future” of the Research Institute of Humanity and Nature.

Literature Cited
 
© 2016 by JAPANESE SOCIETY OF BREEDING
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