# Allele Mining of Seed-Related Genes Reveals Early movement, Selection and Adaptation of Asian Rice Landraces

Cheng-Chieh Wu, Yi-Tzu Tseng, Yuan-Ching Tsai, Chun-Kai Liu, Lin-Tzu Huang, Tze-Fu Hsu, Yi-Fang Chen, Nai-Chiang Dai, Ying-Hao Wang, Jeng-Chung Lo, et al.

► To cite this version:

Cheng-Chieh Wu, Yi-Tzu Tseng, Yuan-Ching Tsai, Chun-Kai Liu, Lin-Tzu Huang, et al. Allele Mining of Seed-Related Genes Reveals Early movement, Selection and Adaptation of Asian Rice Landraces. Rice, 2025, 18 (1), pp.95. 10.1186/s12284-025-00854-9 . hal-05333030

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# Allele Mining of Seed-Related Genes Reveals Early movement, Selection and Adaptation of Asian Rice Landraces

Cheng-Chieh Wu<sup>1,19</sup>, Yi-Tzu Tseng<sup>1,2</sup>, Yuan-Ching Tsai<sup>3</sup>, Chun-Kai Liu<sup>1</sup>, Lin-Tzu Huang<sup>1</sup>, Tze-Fu Hsu<sup>1</sup>, Yi-Fang Chen<sup>4</sup>, Nai-Chiang Dai<sup>5</sup>, Ying-Hao Wang<sup>6</sup>, Jeng-Chung Lo<sup>7</sup>, Dah-Pyng Shung<sup>8</sup>, Chih-Wen Wu<sup>9</sup>, Chen-Hwa Tsang<sup>10</sup>, Kuang-Ti Li<sup>11</sup>, Whei-Lee Chu<sup>11</sup>, Ming-Hsin Lai<sup>5</sup>, Dong-Hong Wu<sup>5</sup>, Shu Chen<sup>12</sup>, Su-Jein Chang<sup>13</sup>, Kai-Yi Chen<sup>14</sup>, Wan-Yi Chiou<sup>15</sup>, Su-May Yu<sup>16</sup>, Tuan-Hua Ho<sup>1</sup>, Laurent Sagart<sup>17</sup>, Jun-Yi Yang<sup>18</sup> and Yue-le C. Hsing<sup>1,19*</sup>

**Abstract**

**Background** Asian cultivated rice is one of the most important crops in the world. According to archaeological studies, carbonated rice grains in Taiwan were quite small before 3300 BP, but rice seeds from excavated sites became much larger later on. In the current study, we explored seed size differences by using genome re-sequencing, followed by allele mining of several seed- and adaptation-related genes to propose the early movement, selection and adaptation of Asian rice landraces.

**Results** Taiwan indigenous people are descendants of early Austronesians. We collected 116 rice accessions from Taiwan indigenous villages and used whole-genome re-sequencing to explore the mutations, early movement, selection and adaptation of world rice landraces together with landraces collected from all rice-growing areas in Asia. The morphology of Taiwan indigenous rice accessions featured huge variations, and the most primitive accessions were tropical *japonica*. Also, some grain size-related genes could explain grain size differences. Allele analyses revealed that some mutations in grain morphology-controlling genes may have been targets for selection. Mutation, movements, selection and adaptation occurred in early Asian rice cultivation. Our findings do not conflict with the "out-of-Taiwan" theory.

**Conclusion** The grain sizes of all rice subgroups studied had been selection targets, and seed size gradually changed along with the improvement of rice cultivation, via introgression and expansion (i.e., by human beings), over thousands of years.

**Keywords** Adaptation, Carbonated rice grain, Genetic diversity, Population structure, Rice grain-related genes, Selection

Full list of author information is available at the end of the article

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Springer Open

© The Author(s) 2025. **Open Access** This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

<footnote>*Correspondence:
Yue-le C. Hsing
bohsing@gate.sinica.edu.tw</footnote>
## Introduction

Asian cultivated rice (Oryza sativa) is one of the most important crops in the world and the most widely consumed. Rice was genetically divided into 2 main subspecies, japonica and indica, by Kato (Kato et al. 1930) and according to many subsequent analyses of morphologic, physiologic and cytogenetic characteristics (reviewed by Matsuo and Hoshikawa 1993). Later on, with the analysis of molecular markers (Liakat Ali et al. 2011) and whole genome sequencing (3KRGP 2014), rice was classified into 5 major subpopulations: aus, indica, temperate japonica, tropical japonica, and aromatic. In 2018, a detailed analysis of the 3 K accessions further classified indica rice into ind1a, ind1b, ind2 and ind3 and japonica rice into tropical (trop1), subtropical (trop2) and temperate (Wang et al. 2018).

According to archaeological studies, 2 early domesticated (i.e., non-shattering) cultivated rice types in China were the japonica type, including rice grown about 8,500 years ago in Baligang in the Yellow River-Huai River-Plain (Deng et al. 2015) and about 9,000 years ago in the lower Yangtze river region (Fuller et al. 2009). Wild rice ancestral to indica, also known as proto-indica, was present in the northern Indian subcontinent before the arrival of domesticated japonica (Silva et al. 2018). The domestication genes from japonica were contributed to cultivated proto-indica rice and domesticated indica rice was present about 4,000 years ago in the Ganges plains of eastern India (Fuller et al. 2010; Choi et al. 2017). PCR amplification followed by sequencing of fragmented products using nuclear and plastic primers of hundreds of archaeological grains excavated from India (2 archaeological sites) and Thailand (4 sites) dated 2500 to 1500 before present (BP) revealed that the rice was predominantly japonica in Thailand and a mixture of japonica and indica, with indica in the minority, in India (Castillo et al. 2016). Thus, the movement of indica rice accessions to Southeast Asia (SEA) and East Asia was relatively late as compared with japonica rice cultivation.

Taiwan indigenous people are the original inhabitants of Taiwan. Their ancestors may have been living in Taiwan for thousands of years before Han Chinese immigration began in the 17th century. Taiwanese aborigines are Austronesian-speaking peoples and were demonstrated as the origin of Austronesian languages in the Philippines, Indonesia, Malaysia, Madagascar, Polynesia and Oceania (Hill et al. 2007).

Archaeological studies revealed many carbonized rice grains in the excavated sites in southern (Hsieh et al. 2011; Tsang and Li 2015), eastern (Wu et al. 2016; Deng et al. 2022), central (Chu 2016; Deng et al. 2022) and northern Taiwan (Huang 1984; Deng et al. 2022) from 4 to 5 thousand years ago. Thus, the archaeological record shows that rice cultivation has gone on since the 5th

millennium BP. The collection of rice accessions from the indigenous villages started from the early 20th century to recent times, including both japonica and indica rice (Wu et al. 2022). Han Chinese immigrated to Taiwan during the late Ming to early Ching Dynasty, and all rice lines brought with them were indica (Teng 1999).

The early movements of rice between Taiwan and nearby regions are an important topic and have been studied for decades. Dispersal of rice into insular SEA may have been suggested to the Austronesian expansion (Bellwood 1997; Diamond 2001; Bellwood 2006). However, many studies showed conflict with this simple "out-of-Taiwan" hypothesis for the spread of rice. For instance, in our recent study involving whole-genome re-sequencing of the regional dispersal of rice in Taiwan and SEA, japonica rice cultivated by Taiwan indigenous peoples consisted of 2 distinct populations: (1) temperate japonica from northeast Asia and (2) tropical japonica from northern Philippines and mainland SEA (Alam et al. 2021). However, only 24 indigenous lines were used in that study. Many of the indigenous rice lines showed typical tropical japonica morphology and some were temperate japonica. There was no definite conclusion about which type was the earlier one in Taiwan. Also, subsequent frequent trade activities might have influenced the diversity of rice accessions.

Taiwan is a small island and is relatively far away from nearby islands and the mainland. Even though many studies have found exchanges of crops, jade, language, etc. with adjacent regions (Bellwood 1997; Pawley 2002), further exchange and reception of new types of seeds were not as frequent as in large continents. The indigenous people in Taiwan had kept the seed resources faithfully for thousands of years and the Han farmers for hundreds of years. Thus, the detailed comparison of Taiwan rice landraces with those in nearby regions is important.

Many indigenous japonica rice seeds were larger than the early carbonated rice grains and the modern temperate japonica varieties. The major aim of the current study was to examine changes in grain morphology, investigating in detail the allele types of several grain size-controlling genes. Rice grain size is a quantitative trait and is controlled by multiple genes (for recent review, see Li et al. 2018; Jiang et al. 2022). The grain size is a 3-D structure, including seed length, width and thickness, so it has an appearance quality as a target during harvesting/ selection. Mutations in the open reading frame (ORF) or promoter sequence changes of genes could lead to changes in grain size. They are transcriptional regulatory factor LGY3 (rice grain yield quantitative trait locus on chromosome 3, Liu et al. 2018), grain weight in chromosome 7 (GW7, Wang et al. 2015), grain length and weight on chromosome 7 (GLW7, Si et al. 2016). grain weight in
chromosome 8 (GW8, Wang et al. 2012), a protein GS3 (grain shape gene on chromosome 3, Fan et al. 2006; Mao et al. 2010), a phytohormone signaling protein qTGW3 (quantitative trait controlling grain size and weight on chromosome 3, Hu et al. 2018), seed width controlling protein qSW5 (quantitative trait controlling seed width on chromosome 5, Shomura et al. 2008; Duan et al. 2017), as well as hormone signaling proteins grain size in chromosome 5 (GS5, Li et al. 2011), TGW6 (thousand grain weight on chromosome 6, Ishimaru et al. 2013) and GW6 (grain weight on chromosome 6, Shi et al. 2020).

Thus, we performed whole-genome re-sequencing and seed morphology analysis of many Taiwan landraces. Additionally, we retrieved genome sequence data and seed size information for landraces from rice-growing regions through the 3 K Rice Genome Project (3KRGP 2014). We established a panel of cultivated rice with >85% landraces and also performed allele mining of several seed-related genes. We aimed to provide data on seed morphology changes and allele variations to give information on the early movement, selection and adaptation of Asian rice landraces.

# Materials and Methods

## Archaeological Materials

Hundreds of carbonized rice grains were collected from 4 excavated sites in Taiwan belonging to 4 cultures by using the flotation method. The sites are Nan-kuan-li East (NKLE) (23°6′58″N, 120°16′35″E, altitude 0.5 m, 4800 BP) of the Dapenkeng culture (4800–3300 BP), Youhsienfang (YHF, 23°06′54.0″N 120°16′25.0″E, altitude 6 m) of the Niuchouzi culture (3800–3300 BP), Wuchiansuo (WCT, 23°05′39.0″N 120°16′25.0″E, altitude 7 m) of the Niaosong culture (1400–500 BP), and Huilaili (HLL, 24°09′42.8″N, 120°38′11.6″E, altitude 73 m, 1300 BP) of the Fanziyuen culture (2000-400BP). In total, 100 carbonized rice samples from each site were used for measuring seed width, length and thickness with a digital ruler (Mitutoyo Co., Japan) with 0.01-mm accuracy. The information, including Chinese translation, of the excavated sites and cultures are in Table S1.

## Sequencing Data

We performed whole-genome re-sequencing of 260 rice accessions collected in Taiwan, including (1) 116 Taiwan indigenous rice accessions. These landraces were collected from the indigenous villages of the mountain regions of Taiwan; (2) 59 landraces that were brought to Taiwan by Hang people during the late Ming to early Ching Dynasty, designated “Ming-Ching”; (3) 72 modern varieties; and (4) 11 weedy rice accessions found in the paddy field. (5) Wild rice (O. rufipogon) accessions were found in northern Taiwan (Oka 1991) and we chose 2 for analyses. All seeds were available in the National Plant

Genetic Resource Center (NPGRC) of the Taiwan Agricultural Research Institute (TARI). The list of all Taiwan rice accessions is shown in Table S2, and sorted by their types. The plants of single seed descent were cultivated until tillering stage in an Academia Sinica greenhouse under natural light. Healthy leaves without insect damage from one single plant were harvested, frozen under liquid nitrogen and stored at -80°C for DNA extraction. The high-quality genomic DNA and the followed-up whole-genome re-sequencing information were prepared as described (Wu et al. 2020a). Sequencing data for these accessions are available from the NCBI Short Read Archive (SRA) and the Bioproject accessions numbers are listed in Table S3.

Several sets of whole-genome re-sequencing data were downloaded from the public domain: the 3 K-RG project (3KRGP 2014; Wang et al. 2018); Asian wild rice (Stein et al. 2018; Zhao et al. 2018b); sequencing data for some modern varieties such as Koshihikari, 9311, IR24 and IR64; and Chinese weedy rice accessions (Qiu et al. 2017). The accession names and seed storage information for all seeds used as well as sequencing data for all these accessions are available from the NCBI Short Read Archive (SRA) under the Bioproject accession numbers listed in Table S3.

## Mining of Loss-of-Function (LOF) Alleles for Seed Trait-Related Genes

Adaptor sequences, low-quality bases and reads <20 bp long were discarded. The trimmed paired reads were then aligned to the reference rice Nipponbare genome sequence (IRGSP v1.0). SAMtools and VCFtools (Danecek et al. 2011) were used to manipulate and transform the Sequence Alignment Map (SAM) and variant call format (VCF) file format (Danecek et al. 2011). We performed allele mining of 10 grain-size related genes, including LGY3 (Liu et al. 2018), GS3 (Fan et al. 2006), qTGW3 (Hu et al. 2018), qSW5 (Shomura et al. 2008), GS5 (Li et al. 2011), TGW6 (Ishimaru et al. 2013), GW6 (Song et al. 2015), GW7 (Wang et al. 2015), GLW7 (Si et al. 2016), and GW8 (Wang et al. 2012). The functional impact of nucleotide variants in the whole genome re-sequencing data was analyzed by using SnpEff (Cingolani et al. 2012). From the genome annotation, sequence variants (single nucleotide polymorphisms [SNPs] and small indels) were classified according to their location (ORF, intron, splice sites, etc.) and predicted functional impact (missense, frame shift, early stop, etc.). We also analyzed genes for other seed-related traits such as red caryopsis (Rc, Sweeney et al. 2006); waxy (wx, Wanchana et al. 2003); 3 non-shattering genes qSH1 (Konishi et al. 2006), qSH3 (Ishikawa et al. 2022) and sh4 (Li et al. 2006); and 2 adaptation-related genes Cold1 (Ma et al. 2015) and
*Heading date* 1 (Hd1Yano et al. 2000; Takahashi and Shimamoto 2011; Wu et al. 2020a).

To detect the fragment (> 35 bp) insertion or deletion, we used the Nipponbare genome sequence or the semi-assembled genome of *indica* rice R498 (Du et al. 2017; for the *qSW5* gene) for alignment. The VCF files for each accession at nearby region were compared by using "vcf-isec" to classify sample-specific or intersection variants. The VCF files were then imported into Integrative Genomics Viewer (IGV) (Robinson et al. 2011; Thorvaldsdóttir et al. 2013) to show alignments.

## Bioinformatic Analyses

Alignment sites shared in all accessions were identified first, and SNPs in the shared alignment sites were used for phylogenetic analyses. To remove low-frequency and low-quality SNPs, we filtered out VCF files with any missing genotypes, mean depth values < 5, and minor allele frequency (MAF) < 5% using VCFtools (Danecek et al. 2011). The SNPs were used to evaluate the genetic distances between different samples. A neighbor-joining tree was constructed on a distance matrix, calculated by the Neighbor-Joining/UPGMA method with Neighbor 3.696 (Felsenstein 2004) and presented with MEGA 5.2 (Saitou and Nei 1987; Kumar et al. 2016) and FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Population structure analysis involved using ADMIXTURE, with the model-based maximum likelihood approach executed with the program (Alexander et al. 2009) and PCA. ADMIXTURE is a clustering software similar to STRUCTURE that infers populations and individual ancestries. SNPs in VCF formats were converted to a plink format by using PLINK (Chang et al. 2015). Linked sites, monomorphic or multiallelic sites, sites with MAF < 5% and Phred quality < 30 were removed by using PLINK. We tested clustering models in the population with presumed cluster number K from 2 to 15. In addition, PLINK was used for PCA on the same genotype likelihood dataset. R 4.03 was used for structure analysis and PCA plots (R Core Team 2013).

For detecting the selection of target gene (GS3), the 280 accessions with type 2 mutation were used. DNA sequences were aligned by using MUSCLE (Edgar 2004a, b). The -1- to +1-kb region of the genes corresponding to the Nipponbare genome was used for Bayesian inference implemented in BEAST2 (v2.7.7) (Bouckaert et al. 2014) to reconstruct the early relationships. The parameters for BEAST running included: site model, HKY (Hasegawa Kishino Yano); molecular clock models, strict clock or fixed local clock; tree priors, as default.

To check the insertions in the tandem genes of flavone glucosyltransferase, IGV (Thorvaldsdóttir et al. 2013) was used to visualize the 30-Mb region of chromosome 1.

To identify the signatures of selective sweeps in the chromosome 1 long arm region, we used Raised Accuracy in Sweep Detection (RAiSD v2.9). Mu (μ) was computed by integrating multiple genomic signals, including the site frequency spectrum (SFS) and linkage disequilibrium (LD) patterns (Alachiotis and Pavlidis 2018). Analysis parameters included a window size of 50 SNPs and evaluation of the μ statistic across 10,000 equidistant grid points per chromosome. Results were visualized as Manhattan plots with custom R scripts.

We used the ± 5-kb region of each gene, including the promoter, ORF, intron, and untranslated region (UTR) for all 17 genes of the 14 subgroups for analyses of genetic distance and diversity. Nucleotide diversity (π) (Cingolani et al. 2012), θw (Watterson 1975), and Tajima's D (Tajima 1989) were calculated by using DNASP v5.0 (Rozas et al. 2017). Insertion, deletion, and unalignable regions were excluded from the analysis.

## Protein Structure Prediction

The protein structures of Os01g0734800 and Os01g0734600 were predicted by using Boltz-1 (Wohlwend et al. 2024), an AlphaFold3 (Abramson et al. 2024)-based tool available on the Neurosnap website (https://neurosnap.ai/). The predicted protein structures were then analyzed for their binding interactions with the flavonoid compound kaempferol.

## Statistical Analysis and Scoring of Grain-Related Traits

To characterize and compare the grain morphologic traits across excavated sites and 12 subpopulations, we implemented a comprehensive statistical analysis framework. Statistical analyses involved using R 4.4.2 (R Core Team 2024) and the analytical approach comprised the following methodology: (1) raw data were processed and grouped by using the tidyverse ecosystem (Wickham et al. 2019); (2) one-way analysis of variance (ANOVA) was conducted individually for each trait; (3) post-hoc analysis involved using Tukey's Honest Significant Difference (HSD) test to identify significant pairwise differences between subpopulations (α = 0.05); (4) for each excavated site or subpopulation and trait combination, we calculated comprehensive summary statistics including mean values, quantiles (25%, 50%, 75%), maximum values, and sample sizes (n); and (5) distribution patterns were visualized with violin plots created with ggplot2 (Wickham 2011).

Several grain-related traits of the Taiwan landraces were screened and recorded. Long awn: awn > 3 cm. Red caryopsis: palea and lemma were removed, and the caryopsis color was recorded. Glutinous phenotype: 0.2 ml of 0.2% iodine reagent was added to the endosperm powder from 3 polished seeds. The color change (if any) was recorded 5 min after. Seed width and seed length: a
digital ruler (Mitutoyo Co., Japan) was used to measure.
Twenty seeds each samples were scored.

## Results

### Very Early Rice Grown in Taiwan Had Small Grains

Figure 1A and C shows details of carbonated rice seeds
from the excavated sites in Taiwan. Using 100 carbon-
ized seeds from 4 excavated sites, we studied seed size
changes by measuring the seed length, width and thick-
ness (Fig. 1D, Table S4). ANOVA results showed signifi-
cant differences in size parameters from the 4 excavated
sites (Table S5). Seed length before 3,300 BP was < 4 mm,
much smaller than the seeds collected after, with an aver-
age of 5.3 and 6.2 mm from HLL and WCT (Table S6).
Some of these seeds were twice the size of seeds from
the 2 earlier sites, shown in Fig. 1D. Early rice seed size

in Taiwan has changed over time, from small and round
shaped to large with an oblong shape.

### Specific Rice Landraces from Taiwan

Many rice heritage landraces had been cultivated by
indigenous people living in the hill and mountainous
regions, with an upland practice, from about 4500 years
ago (Hsieh et al. 2011). A total of 60 upland rice acces-
sions were collected from indigenous villages from 1895
to the early 1910s. These accessions were since propa-
gated (renewed) every 5 years by rice breeders, and seeds
were stored in TARI (Teng 1999). There were only names
for this seed resource, without other information such as
collection year, villages and tribes. In cooperation with
linguists, the possible tribe information for 10 accessions
were proposed and these are listed in Table S7. Some

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Carbonated rice seeds excavated in Taiwan. **A** A group of carbonated rice grain, bar = 5 mm. **B** Scanning electron microscope (SEM) image of a complete carbonated rice grain, bar = 2 mm. **C** Zoom-in SEM image of the tip of a carbonated grain showing the base of an awn, bar = 500 µm. **D** Distribution of seed length, width and thickness (in mm) of 100 seeds from each site. Green: Nankuanli East (NKLE), ~4800 BP at Tainan; purple: Youhsienfang (YHF), ~3800 BP at Tainan; cyan: Wuchiantsuo (WCT), ~1400–500 BP at Tainan; orange: Huilaili (HLL), ~1300 BP at Taichung</figcaption></figure>
rice breeders continued to visit mountain regions and collected more accessions in recent years. They added another 56 lines, with information on tribes collected (Table S7).

There are 16 indigenous tribes in Taiwan. The rice accessions collected in these villages are highly diverse in seed morphology, plant height, plant stature, and heading behavior. For instance, 16 accessions were collected from a “CD” village of the Tsou tribe. Figure S1 illustrates the plant architecture during heading, the mature seeds and caryopsis of 4 accessions, CD1, CD5, CD7 and CD10. These plants were transplanted into the open field on the Academia Sinica campus at the same time, but the head- ing period of these 4 accessions varied from early head- ing to seed mature (i.e., panicle bent down) (Fig. S1A to D). The grain shape, grain color and length of awns (Fig. S1E to H) as well as the caryopsis color (Fig. S1I to L) also show variations. Supplementary Table 8 illustrates the subspecies (japonica or indica), awn length, awn color, grain color, caryopsis color, glutinous type, and grain length, width, length/width ratio and area index (length x width) for each of the 10 lines from the CD village. The data indicate that grain-related traits such as the pres- ence and length of awns, grain type, color, size and cary- opsis color, glutinous trait, etc. show large variations. Similar variations are present from lines collected within or between tribes.

We received 16 accessions that were stored under ambient temperature for decades from this CD village, and only 10 were rescued (germinated) successfully. Sim- ilar problems existed in the seeds collected from many other villages. Together, it was estimated that we lose (no germination) 30–40 accessions.

The Han people migrated from the southeast coastal area in China, mainly Fujian and Guangdong, to Taiwan during the late Ming to early Ching Dynasty. Accord- ing to the literature, there were 1,679 Ming-Ching rice accessions during the survey in 1906, and the breeders reduced these to 547 lines after screening and elimina- tion according to similarity in morphology (Iso 1944). These resources are also stored in NPGRC. They were representatives of the early landraces existing about 400 years ago in southern China. Weedy rice, also known as red rice, has been a problem in rice production in Tai- wan in recent years. All these weedy rice lines are indica but grew in/contaminated the japonica field (Wu et al. 2020b; Huang et al. 2021). In addition, 2 wild rice acces- sions existed in northern Taiwan were used for the cur- rent study.

We then compared the grain shape of heritage land- races and modern varieties. Figure 2 illustrates seeds of 6 indigenous japonica lines, 5 japonica modern variet- ies, 3 indigenous indica lines, 5 Ming-Ching indica lines and 5 modern indica lines. Of note, (1) 2 types of Taiwan

indigenous japonica seeds were present (e.g., the 4 on the left are larger than the 2 on the right) and the latter 2 show similar seed size as modern temperate japonica varieties and (2) the Ming-Ching indica rice grains were smaller, both in length and length/width ratio, than the modern indica seeds. Thus, several questions were raised: (1) Did qSW5, a seed width-controlling gene, have other LOF mutation(s) in addition to the 1.2-kb deletion?; (2) Which type(s) of the Taiwan indigenous japonica rice were, and what were the reason(s) for their larger size as compared with the early carbonated seeds?; and (3) What were the reasons for the short seed length of the Ming- Ching rice accessions?

To solve these questions, in addition to the Taiwan landraces, we included 855 rice 3 K data marked as “T” and the weedy rice (also known as red rice, Huang et al. 2021) sequencing data from Taiwan and China in the analysis panel. Data for 78 modern varieties and 20 wild rice accessions were also added. Together, < 15% of the lines used had been improved by modern breeding pro- grams. The information on the accessions’ ID, names, collected regions, seed storage centers, DNA sequenc- ing accessions, etc. is available in Table S3. The GPS information for the collection sites for most accessions is also available in the same table. That information was obtained from Gutaker et al. (Gutaker et al. 2020), and we added the data for accessions from Taiwan.

The grain size information for about 3 K rice acces- sions is from the IRRI SNP-Seek Database (Mansueto et al. 2016). We measured the grain size information for the accessions from Taiwan. Thus, we had grain size informa- tion for 1196 of the 1365 accessions, listed in Table S9. With the morphologic information, we also performed allele mining of 10 seed size-/shape-related genes fol- lowed by in-depth comparisons. We also checked another 5 grain morphology-related genes and 2 adapta- tion-related genes.

## Genome Sequence Analysis Results

The average sequencing depth of the panel was about 16 X, with 0.7 million SNPs per accession. Structure (also known as ADMIXTURE) analysis at K=2 revealed 2 major groups, corresponding to the 2 subspecies japonica and indica, and the charts for K=2 to K=15 are shown in Fig. S2. Figure S3 illustrates the cross-validation (CV) error versus K in the admixture analysis and we chose K=14 because of its low CV error. Figure 3A shows the K=14 structure analysis and Fig. 3B the phylogenetic tree analysis. Indica rice were grouped into 6 categories: V8 are accessions mainly from the Indian subcontinent; V12 are accessions mainly from the Indochina Peninsula; V11 are accessions mainly from insular SEA; V6 are indica rice, with more than half from Taiwan Ming-Ching and indigenous villages, the remaining being Chinese weedy
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Grain shapes of several landraces and modern rice varieties. Six Taiwan indigenous *japonica* lines, 5 modern *japonica* varieties, 3 Taiwan indigenous *indica* lines, 5 Ming-Ching *indica* lines and 5 modern *indica* varieties. Scale bar: 10 mm. Several accession names are shown in abbreviation: TC194, Tai-chung 194; TNG67, Tainung 67; TNG71, Tainung 71; TC65, Taichung 65; KH 145, Kaohsiung 145; SML, SzuMingLuTao; DGWG, Dee Geo Woo Gen; TNGS20 Tainung Sen 20; TNGS22 Tainung Sen 22; TCS10, Taichung Sen 10; TN17, Taichung Sen 17; Tainung Sen Naw 2 TNGSW2</figcaption></figure>

rice; V13 are accessions also with about half from Tai-
wan Ming-Ching and a few from indigenous villages,
and another half were Chinese landraces and weedy rice;
and V9 are aus. *Japonica* rice were also grouped into 6
categories: V2 are temperate *japonica* including modern
varieties and some Taiwan indigenous accessions; V1
are *japonica* accessions all collected from Taiwan indig-
enous villages; V4 are trop2 *japonica* rice mainly from
the Indochina Peninsula. Trop1 *japonica* were grouped
into 3 types: V5 are collected from Japan, Korea and
Taiwan indigenous villages, that is, from the northern
region; both V7 and V10 are trop1 *japonica* from insular
SEA; 89% of the V7 were collected from western Indo-
nesia, including Sumatra and Java; and V10 are lines col-
lected from all tropical insular regions, including eastern
Indonesia. Most wild rice (V3) or weedy rice (V14) were
grouped separately. The brief and detailed description of
these 14 subgroups are shown in Table 1 and Table S10.
The color code for each group was kept the same in all
figures, as shown in Table 1. The detailed subgroup infor-
mation for all 1365 accessions is in Table S3.

The phylogenetic analyses revealed clear differentiation
of the panel into 2 major groups – *indica* and *japonica*

(Fig. 3B, with each subgroup indicated). Trop1 *japonica*
was divided into 4 clades: V7 (violet), V10 (peru), V5
(cornflower blue) and V1 (light salmon). V4 (sky blue)
and V2 (medium aquamarine) were in another clade.
A total of 12 wild rice accessions were clustered with
*japonica* rice: W1944, W0104, W1739, W2012, W1943,
W3095, W3078, W1777, W1979, TWR1, TWR72, and O.
*barthii*. For *indica* rice, V9 (pale violet red) were *aus* and
were distinct from other *indica* clades. V11 (light coral),
V12 (yellow green) and V8 (orchid) were relatively close,
and V6 (thistle), V13 (dark sea green) and V14 (dark olive
green) were relatively close. Four wild rice accessions,
W0170, W1698, W1687 and W1718, were clustered
with *indica* rice. The colors for structure and phyloge-
netic analyses fit well, indicating that the classification is
reasonable.

The Taiwan indigenous rice accessions were grouped
into several subgroups. For *japonica*, all 43 lines in V1 are
indigenous and have relatively primitive traits, that is, tall
plant, very long awn (e.g., some > 8 cm), mostly red cary-
opsis and relatively shattered, etc. V2 (temperate *japon-
ica*) consisted of 20 accessions collected from indigenous
villages. They are relatively modern, have no awn, few
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3** (See legend on next page.)</figcaption></figure>
(See figure on previous page.)

**Fig. 3** Population structure of Asian rice landraces. **A** Structure analysis showing k= 14 subgroups; **B** phylogenetic analysis of all subgroups. Color code for each subspecies, subgroup, and area: *japonica*: V7, trop 1, insular, violet; V10, trop 1, insular, peru; V5, trop1, northern, cornflower blue; V1, trop 1, Taiwan indigenous, light salmon; V4, trop2, Indochina, sky blue; V2, temp, medium aquamarine. *indica*: V6, primitive, thistle; V14, weedy, dark olive green; V13, primitive, dark sea green; V11, insular, light coral; V12, Indochina, yellow green; V8, India, orchid; V9, *aus*, pale violet red. Other Oryza species, V3, light slate gray

with red caryopsis, and are less shattering; V4, V5 and V10 consist of 10, 6 and 2 indigenous lines, respectively, and are relatively primitive. No Taiwan indigenous rice accessions are found in subgroup V7, the insular trop1 group at the western SEA. For the *indica* type, V6, V8 and V13 consist of 34, 1 and 4 indigenous lines, respectively, and all Taiwan indigenous *indica* rice accessions had no awn and most had white caryopsis and were less shattering. Also, subgroups V11 and V12, the insular SEA and Indochina *indica* subgroups, contained no indigenous *indica* rice accession.

The PCA results indicated *japonica* lineages positioned on the left and *indica* lineages on the right along principal component 1 (PC1) (Fig. S4A, B). PC2 further distinguished *indica* accessions (Fig. S4A), and PC3 separated *japonica* accessions (Fig. S4B), with each subgroup circled and labeled. The wild relatives (V3) located at the center of PC1. The *aus* (V9) located at the top and could separate well from the *indica* of the Indian subcontinent (V8). Those from Indochina (V12) and from insular SEA (V11) could be distinguished, whereas those from China and Taiwan (V6 and V13) as well as weedy rice (V14) were scattered. The temperate *japonica* (V2) located at the very top of PC3, followed by V1, the trop1 of primitive Taiwan indigenous accessions. Of note, V1 was closer to V2 but not V4. Trop2 *japonica* lines from Indochina (V4) and trop1 lines from insular SEA (V7 and V10) were at the bottom. The northern trop1 accessions (V5) were separated from other trop1 accessions (V1, V7 and V10). Taiwan indigenous *japonica* accessions are scattered from the top (V2, V1) to the bottom (V4, V5, V10) along the PC3 scale (Fig. S4, and Table S2).

**Grain Size Varied in these subgroups; Many Rice Accessions Contained Different Alleles in Grain-Controlling Genes**

Rice grain size is a quantitative trait and is controlled by multiple genes. Sequence changes of several grain-controlling genes led to variations in seed size. We searched for different alleles mentioned in Methods. For instance, a G-to-T SNP occurring in *GS3* led to an early stop and thus a larger grain (Fan et al. 2006). We found another 5 mutations leading to a frameshift, early-stop or splicing site change, also leading to LOF *GS3* gene, in the panel we used. Table S11 shows the gene names, locus ID, references, mutation positions and types, and sequence changes for all *GS3* LOF mutations. Likewise, the allele information for all genes are listed in the same table. The detail information of the mutated alleles of each

gene for each accession are in Table S3. If the alleles are heterozygous, they are marked as a reference sequence. Other grain size-related genes such as *WTG1* (Huang et al. 2017), *GL2* (Che et al. 2015), *GW2* (Song et al. 2007), *qGSL3-1* (Qi et al. 2012), *GL6* (Wang et al. 2019) and *GS9* (Zhao et al. 2018a) were checked, but no mutation was found in this panel, possibly because it contained traditional landraces.

ANOVA results (Table S12) indicated significant differences among the grain size parameters in the 1196 accessions listed in Table S9. The statistical analysis for seed length, width, length/width ratio and area is shown in Table S13 and violin plots are in Figure S5, with the significance level and population size indicated. The means for each subgroup and the significant levels are in Table 2. For seed width, *japonica* rice (V7, V10, V5, V1, V4 and V2) was wider than all *indica* accessions. For seed length, only temperate *japonica* (V2) and 2 relatively primitive *indica* groups (V6 and V13) were <8 mm. These two subgroups mainly composed of Taiwan indigenous lines and Ming-Ching. The slender seed shape (l/w > 2.8) was present only in V11, V12, V8 and V9, the subtropical and tropical *indica* lines. Small seeds (less length x width) were mainly in temperate *japonica* (V2) and the relatively primitive *indica* landraces (V6 and V13).

With the obvious seed size differences, we then compared the different alleles of the 10 grain-size related genes by using pie charts. The allele mining results shown in Table S3 were reformatted into percentages due to different sample size. The functional alleles (in grey) and mutated alleles (other colors, with different colors if several kinds of mutations were present) of the 10 grain-size related genes for each subgroup are illustrated in Fig. 4.

There were 2 kinds of mutations in *LGY3* gene: type 1 was local, present in 1.0% of V9; type 2 was present in all *indica* subgroups: 5.2, 5.2, 24.8, 13.7, 17.6 and 5.9% for subgroups V6, V13, V11, V12, V8, and V9, respectively. For the 6 *japonica* subgroups, type 2 mutation was present as 44.2, 40.1, 34.8, 18.6, 46.5 and 11.4% for V7, V10, V5, V1, V4 and V2, respectively. There were 5 types of LOF alleles for the *GS3* gene: type 1 is local, present in 2.0% of V9. Type 2 was present in all *indica* accessions: 28.9, 61.9, 5.1, 15.14, 25.4, 68.6% for V6, V13, V11, V12, V8, and V9, respectively. Type 3 was present in all *indica* and *japonica* groups: 25.8, 6.2, 29.3, 56.0, 13.4 and 1.0% for V7, V10, V5, V1, V4 and V2, respectively, of *indica*; and 88.5, 65.5, 4.4, 95.4, 83.1 and 3.4% for V6, V13, V11, V12, V8, and V9, respectively, of *japonica*. Type 5 is local,
<table><caption>**Table 1** The general descriptions for the 14 subgroups</caption><thead><tr><th>subgroup</th><th>subtype</th><th>description</th><th>color</th></tr></thead><tbody><tr><td>V1</td><td>trop1 jap</td><td>Taiwan primitive indigenous</td><td>light salmon</td></tr><tr><td>V2</td><td>temp jap</td><td>temperate japonica</td><td>medium aquamarine</td></tr><tr><td>V3</td><td>wild rice</td><td>Other <i>Oryza</i> species</td><td>light slate gray</td></tr><tr><td>V4</td><td>trop2 jap</td><td>mainly from Indochina</td><td>sky blue</td></tr><tr><td>V5</td><td>trop1 jap</td><td>mainly from northern Asia</td><td>cornflower blue</td></tr><tr><td>V6</td><td>ind</td><td>mainly Taiwan indigenous + Ming-Ching</td><td>thistle</td></tr><tr><td>V7</td><td>trop1 jap</td><td>insular SEA, most are from western</td><td>violet</td></tr><tr><td>V8</td><td>ind</td><td>mainly from Indian subcontinent</td><td>orchid</td></tr><tr><td>V9</td><td>aus</td><td>mainly from Indian subcontinent</td><td>pale violet red</td></tr><tr><td>V10</td><td>trop1 jap</td><td>mainly from insular SEA, most are from eastern</td><td>peru</td></tr><tr><td>V11</td><td>ind</td><td>mainly from insular SEA</td><td>light coral</td></tr><tr><td>V12</td><td>ind</td><td>mainly from Indochina</td><td>yellow green</td></tr><tr><td>V13</td><td>ind</td><td>mainly Taiwan Ming-Ching + China</td><td>dark sea green</td></tr><tr><td>V14</td><td>ind</td><td>mainly weedy rice from Jiangsu, China</td><td>dark olive green</td></tr></tbody></table>

<table><caption>**Table 2** The mean of seed size parameters for each subgroup</caption><thead><tr><th>Sub-population</th><th>Sample size</th><th>Length (mm)</th><th>Width (mm)</th><th>I/w ratio</th><th>Index</th></tr></thead><tbody><tr><td>V1</td><td>31</td><td>8.59 <sup>abcd</sup></td><td>3.60 <sup>a</sup></td><td>2.41 <sup>fg</sup></td><td>30.71 <sup>ab</sup></td></tr><tr><td>V2</td><td>97</td><td>7.32 <sup>e</sup></td><td>3.24 <sup>cd</sup></td><td>2.31 <sup>g</sup></td><td>23.53 <sup>d</sup></td></tr><tr><td>V4</td><td>70</td><td>8.99 <sup>a</sup></td><td>3.40 <sup>ab</sup></td><td>2.65 <sup>def</sup></td><td>31.07 <sup>a</sup></td></tr><tr><td>V5</td><td>22</td><td>8.23 <sup>bcd</sup></td><td>3.38 <sup>abc</sup></td><td>2.46 <sup>efg</sup></td><td>27.78 <sup>abc</sup></td></tr><tr><td>V6</td><td>60</td><td>7.98 <sup>d</sup></td><td>2.93 <sup>efg</sup></td><td>2.74 <sup>cde</sup></td><td>23.32 <sup>d</sup></td></tr><tr><td>V7</td><td>52</td><td>8.83 <sup>abc</sup></td><td>3.30 <sup>abc</sup></td><td>2.66 <sup>def</sup></td><td>29.51 <sup>ab</sup></td></tr><tr><td>V8</td><td>139</td><td>8.61 <sup>abc</sup></td><td>3.04 <sup>e</sup></td><td>2.90 <sup>bc</sup></td><td>25.97 <sup>c</sup></td></tr><tr><td>V9</td><td>98</td><td>8.33 <sup>cd</sup></td><td>3.05 <sup>ef</sup></td><td>2.82 <sup>cd</sup></td><td>24.95 <sup>cd</sup></td></tr><tr><td>V10</td><td>137</td><td>8.73 <sup>abc</sup></td><td>3.30 <sup>bc</sup></td><td>2.73 <sup>cde</sup></td><td>28.51 <sup>b</sup></td></tr><tr><td>V11</td><td>153</td><td>8.45 <sup>c</sup></td><td>2.80 <sup>g</sup></td><td>3.02 <sup>ab</sup></td><td>23.88 <sup>d</sup></td></tr><tr><td>V12</td><td>218</td><td>8.88 <sup>ab</sup></td><td>2.90 <sup>fg</sup></td><td>3.12 <sup>a</sup></td><td>25.83 <sup>c</sup></td></tr><tr><td>V13</td><td>76</td><td>7.98 <sup>d</sup></td><td>3.10 <sup>de</sup></td><td>2.61 <sup>ef</sup></td><td>24.31 <sup>cd</sup></td></tr></tbody></table>

Grain size data of 1196 accessions were used. The subpopulations were grouped by using structure. The units for width and length are mm. The ratio is length/width. The index is length x width. The population size, variation and statistics significant level were shown in Figure S6 as violin plots

present in 0.6 and 0.5% of for V11 and V12, respectively. Type 6 is also local, present in 1.0% for V9 only. There were 3 types of *qTGW3* mutations. Type 1 was present in wild rice. Type 2 was local, present in 28.2% for V4. Type 3 was also local, with 32.6 and 1.4% for V1 and V4, respectively. A total of 3 deletions were found in the *qSW5* gene region (Fig. 4, and Table S6). The 0.95-kb deletion was present as 79.4, 88.5, 40.4, 42.2, 45.0, 19.4% for V6, V13, V11, V12, V8, and V9, respectively, of *indica*; and 63.5, 4.9, 4.3, 0, 2.3 and 0.9% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*. The 1.2-kb deletion was present as 1.5, 1.3, 0, 0.46, 2.9 and 0% for V6, V13, V11, V12, V8, and V9, respectively, of *indica*; and 0, 18.3, 91.3, 90.7, 54.3 and 89.0% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*. The 13.4-kb deletion was present as 1.9 and 2.0% for V11 and V9, respectively of

*indica*; and 23.1, 15.5, 4.4, 0, 5.6 and 3.4% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*. However, the 0.95-kb deletion was 593 bp 3' downstream of the *qSW5* gene (Fig. S6). ANOVA results indicated significant differences between different deletion lengths (Table S14). Grain width was significantly larger for accessions with a 13.4-kb deletion than a 1.2-kb deletion, and those with a 13.4- or 1.2-kb deletion than a 0.95-kb deletion. Grain width was larger for accessions with a 0.95-kb deletion than the wild type, that is, without any deletion, but to a lesser extent (Figure S7 and Table S15). Only one type of *GS5* mutation was found, and in *indica* rice only: present in 23.7, 6.2, 70.1, 76.2, 14.1 and 3.9% for V6, V13, V11, V12, V8 and V9, respectively. There were 2 types of *TGW6* mutation: type 1 was local, present in 1.7% for V2 only. Type 2 was found only in *indica* rice: 3.1, 38.1, 21.7, 5.1, 3.5, and 7.8% for V6, V13, V11, V12, V8 and V9, respectively. *GW6a* mutation occurred at the promoter region in 2 positions, i.e. types1 and 2 with 300 bp apart. The type 1 only occurred at local, present in 0.5 and 1.1% for V12 and V8, respectively. All others were types 1 and 2 occurred together, and present in 79.4, 74.2, 96.8, 60.1, 0, and 35.3% for V6, V13, V11, V12, V8 and V9, respectively, of *indica*; and 7.7, 0.7 and 2.8% for V7, V10 and V4, respectively, of *japonica*. There were 4 types of *GW7* mutations: type1, type 2, type 3, and both types 1 and 3 together. Type 1 was present as 19.6, 2.1, 0.6, 0, 0.1, 2.9% for V6, V13, V11, V12, V8 and V9, respectively, of *indica*; and 0, 0.7, 8.7, 13.6, 1.4 and 50.3% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*. Type 2 was present in 72.2, 87.6, 96.2, 99.1, 95.1 and 61.8% for V6, V13, V11, V12, V8 and V9, respectively, of *indica*; and 0.6% for V2 of *japonica*. Type 3 was only present in *japonica*: 1.9, 2.1, 5.6% for V7, V10 and V4, respectively. Both type 1 and 3 mutations were present only in 2 *indica* subgroups: 3.1 and 0.7% in V6 and V8, respectively. Both mutations were found in all *japonica*: 98.1, 95.8, 87.0, 86.1, 91.6 and 5.1%
<figure><img src="image_8.png" /><figcaption>**Fig. 4** Pie chart analysis of 10 grain-size related genes in the Asian rice populations. Gray indicates the functional type and other colors indicate different mutations, shown at the top of each gene</figcaption></figure>

for V7, V10, V5, V1, V4 and V2, respectively. For *GLW7* gene, the type 1 mutation was present in 85.6, 93.8, 77.7, 81.2, 84.5 and 86.3% for V6, V13, V11, V12, V8 and V9, respectively, of *indica*; and 13.5, 58.5, 34.8, 18.6, 28.2, and 2.3% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*. For the *GW8* gene, the type 1 mutation was present in 95.9, 100, 96.2, 98.2, 97.2 and 67.7% for V6, V13, V11, V12, V8 and V9, respectively, of *indica*; and 0, 27.5, 82.6, 2.3, 42.3 and 1.7% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*.

Together, it was illustrated that few mutations related to the grain-size related genes were local, such as *qTGW3*. However, most of them were present in relatively high percentages of *indica* accessions only, such as *GS5*, *TGW6*, *GW6a*, or all over the *indica* and *japonica* accessions, from Taiwan, Indochina, insular SEA to Indian subcontinent for another, such as *LGY3*, *GS3*, *qSW5*, *GW7*, *GLW7* and *GW8*. The data illustrated that rice grain size has been a selected target for long time.

## Allele Mining of Other Genes Related To Seed Morphology and Adaptation

In addition to the grain-size controlling genes, we performed similar allele mining analyses on seed morphology-related genes. The proportion of each allele type is

also shown in pie charts (Fig. 5). The allele information is in Table S11 and the allele type of each accessions in Table S3. The LOF mutation of *Rc* gene, i.e. white caryopsis, were present in 79.4, 77.6, 93.5, 95.4, 64.2, and 3.1% for V6, V13, V11, V12, V8, and V9, respectively, of *indica*; and 93.9, 80.1, 100, 55.8, 83.1 and 93.2% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*.

The LOF *wx* mutation led to glutinous (sticky) grain (Wanchana et al. 2003). The 22-bp deletion was present in 19.4, 6.5, 5.9, 30.0, 0.7 and 0% for V6, V13, V11, V12, V8, and V9, respectively, of *indica*; and 34.0, 8.5, 50.0, 88.1, 41.8 and 6.1% for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*.

Three non-shattering seed genes have been cloned and the mutated SNP information is available: *qSH1*, Konishi et al. 2006; *qSH3*, Ishikawa et al. 2022; *sh4*, Li et al. 2006. *qSH1* mutation was present in 4.1 and 0.5% for V6 and V12 of *indica*, and 12.4% for V2 of *japonica*. The *qSH3* mutation was present in 100, 99.0, 99.4, 100, 95.1 and 12.2% for V6, V13, V11, V12, V8, and V9, respectively, of *indica*; and 100, 95.0, 100, 100, 100, and 100 for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*. For *sh4*, it was present in 81.3, 98.0, 99.4, 100, 98.6 and 97.1% for V6, V13, V11, V12, V8, and V9, respectively, of *indica*; and 100% for all the *japonica* subgroups.
<figure><img src="image_9.png" /><figcaption>**Fig. 5** Pie chart analysis of grain morphology- and adaptation-related genes in the Asian rice populations. Gray indicates the functional type and other colors indicate loss-of-function ones or different mutations, shown at the top of each gene</figcaption></figure>

We also performed allele mining of 2 adaptation-
related genes: *Cold1* (Ma et al. 2015) and *Heading date
1* (*Hd1*; Yano et al. 2000; Takahashi and Shimamoto
2011; Wu et al. 2020a). *Cold1* was found related to chill-
ing tolerance; that is, an SNP located at exon 4 of *Cold1*
associated with the adaptation to cold environment in
temperate *japonica* rice. Specific SNPs could distinguish
different subgroups well (Fig. 5): A was present in 95.5,
98.7, 100, 100, 85.1 and 27.4% for V6, V13, V11, V12, V8,
and V9, respectively, of *indica*; and 37.3, 72.7, 86.4, 37.2,
72.9 and 1.7% for V7, V10, V5, V1, V4 and V2, respec-
tively, of *japonica*. T was present in 4.5, 1.3, 0, 0, 0.7 and
0% for V6, V13, V11, V12, V8, and V9, respectively, of
*indica*; and 62.8, 27.3, 13.6, 62.8, 27.1 and 98.3% for V7,
V10, V5, V1, V4 and V2, respectively, of *japonica*. G was

present in *indica* in Indian subcontinent only, with 13.6
and 72.6% for V8 and V9, respectively.

Rice is a short-day plant; its sensitivity to photoperiod
is important for adaptation, especially for those grown in
sub-tropical or tropical regions. As one of the important
genes for photoperiod sensitivity, *Hd1* contains several
LOF mutations leading to loss of sensitivity (Yano et al.
2000; Takahashi and Shimamoto 2011; Wu et al. 2020a).
The definition of these alleles follows the last 2 papers.
Figure 5 shows 6 different mutations and the wild type in
these subgroups. Type 13 was present in 8.8, 10.3, 18.0,
0.9, 4.3 and 72.5% for V6, V13, V11, V12, V8, and V9,
respectively, of *indica*; and 63.5, 90.9, 4.4, 2.3, 0 and 5.1%
for V7, V10, V5, V1, V4 and V2, respectively, of *japonica*.
Type 3 was local, it was present in 1.5 and 20.5% of V6
and V13, respectively. Type 7 was present in *indica* only, in 16.2, 42.3, 10.9, 2.8, 4.3, and 0% for V6, V13, V11, V12, V8, and V9, respectively. Type 19 was present in *japonica* only in 0, 0.7, 0, 2.3, 0 and 42.4% for V7, V10, V5, V1, V4 and V2, respectively. Type 12 was local, present in 14.1% of V4. Type 20 was also local, present in 5.7% of V8.

## Some Indigenous Japonica Rice Accessions Showed Selection Related To UV Tolerance

By using 24 Taiwan indigenous *japonica* accessions together with other SEA landraces, we previously showed that some Taiwan *japonica* lines associated with genomic regions under heavy selection (Alam et al. 2021). The gene was suggested to be the OsUGT706D1 and could be related to UV tolerance (Peng et al. 2017). In fact, there were 6 tandem flavone glucosyltransferase genes in the region, including Os01t0734600, Os01t0734800, Os01t0735300, Os01t0735900, Os01t0736100, Os01t0736300 (also known as OsUGT706D1), at the 30.7-Mb region of chromosome 1.

In the present study, we performed whole genome re-sequencing of all Taiwan indigenous lines including 77 *japonica* and 39 *indica* accessions. RAiSD (Alachiotis and Pavlidis 2018) was used to survey positive selection of the long-arm region of chromosome 1 in 6 Taiwan rice populations: indigenous *japonica*, indigenous *indica*, Ming-Ching (*indica*), modern *japonica*, modern *indica* and weedy rice (all *indica*). Figure S8 indicates a high selection peak at the 30.7-Mb region of chromosome 1 in indigenous *japonica* only. (1) IGV analysis was used to determine any insertion near this region and the results indicated no newly transposed transposable element. (2) Amino acid changes of these 6 proteins in indigenous lines were checked by using SNPeff (Cingolani et al. 2012). Many randomly distributed missense mutations occurred in these lines, but only 2 proteins had specific mutations. Data mining suggested that the selected genes were Os01t0734600 and Os01t0734800, and 20 indigenous *japonica* accessions were the selected accessions (Table S16). These included 4 trop1, 11 trop2 and 5 temperate *japonica* lines. These accessions consisted of specific amino acid mutations in the ORF of the 2 proteins (listed in Tables S17 and S18).

Uridine diphosphate-dependent glycosyltransferases (UGTs) generally adopt a two-domain architecture, consisting of N-terminal and C-terminal domains arranged in a left-right opposition to one another. A catalytic pocket is formed between these two domains, serving as the site for interactions with both the glycosyl donor and acceptor substrates. The glycosyl donor typically interacts with the C-terminal domain, while the acceptor substrate binds to the N-terminal domain (Liu et al. 2025). Among the five mutation sites identified in Os01g0734600, four are located within the glucosyl donor binding region,

suggesting potential effects on the affinity between the protein and the glucosyl donor (Fig. S9). All mutation sites in Os01g0734800 are located in the glucosyl acceptor binding region, specifically on the outer sides of two peripheral helices (helix wheel positions) (Fig. S10). These mutations may affect the interaction between the protein and the glucosyl acceptor, or with other molecules. The finding remains for further experimental examination.

## Discussion

The current study details the story behind the early carbonated rice grain versus the current indigenous rice cultivated in Taiwan. We performed whole-genome re-sequencing and analyzed the current seed morphology information by using a panel of 1365 rice accessions, about 85% landraces, weedy and wild rice. We show the sequence changes and the fluctuation of early seed size dimensions and how genes related to grain size have been changed. We also show Taiwan indigenous rice accessions consist of important traits, including tolerant to UV, drought and flooding.

## Several Mutations Could Lead To Larger Seeds as Compared with Very Early Rice

In one of our previous studies, very early (before 3300 BP) carbonated rice grain from 5 excavated sites in southern Taiwan had small size: average length, width and length/width ratio 3.91 mm, 2.35 mm, and 1.68, respectively (Hsu et al. 2019). In the current study, we performed statistical analyses of 100 seeds from 4 excavated sites and also showed that rice seeds collected before 3300 BP were tiny (Fig. 1D, Tables S4 and S5). For instances, the grain length average of NKLE site was 3.83 mm, with min 2.92, max 5.00, mode 4.05, median 3.79 and the coefficient variation value 10.29%. The value was quite high compared with modern varieties, indicating huge variations in grain size at ancient time. Our previous studies indicated that early Taiwan agriculture came from northern China (Sagart et al. 2018), so we checked the rice seed size of early northern China (before 5,000 BP) and Korea (around 3300 BP). The average carbonated rice grain length was 4.1 mm in the Yuezhuang site (6060–5,750 BP), northern China (Crawford et al. 2006). For the carbonated rice grain in the Baligang (northern China) site, there were 4 grains in the excavated site of the pre-Yangshao period (8700-8300 BP), with length from 4.0 to 4.8 mm, and 119 grains in the excavated site of the Yangshao period (6300-5000 BP), with length from 3.1 to 5.0 mm (Deng et al. 2015). In the early Mumun period (~3,00 BP) in Korea, with a total of 7 excavated sites, the average length of carbonated rice grains was 3.3 mm (Ahn 2010). Hence, very early rice grains in northern China, Korea and Taiwan were tiny, ranged from 2.9 to 5.0 mm in grain length, as compared with later grains.
Many studies focused on reproducing charring experi-
ments and checked the grain size changes on carbon-
ated grains from excavated sites. Several cereals showed
a decrease in grain length during the process, such as
rice (Chuenwattana 2010), wheat (Renfrew 1973), barley
(Renfrew 1973), and oat (Renfrew 1973). A recent study
of the natural charring processes aiming for the preser-
vation biases that occurred in prehistoric contexts of
carbonization showed that grain length had a shrinkage
of 21.05% for rice, 25.0% for proso millet, and 18.9% for
foxtail millet, etc. (Castillo 2019). Thus, the estimated
original grain length of early Taiwan rice was 4.8 mm
before 3300 BP and then became larger later on, includ-
ing 6.4 mm at HLL and 7.5 mm at WCT. For the present
indigenous trop1 accessions, the grain length was 8.6 mm
(Table 2).

All carbonated rice seeds used for size measurements
were caryopses, as the images in Fig. 1A show. In the
present work, we used the genome sequencing informa-
tion coding for the grain size trait to interpret differences
between carbonated grains and current grains. Accord-
ing to the studies on rice grain development, there was
a high correlation between the size of caryopsis and the
whole grain (with palea and lemma) (Takeda and Taka-
hashi 1970; Takita 1988), and thus allele analysis could
be used to explain differences in ancient seeds excavated
from archaeological sites.

According to the sequencing analysis, the most primi-
tive Taiwan trop1 indigenous lines, V1, contained alleles
leading to larger grains, including LGY3, 19%; GS3, 96%;
qTGW3, 33%; GLW7, 19% and GW7, 100%. The addi-
tive effects could be one of the reasons leading to larger
grains.

In the current study, we investigated different muta-
tions present in 10 grain-size controlling genes (sum-
marized in Fig. 4). However, these mutations could
not explain well why the grain size of Ming-Ching rice
accessions were smaller than most of the other indica
rice accessions in Asia. Further detail studies are still
requisite.

Landraces are Precious Genetic Resources

Taiwan is a small island, however, there are 16 different
tribes living here for more than 4000 years. We've col-
lected 116 indigenous rice accessions and 546 Ming-
Ching accessions. All of them have been kept well in
the national seed stock center. In the current study, we
performed re-sequencing of 116 indigenous lines and 59
Ming-Ching lines, all the sequencing data were submit-
ted to the public domain. The biological materials and
genome sequencing data are important resources for any
further studies.

All Taiwan rice accessions used in the current study
are listed in Table S2, sorted by types. The most primitive

indigenous trop1 group (V1) consisted of 43 acces-
sions, with 67.4% of them collected after WWII. Thus,
it indicated the importance of continuous visit/collect in
mountain regions. All lines unable to germinate belonged
to this collection, point out the collection should be pro-
ceeded as soon as possible.

During the breeding process, a famous rice variety
Taichung 65, bred in 1929, received LOF mutations of 2
important flowering-time related genes *Hd1* and *early
heading date 1* (*Ehd1*) from 2 Taiwan indigenous acces-
sions (Wei et al. 2016). The indigenous rice lines have
been used for breeding programs, and Tunglu 1, Tunglu2
and Tunglu3 were designated in 1964. The other parental
lines for the 3 varieties were temperate *japonica* variet-
ies. According to the current sequencing data, Tunglu3 is
trop1 and the other 2 are temperate *japonica*. All 3 lines
are resistant to rice blast, lodging and drought stress and
have been cultivated in an upland practice in the indig-
enous villages.

We performed a genome-wide association study of 2
panels previously: the *indica* panel contained 268 acces-
sions and the *japonica* panel 238 accessions, for pheno-
types of resistance to drought, flooding and abscisic acid
treatments (Wu et al. 2022). All Taiwan indigenous lines
were in the panel. Table S19 lists the indigenous line
resistant to drought treatment: 16 were *indica* accessions
and 3 *japonica*. Table S20 lists the indigenous lines whose
shoots still grew well after 7-day flooding treatment: 3
were *indica* accessions and 13 *japonica*. Table S21 lists
the indigenous lines whose roots still grew well after
7-day flooding treatment: 4 were *indica* accessions and
12 *japonica*. Of note, 4 lines grew well with both shoots
and roots under flooding.

The Taiwan indigenous people have lived in hill to
mountain regions, at elevations ranging from 500 to
1500 m, for thousands of years. Our studies illustrated
that many of the indigenous rice accessions contained
resilient traits such as tolerance to drought, flooding or
UV. They certainly provide very precious resources in the
current climate change era.

The semidwarf 1 (SD1) mutation played an impor-
tant role in the rice green revolution. The miracle rice
IR8 and one of its parental lines, Dee Geo Woo Gen
(DGWG), contained a 383-bp deletion on GA20ox-2 from
part of exon 1 to exon 2 and led to the LOF (Sasaki et
al. 2002; Spielmeyer et al. 2002). This DGWG was one
of the Ming-Ching accessions. In addition, another 4
Ming-Ching lines, including Hsinchu-Ai-Chueh-Chien,
Ti-Chueh-Wu-K'o, Ai-Tzu-Ch'ung and Liu-Tou-Tzu,
were also semidwarf and contained the same mutation.
W1718, one of the wild rice accessions in the current
panel, also had the same allele. This W1718 was col-
lected from southern China. All 6 accessions contained
the DGWG sd1 allele and were originally from southern
China. However, this allele was not present in the south-
ern China landrace collections in the China National Rice
Research Institute. Somehow Ming-Ching lines could
have been abandoned in China but kept in Taiwan.

Bayesian Evolutionary Analysis Indicated GS3 Gene Mutation Occurrence, Introgression and Expansion

The distribution of type 2 mutation of GS3 gene were relatively wide, from Taiwan, Indochina, insular SEA to Indian subcontinent, and including *japonica*, *indica* and *aus* accessions, as shown in Fig. 4. Thus, we further examined the gene by using Bayesian Evolutionary analysis sampling Trees. We used the fragments containing the whole gene and –1-kb to +1-kb up- and downstream regions for elucidating the relation. The 280 accessions carrying the mutation and all 20 wild rice accessions were used in the analysis.

The results of BEAST analysis could be separated into
6 sessions (Fig S11). Wild rice accessions were group
into Wa and Wb. Session 1 were closed to Wa, indicat-
ing the mutation might occur in trop1 *japonica* of insular
SEA (V10). It was followed by sessions II that consisted
of mainly V13 and with few V8. The next clade started
with another group of wild rice, Wb. Session III was a
small clade with mainly V6, and followed by session IV,
all are V9. Together, it was suggested that *GS3* type 2
mutation occurred in trop1 *japonica* from insular SEA
(V10). However, most of the remaining accessions were
*indica* rice, including V13, V8, V12, V9 (*aus*), and V6.
From pie chart analysis (Fig. 4), type 2 mutations were
mainly in *indica* rice, and only one *japonica* subgroup
(V10, insular trop 1) carried this mutation. Thus, the
mutation occurred at insular SEA then introgressed and
expanded (i.e., brought by human beings) to *indica* rice
in TW (V13), Indochina (V12) and the Indian subconti-
nent (V8), as well as *aus* (V9).

Mutations, Movements, Selections and Adaptations
Occurred in Early Rice Cultivation

There were frequent trade networks around the south
China sea since ~2500 BP (Hung et al. 2007, 2013; Alam
et al. 2021; Wang et al. 2022, 2023). The early Austro-
nesian trading area included Taiwan, southern China,
the Indochina peninsula, insular SEA, and India to the
west, with high activities from ~2500 to ~1800 BP. For
instance, Fengtian jade was found in the excavated sites
around these areas, thereby indicating a jade trade (Hung
et al. 2007, 2013; Alam et al. 2021), whereas both min-
ing and manufacturing were found only on the east side
of Taiwan (Hung et al. 2007, 2013). Thus, back-and-forth
movement between these areas was relatively frequent in
early times.

We showed that mutations and movements occurred
heavily in eastern, southeastern and southern Asia. We

also used Tajima's D analysis (Tajima 1989) to check for
selection across all 17 genes tested in the studies. D val-
ues were significant or negative (although not significant)
for most genes except subgroup V14 (wild rice) (Table
S22). Thus, these genes have indeed been under selection
in the landraces. The current Taiwan indigenous millet
farmers still performed panicle selection at harvesting
stage (Fig. S12), thus illustrating the importance of selec-
tion also in current agriculture.

The movement of rice into insular SEA has been sug-
gested to Austronesian expansion (Bellwood 1997; Dia-
mond 2001; Bellwood 2006). Austronesian farmers from
southern Taiwan started to migrate to the northern Phil-
ippines about 4,000 years ago (Gray and Jordan 2000;
Diamond and Bellwood 2003), then expanded across
insular SEA (Sagart 2004; Bellwood 2011). The Austro-
nesian vocabulary of rice cultivation is shared by Taiwan
and the Malayo-Polynesian languages (Proto-Austrone-
sian *pajay* 'rice plant', *beRas' husked grain', *S₁emay' rice
as food'; Sagart et al. 2018). This finding argues that the
first Austronesians who migrated out of Taiwan brought
with them rice cultivation and the attendant vocabu-
lary. The previous whole-genome re-sequencing stud-
ies suggested that Taiwan indigenous tropical japonica
could came from northern Philippines and mainland
SEA (Alam et al. 2021). We hypothesized that once the
rice lines arrived in the Philippines, their landraces were
out-competed by better-adapted tropical landraces from
the southern mainland (Indochina Peninsula), while the
vocabulary remained unchanged. From the current allele
mining of grain-size related genes, we suggested that rice
accessions with larger seeds were then brought back to
Taiwan via the trade networks and replaces the original
small ones. Together, the genome re-sequencing and lin-
guistic analyses fit well with the out-of-Taiwan theory.

Conclusion

Crop cultivation is influenced by social and cultural
exchanges with nearby regions. The history of dispersal/
exchange of rice in Asia has been an interesting research
target and has been studied by archaeological and lin-
guistic research as well as whole genome re-sequencing.
In the current study, we showed that ancient seed size,
for example, carbonated seeds before 3,300 BP in Taiwan,
were tiny as compared with the size of recent accessions
grown in indigenous villages. Use of whole-genome re-
sequencing and survey of grain size revealed that some
mutations in grain morphology-controlling genes might
be targets for selection, leading to great size changes in
carbonated rice grains versus recent cultivated ones.
We also performed allele mining of 2 adaptation-related
genes. Similar to the current small-scale farming of fox-
tail millet in Taiwan indigenous villages, ancient people
may have selected rice during harvest for the following
growing seasons. The grain sizes of all subgroups studied have been selection targets, and seed size gradually changed along with the improvement of rice cultivation, via introgression and expansion, over thousands of years.

### Abbreviations

<table><tr><td>ANOVA</td><td>Analysis of variance</td></tr><tr><td>BEAST</td><td>Bayesian evolutionary analysis by sampling trees</td></tr><tr><td>BP</td><td>Before present</td></tr><tr><td>CV</td><td>Cross-validation</td></tr><tr><td>DGWG</td><td>Dee Geo Woo Gen</td></tr><tr><td>GS3</td><td>Grain shape gene on chromosome 3</td></tr><tr><td>GS5</td><td>Grain shape gene on chromosome 5</td></tr><tr><td>GW6</td><td>Grain weight on chromosome 6</td></tr><tr><td>GW7</td><td>Grain weight on chromosome 7</td></tr><tr><td>GLW7</td><td>Grain length and weight on chromosome 7</td></tr><tr><td>GW8</td><td>Grain weight on chromosome 8</td></tr><tr><td>HLL</td><td>Huilaili</td></tr><tr><td>IGV</td><td>Integrative Genomics Viewer</td></tr><tr><td>IRRI</td><td>International Rice Research Institute</td></tr><tr><td>LGY3</td><td>Rice grain yield quantitative trait locus on chromosome 3</td></tr><tr><td>LOF</td><td>Loss-of-function</td></tr><tr><td>NKLE</td><td>Nan-kuan-li East</td></tr><tr><td>NPGRC</td><td>National Plant Genetic Resources Center</td></tr><tr><td>ORF</td><td>Open reading frame</td></tr><tr><td>qSW5</td><td>Quantitative trait controlling seed width on chromosome 5</td></tr><tr><td>qTGW3</td><td>Quantitative trait controlling grain size and weight on chromosome 3</td></tr><tr><td>SAM</td><td>Sequence Alignment/Map</td></tr><tr><td>SEA</td><td>Southeast Asia</td></tr><tr><td>TARI</td><td>Taiwan Agricultural Research Institute</td></tr><tr><td>TGW6</td><td>Thousand grain weight on chromosome 6</td></tr><tr><td>WCT</td><td>Wuchiansuo</td></tr><tr><td>YHF</td><td>Youhsienfang</td></tr></table>

### Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12284-025-00854-9.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

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Supplementary Material 13

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Supplementary Material 19

Supplementary Material 20

### Acknowledgements

We are grateful to the Taiwanese indigenous people and traditional farmers for their stewardship of traditional rice landraces. We thank Ms. Lie-Hong Wu for maintaining the greenhouse plants and Laura Smales (BioMedEditing, Toronto, Canada) for English editing. We also wish to acknowledge the contributions of Dr. Yung-Pei Wu, who passed away two years ago while conducting fieldwork.

### Author Contributions

YICH conceived and designed the study, CCW, KYC, YFC, LTH, SMY, THH generated sequencing data. YHW, YCT, TFH, YTT analyzed the carbonized grains. YCT, YTT, NCD, JCL, DPS, CWW, MHL, DHW, SC, SJC collected rice accessions. CCW, CKL, WYC performed bioinformatics analysis. CHT, KTL, WLC provided archaeological materials. WYC, JYY performed the protein modelling analysis. SL provided linguistic and historic information. YICH wrote the manuscript with input from all authors.

### Funding

This work was supported by the MOST grants (110-2313-B-001-005 and 109-2313-B-001-008 to YIH) as well as ITAR grants (AS-ITAR-110-TD06, AS-109-ITAR-TD08 and AS-108-ITAR-TD08 to YIH).

### Data Availability

The data underlying this article are available in the NCBI Short Read Archive (SRA) database as indicated in Table S3.

### Declarations

### Competing Interests

The authors declare no competing interests.

### Author details

<footnote n="1">¹Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan</footnote>

<footnote n="2">²Institute of Plant Biology, National Taiwan Normal University, Taipei, Taiwan</footnote>

<footnote n="3">³Department of Agronomy, National Chiayi University, Chiayi, Taiwan</footnote>

<footnote n="4">⁴Agriculture and Food Agency, Ministry of Agriculture, Taipei, Taiwan</footnote>

<footnote n="5">⁵Crop Science Division, Taiwan Agricultural Research Institute, Taichung, Taiwan</footnote>

<footnote n="6">⁶Institute of Electro-Optical Engineering, National Taiwan Normal University, Taipei, Taiwan</footnote>

<footnote n="7">⁷Taiwan District Agricultural Research and Extension Station, Tainan, Taiwan</footnote>

<footnote n="8">⁸Hualien District Agricultural Research and Extension Station, Hualien, Taiwan</footnote>

<footnote n="9">⁹Kaohsiung District Agricultural Research and Extension Station, Pingtung, Taiwan</footnote>

<footnote n="10">¹⁰Institute of Anthropology, National Tsing Hua University, Hsinchu, Taiwan</footnote>

<footnote n="11">¹¹Department of History, Tung Hai University, Taichung, Taiwan</footnote>

<footnote n="12">¹²National Plant Genetic Resource Center, Taiwan Agriculture Research Institute, Taichung, Taiwan</footnote>

<footnote n="13">¹³Miaoli District Agricultural Research and Extension Station, Maioli, Taiwan</footnote>

<footnote n="14">¹⁴Department of Agronomy, National Taiwan University, Taipei, Taiwan</footnote>

<footnote n="15">¹⁵Department of Agronomy, National Chung Hsing University, Taichung, Taiwan</footnote>

<footnote n="16">¹⁶Institute of Molecular Biolog, Academia Sinica, Taipei, Taiwan</footnote>

<footnote n="17">¹⁷Centre de Recherches Linguistiques sur l'Asie Orientale, Centre National de la Recherche Scientifique, Paris, France</footnote>

<footnote n="18">¹⁸Graduate Institute of Biochemistry, National Chung Hsing University, Taichung, Taiwan</footnote>

<footnote n="19">¹⁹Present address: Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan</footnote>
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