Population structure dynamics of Taiwan rice accessions over thousands of years as revealed by archaeological, morphological and genome sequencing information

doi:10.21203/rs.3.rs-3218983/v1

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Population structure dynamics of Taiwan rice accessions over thousands of years as revealed by archaeological, morphological and genome sequencing information

https://doi.org/10.21203/rs.3.rs-3218983/v1

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Background

Cultivation of rice (Oryza sativa) started in Taiwan about 5000 years ago. Here we studied changes in the rice population during this period by using archaeological, morphological, genetic and genomic strategies. We studied the grain size changes of carbonized rice from excavated sites. We also revealed the variations in landraces collected from the indigenous villages and landraces that arrived in Taiwan from southern China about 400 years ago. Some modern varieties were also used in the current study.

Results

The very early cultivated rice must have been temperate japonica type, and the seeds were relatively small. Rice seeds became relatively bigger around 1500 BP, with some indica or tropical japonica ones. Most, if not all, of the indica rice were not primitive types, suggesting they arrived in Taiwan rather late. Together, temperate, subtropical, tropical japonica and indica rice have been cultivated by indigenous people for a long period, with all in upland practice. However, only indica landraces were cultivated in the plain region from the early 17th century to about 100 years ago, when japonica rice accessions become dominant. We illustrated huge differences in genetic diversity among the subpopulations of Taiwan rice accessions, and many of these lines showed stress resistance to drought, flooding and ABA treatments.

Conclusion

We show how civilization, human migration, taste preference, natural introgression and breeding programs have shaped the population structure of Taiwan rice accessions over thousands of years. We also indicate that Taiwanese indigenous peoples and traditional farmers have kept the rice landraces for hundreds and up to thousands of years. With many old traits preserved, they are good resources for future breeding programs.

Excavated rice grain

genetic diversity

population structure

stress tolerance

Asian cultivated rice (Oryza sativa) is one of the most important crops in the world and the most widely consumed. The rice was genetically divided into 2 main subspecies, japonica and indica, with distinct morphological and physiological characteristics (reviewed by atsuo and Hoshikawa, 1993). Later on, with the analysis of molecular markers nd whole genome sequencing (3KRGP 2014), rice was found to contain 5 major subpopulations: aus, indica, temperate japonica, tropical japonica, and aromatic. In 2018, a detailed analysis of the 3K accessions further classified indica rice into ind1a, ind1b, ind2 and ind3 and japonica rice into tropical, subtropical and temperate japonica (Wang et al. 2018). According to archaeological studies, the early domesticated (i.e., non-shattering) cultivated rice in China was the japonica type, about 8,000 years ago, collected from Baligang in the Yellow River-Huai River-Plain (Deng et al. 2015). Although there was rice collected from the Shangshan site about 9,000 years ago in the Lower Yangtze region (Liu et al. 2007; Zuo et al. 2017), most of them are still shattering. Wild rice ancestral to indica was present in the northern Indian subcontinent before the arrival of non-shattering japonica (Silva et al. 2018). Indica rice was domesticated after the arrival of domesticated japonica rice, about 4,000 years ago, in the Ganges plains of eastern India (Fuller et al. 2010). 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 (EA) was relatively late as compared with japonica rice cultivation.

According to the archaeological studies, there are thousands of carbonized rice grains found in Nan-kuan-li (NKL) and Nan-kuan-li East (NKLE) excavated sites in southern Taiwan. Ten ¹⁴C dates obtained from these sites indicate that they were there between 5,000 to 4,500 BP. Other crop remains such as foxtail millet (Setaria italica) and broomcorn millet (Panicum miliaceum) as well as farming tools such as shell knives and stone hoes were discovered (Hsieh et al. 2011; Tsang et al. 2017). Thus, rice and millet farming may have been an important food source for people living about 5000 years ago in southern Taiwan.

Taiwan indigenous people are the original inhabitants of Taiwan. Their ancestors may have been living in Taiwan for thousands of years before the Han Chinese immigration began in the 17th century. Taiwanese aborigines are Austronesian-speaking peoples and were demonstrated as the origin of Austronesian languages located in the Philippines, Indonesia, Malaysia, Madagascar, Polynesia and Oceania (see e.g., Hill et al 2007). The dispersal is presumed to have led to the spread of Austronesian languages, cultural similarities, and agriculture and so on (Blust 1995; Bellwood 1997; Bellimod 2004; Bellwood 2023). Thus, the Taiwanese indigenous people have played important roles as the bridge of the mainland and the Pacific, and the analyses of early agriculture are critical. According to this “Out-of-Taiwan” hypothesis, rice, millet and taro are the three among many others important for the dispersal. The linguistic studies also coincide well with the flow as most people, if not all, in this area called rice *pajay before Columbus’ time (Sagart 2011).

The population structure of domesticated rice species has been influenced by civilization, human migration, taste preference, plant introgression, etc., during the history of the cultivation. Since the 20th century, plant breeding programs have played critical roles in crop production; thus hybridization between parental cultivars with desirable traits followed by intensive artificial selection are important for the development of new varieties.

In the current study, we used carbonized rice grains from several excavated sites in Taiwan to reveal the seed size changes across thousands of years on the island. We also used whole genome sequencing to reveal the population structure differences among the current Taiwan rice accessions with the view that 1) the early rice accessions in Taiwan had been cultivated for thousands of years, 2) rice cultivation changed with the arrival of the Han people about 400 years ago, 3) changes from indica to japonica rice during the Japanese colonial period started about 100 years ago, and 4) the modern breeding period has existed since the early 20th century. The questions we asked included 1) Which type was the early rice that arrived in Taiwan in ancient times, 2) What are the reasons for changes in major rice populations, 3) What are the relationships in rice populations with the nearby regions, and 4) How did the domestication/adaptation alleles change in different populations. We show how civilization, human migration, taste preference, natural introgression and breeding programs have shaped the population structure of Taiwan rice accessions over thousands of years.

Archaeological materials

Hundreds of carbonized rice grains were collected from 4 excavated sites belonging to 4 cultures by using the floating method. The sites are NKLE (23º6'58"N, 120º16'35"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), Wuchiantsuo (WCT, 23°05'39.0"N 120°16'25.0"E, altitude 7 m) of the Niaosong culture (1400 − 500 BP), and Huilaili (HLL, 24º09'42.8"N, 120º38'11.6"E, altitude 73 m, 1300 BP) of the Fanziyuen culture (2000-400BP). In total, 100 carbonized rice samples from each site were used to estimate seed width, length and thickness by using a digital ruler (Mitutoyo Co., Japan) with accuracy of 0.01 mm. The Chinese translation of the excavated sites and cultures are listed in Table S1.

Plant materials and growth condition

The seeds of the indigenous rice accessions, landraces and modern varieties were obtained from the National Plant Genetic Resources Center (NPGRC), Taiwan Agricultural Research Institute (TARI), Taiwan. The seeds were collected from the indigenous peoples of the mountain regions of different tribes. Part of the samples were collected around 1900 and the rest in recent years. The accession names for all seeds are in Table S2. 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℃ for DNA extraction.

Whole genome sequencing and single nucleotide polymorphism (SNP) calling

Genomic DNA was extracted from leaves by using a DNeasy Plant Mini Kit (Qiagen) following the manufacturer’s protocol. DNA was prepared for Illumina genome sequencing using the Illumina Hiseq2000 instrument with a 2 x 150-bp read configuration. A few of the Taiwan rice accessions were sequenced in the 3K rice project (3KRGP 2014). Sequencing data for these accessions are available from the NCBI Short Read Archive (Bioproject accession nos. PRJNA485658, PRJNA373799, PRJNA623980, and PRJEB6180).

The paired reads were mapped against the Os-Nipponbare-Reference-IRGSP-1.0 database (IRGSP 2005; Kawahara et al. 2013). SAMtools (Li et al. 2009) and VCFtools (Danecek et al. 2011) were used to handle the sequence alignment/map format (SAM) and variant call format (VCF) of the file. To detect SNPs and small indels, we used the command lines in the section “Variant Calling” in “Workflows” of the SAMtools manual. The information on SNPs and small indels was recorded in VCF files. The detailed methods were similar to those described previously (Wei et al. 2016a; Wu et al. 2020a).

Scoring of grain-related traits

Several grain-related traits were screened: long awn, red caryopsis, glutinous, seed width and seed length. Awns > 2 cm were defined as long awns. Ground rice grain was treated with 0.2% iodine reagent and scored 5 min later for the glutinous (sticky) phenotype.

Estimation of diversity of domestication- or adaptation-related genes

Twenty domestication- or adaptation-related genes were used to evaluate the gene diversity. They are An1 (Luo et al. 2013), An2 (Gu et al. 2015; Hua et al. 2015), Bh4 (Zhu et al. 2011), Cold1 (Ma et al. 2015), Ehd1 (Doi et al. 2004), Gn1 (Ashikari et al. 2005), Hd3a (Tamaki et al. 2007), IPA1 (Lu et al. 2013), Lg1 (Zhu et al. 2013), OsLg1 (Ishii et al. 2013), OsC1 (Saitoh et al. 2004), Phr1 (Yu et al. 2008), Prog1 (Jin et al. 2008), qSH1 (Konishi et al. 2006), qSW5 (Shomura et al. 2008), RAE2 (Bessho-Uehara et al. 2016), Rc (Sweeney et al. 2006), TGW6 (Ishimaru et al. 2013), TT1 (Li et al. 2015) and Waxy (Wang et al. 1995). These genes are related to plant stature, grain phenotypes, temperature tolerance or photoperiod sensitivity. Detailed information for these genes is in Table S3.

Genomic sequences were aligned by using MUSCLE (Edgar 2004a, b). The − 1 Mb to + 1 Mb region of each of these genes was used for the analysis. Statistical analysis involved using DNAsp.v6 (Rozas et al. 2017). Items analyzed included number of polymorphic (segregating) sites (S), total number of mutations (Eta), average number of nucleotide differences (k), nucleotide diversity (π) (Tajima 1983), theta (per sequence) from Eta θ, and Watterson’s estimator of theta (per site) from Eta θw (Watterson 1975). The neutrality test, with Tajima's D value, was used to test the neutral mutation hypothesis (Tajima 1989). The D value was based on the discrepancy between π and θw; thus, negative values indicate excess low-frequency polymorphism.

Continuous rice cultivation in Taiwan from 5000 years ago

The earliest Taiwan carbonized rice grains were found in the excavated sites about 5000 years ago. Thousands of carbonized rice grains were found in NKL and NKLE in southern Taiwan from 5000 − 4500 BP (Hsieh et al. 2011; Zang and Li 2015). Rice was found continuously in the southern part in YHF (3800 − 3300 BP, Zang and Li 2015), WCT (1800 − 500 BP, Zang and Li 2015) and Siliao (2300 − 600 BP, Liu 2011. There were also reports of early rice in Fushan (4500 − 3500 BP) and Chaolaiqiao (4500 − 3500 BP) during the middle Neolithic period in eastern Taiwan (Wu et al. 2016; Deng et al. 2022). In central Taiwan, early rice was found in Anhe (4800 − 4000 BP, Deng et al. 2022) and HLL (1300 BP, Chu 2016). In northern Taiwan, carbonized rice grains were found in the Zhiwuyuan site (Taipei Botanical Garden) (4500 BP, Deng et al. 2022) and Chishanyan site (4000 − 3000 BP, Huang 1984). Thus, archaeological studies indicated continuous rice cultivations in Taiwan from about 5000 years ago to the present.

Using 100 carbonized seeds from 4 excavated sites, we studied the seed size changes by measuring the seed length, width and thickness. Figure 1 shows that early rice seeds were relatively small: with length 3 to 4.5 mm, width 1.8 to 2.9 mm and thickness 1.1 to 2 mm at NKLE and YHF, both located in southern Taiwan and before 3300 BP. The seed size variations during this period were rather small. For the seeds with a wide range of time (~ 1400 − 500 BP) in the south (i.e., WCT), the length varied from 4.5 to 7 mm, width 2.2 to 3.6 mm and thickness 1.3 to 2.9 mm. Thus, the seed size was significantly larger than earlier ones and with large variation. Some of these seeds were double in size as compared with the two earlier ones. About the same time (1300 BP) in central Taiwan (i.e., HLL), seed length was 3.8 to 6 mm, width 1.8 to 3 mm, and thickness 1.2 to 2.1 mm. The seeds were larger than seeds from NKLE and YHF and smaller than those from WCT, again with large variation. Therefore, rice seed size in Taiwan has changed over thousands of years, from relatively small and round to large with an oblonga shape.

Total of 265 accessions were used in the current genomic study

A total of 265 rice accessions collected in Taiwan were used in the study (Table 1, Table S2). These included 129 accessions collected from indigenous villages, 58 belonging to those brought to Taiwan about 400 years ago, 17 breeding lines, 58 modern varieties and one weedy rice. In addition to these 263 lines, 2 wild rice accessions were collected in Ba-der, Taoyuan. All of the 263 + 2 accessions (listed in Table S2) underwent whole genome sequencing followed by further analysis.

Table 1

The subgroups of Taiwanese rice accessions used int the study. No *aus* or *aromatic* types were found in local lines.
	Temperate japonica	Subtropical japopnica	Tropical japonica	indica	Admixture	Total
Indigenous	58	17	12	39	3	129
Mingching	0	0	0	58	0	58
Modern	54	0	4	16	1	75
Red rice (weedy)	0	0	0	1	0	1
total	112	17	16	114	4	263

Sum: 263 + 2 wild relatives

Many rice landraces had been cultivated by indigenous peoples in the mountainous regions with an upland practice. A total of 60 upland rice accessions were collected from the indigenous villages from 1895 to the early 1900s. These accessions were since propagated (renewed) about every 10 years by rice breeders, and seeds were stored in NPGRC, TARI. There are only names for this seed resource, without other information such as collecting time, villages and tribes. Some rice breeders visited mountain regions and collected more accessions in recent years and added another approximately 70 lines, with information on tribes collected. These accessions are highly diverse in seed morphology, plant height, plant stature, heading behavior, etc., as shown in our previous studies (Hsieh et al. 2011; Sagart et al. 2018; Wu et al. 2020a).

The Han people migrated to Taiwan from the southeast coastal area in China, mainly Fujian and Guangdong, to Taiwan during the late Ming to early Ching Dynasty about 400 years ago. The rice accessions they brought were designated “Ming-Ching” in the current study. According to the literature, there were 1,679 Ming-Ching accessions during the survey in 1906, and the breeders reduced these to 547 lines after screening and elimination according to similarity in morphology (Iso 1944). All these resources were also stored in NPGRC. We chose 58 accessions from this collection for the current analysis. These accessions represented the old landraces in southern China about 400 years ago.

Modern breeding programs have been applied for improving rice varieties, including japonica and indica, in Taiwan since the 1920s. Both breeding lines (the intermediate lines during the breeding process for new varieties) and modern varieties (finished the complete breeding process and were assigned names) were chosen for this study; they belong to the category “modern” rice. Red rice, also known as weedy rice, has been a problem in rice production practice in Taiwan in recent years (Huang et al. 2021; Wu et al. 2020b). One red rice accession was also used for the present analysis. A wild rice (Oryza rufipogon) population existed in northern Taiwan previously and had become extinct on the site around 1978 (Kiang 1979). Some of these accessions were kept by rice breeders in the Miaoli Agriculture Research Station, and we received 2 lines for the current study.

Classification of the indigenous upland rice accessions

We prepared a rice genomics and phenomics resource of 500 accessions primarily with Taiwan rice accessions previously described (Wu et al. 2022). This resource consists of the genome sequencing data for 265 Taiwan accessions along with temperate japonica, subtropical japonica, tropical japonica, indica, aus, and aromatic accessions collected from other Asia countries where rice is the major staple food. We used 500 accessions for phylogenetic, structure and genome-wide association analyses. Structure analysis at K = 9 (Fig. 2, Wuet al. 2022) could separate the accessions into 9 categories: japonica accessions into 4 groups (i.e., V1, V2, V4 and V7), indica into 4 groups (V3, V5, V6 and V8) and wild rice accessions as V9. The 265 lines in the current study were classified into these 9 groups (Table 2), with the detailed information for each accession presented in Table S2.

Table 2

Detail information of the V1 to V9 groups classified by using structure analysis.
Group #	Subtype	Total number	Taiwan number*	Descriptions
V1	Temp jap	40	40 (40)	All accessions were from Taiwan, with primitive traits such as long awn and shattered.
V2	Subtrop jap	35	17 (17)	Most were from Indochina.
V3	Indica	48	4 (1)	Most were from Indian Subcontinent.
V4	Temp jap	87	73 (18)	Most were modern varieties from Taiwan and Japan
V5	Indica	99	9 (2)	Most were from Indochina and insular southeast Asia.
V6	Indica	55	44 (5)	Most were from indigenous and Mingching of Taiwan
V7	Trop jap	72	16 (12)	Most were from insular southeast Asia.
V8	Indica	65	61 (34)	Most were from indigenous and Mingching of Taiwan
V9	Wild rice	14	1	Wild rice collected in Asia

*Accessions numbers collected from Taiwan, with the indigenous ones in brackets.

To summarize: the indigenous temperate japonica accessions were grouped into V1 and V4, with the primitive types in V1 and modern ones in V4. For the follow-up analysis, V1 and V4 were assayed differently. Indigenous rice accessions in V2 were subtropical japonica and in V7 tropical japonica. For the indica rice, those in V3 were classified with accessions from the Indian subcontinent and V5 with accessions from the mainland- and insular-SEA. It is intriguing to note that those in V6 and V8 were grouped with Taiwan landraces themselves.

We used several early primitive traits such as long awn and red caryopsis to postulate which subtype of rice arrived Taiwan at an early time, that is, indigenous accessions. Table 3 lists the accessions numbers and percentage of these traits. In the indigenous temperate japonica, 4- or 5-fold more accessions have long awns and red caryopsis in V1 versus V4. About half of the indigenous subtropical japonica accessions contained long awns, with none in the tropical japonica lines. In all, 5.9% and 16.7% of the subtropical and tropical japonica accessions had a red caryopsis. Because indica rice arrived from EA and SEA relatively late (Castillo et al. 2016), the early cultivated rice in Taiwan must be the japonica type. From the phenotype of indigenous rice lines, the temperate lines may have arrived the earliest, followed by the subtropical and then tropical lines. However, indica indeed arrived quite late because only 5.1% had long awns. About half (46.2%) of the indigenous indica rice accessions had a red caryopsis, probably because the early indica in nearby regions was still colored rice during that time.

Table 3

Phenotyping information of grain-related traits in Taiwan indigenous rice accessions.
	Long awn	Red caryopsis	Glutenous endosperm
Indigenous
Temperate jap (V1)	23/40 = 57.5%	19/40 = 47.5%	36/40 = 90%
Temperate jap (V4)	2/18 = 11.1%	2/18 = 11.1%	3/18 = 16.7%
Subtropical jap (V2)	9/17 = 52.9%	1/17 = 5.9%	5/17 = 29.4%
Tropical jap (V7)	0/12	2/12 = 16.7%	5/12 = 41.7%
Indica (V3 + V5 + V6 + V8)	2/39 = 5.1%	18/39 = 46.2%	13/39 = 33.3%
Admixture	0/3	1/3 = 33%	0/3
Mingching
Indica (V3 + V5 + V6 + V8)	0/58	6/58 = 10.3%	4/58 = 6.9%
Modern
Admixture	0/1	0/1	0/1
Tropical jap (V7)	0/4	0/4	0/4
Temperate jap (V4)	0/54	0/54	3/54 = 5.6%
Indica	0/16	0/16	5/16 = 31%
Red rice (weedy)
Indica (V6)	0/1	1/1	0/1

Glutinous rice has been important in indigenous villages because it is used to make wine and rice pudding. The wine is important for sacrifice ceremonies as well as at entertainment parties. Thus, we checked the sticky grains: 90%, 16.7%, 29.4%, 41.7% and 33.3% accessions are the glutinous type for temperate V1, temperate V4, subtropical V2, tropical V7 japonica and indica rice, respectively. For comparison, Ming-Ching and modern rice each have much less sticky rice (about or < 10%). Thus, glutinous rice is specifically popular in the indigenous accessions.

Only indica landraces were cultivated in the plain region since the Han people arrived in the early 17th century

The Han people had been living in the plain region since their arrival and most indigenous villages were moved to high mountain regions. All rice accessions grown in the plain region since then were indica rice according to the history book related to Taiwan rice cultivation (DAFTPG 1989; Teng 2003) and sequencing information (Wu et al. 2022), so most, if not all, landraces cultivated in southern China must have been indica rice during the late Ming Dynasty. In a survey of rice accessions preserved by TARI (searchable at the NPGRC website, https://www.npgrc.tari.gov.tw/npgrc1/index_e.html), 54 of 450 of these Ming-Ching lines are glutinous and all others are wild type. In addition, 19 of the 450 lines have a red caryopsis and all others are white, and 2 of these lines have long awns. As compared with the indigenous rice accessions, no or only a small proportion of the rice grains of these lines have long awns or red caryopsis.

One of the most important traits for the Ming-Ching accessions was semidwarf (sd). This trait was used in the breeding of IR8, the miracle rice, and played important roles in indica rice breeding worldwide since the 1960s (Khush 1995, 1999). This trait came from the Dee-Geo-Woo-Gen (DGWG) sd1 allele, one of the Ming-Ching accessions. The mutation was caused by a 383-bp deletion in the gene GA₂₀oxidase-2 (Os01t0883800) (Sasaki et al. 2002), which led to the abolishment of this GA₂₀ oxidase function. We checked all Ming-Ching accessions and found that in addition to DGWG, another 4 accessions also contained the sd1-DGWG allele: Hsinchu-Ai-Chueh-Chien, Ti-Chueh-Wu-Ko, Ai-Tzu-Chung, and Liu-Tou-Tzu.

Major modern rice accessions were japonica rice due to the taste preference during Japanese colonial times

During the Japanese colonial period (1895 to 1945), Taiwan rice cultivation had gradually shifted to temperate japonica accessions. According to several reviews, including DAFTPG (1989) and Teng (2003), this huge change was due to the taste preference of Japanese people. All the cultivated accessions in the plain region before 1920 were indica type and there were few japonica types since 1925, with the percentage of indica and japonica being 87.5% and 12.5%, respectively. The earliest japonica variety was Taichung 65, which was designated in 1929 (Iso 1944; Wei et al. 2016b). The percentage of indica type then gradually decreased to 32.9% in 1944. There was a small increase during 1945 and 1946, with the ratio being 47.1% and 65.4%, respectively. The indica rice proportion then gradually decreased again (Teng 2003) and has been less than 10% in the recent decade (data from the Council of Agriculture, Taiwan). By using the sequencing information for new accessions since breeding was applied to rice cultivation, only 4 and 12 breeding lines and new varieties, respectively, for indica rice, versus 13 and 46, respectively, for japonica rice (Table S2). Thus, japonica varieties gained more attention in the breeding programs.

The sd1 trait has been used in more than 90% of the modern rice varieties worldwide. This DGWG 383-bp deletion was present in all Taiwan modern indica varieties as well as the weedy rice tested. We also checked its presence in the modern japonica varieties. Taikeng 9 had an indica type (IR5470) as one of its parental lines; however, sequence analysis revealed that it did not contain this mutation in the SD1 locus. The same is true for all other japonica varieties without indica rice in their pedigree. Thus, even though the DGWG sd1 allele has been used in most indica and some japonica varieties worldwide, it was not present in any modern Taiwanese japonica variety.

Changes in phenotypes related to stress tolerance

Previously, we established a resource for rice genome-wide association study with about 500 accessions of selected upland rice and landraces from Taiwan and Asia, along with some modern varieties (Wu et al. 2022). We performed phenotyping studies of seedlings including study of resistance to flooding, drought and abscisic acid (ABA) treatments. Together, information for 19 phenotypes was obtained, including 1) drought survival rate after 25% PEG treatment (severe osmotic stress), 2) ratio of shoot length after flooding treatment for 7 days (compared with control), 3) ratio of root length after flooding treatment for 7 days (compared with control), 4) ratio of total root length after 0.5 µM ABA treatment (compared with control), 5) ratio of crown root length after 0.5 µM ABA treatment (compared with control), 6) ratio of primary root length after 0.5 µM ABA treatment (compared with control), 7) ratio of crown root number after 0.5 µM ABA treatment (compared with control), 8) root length under the control condition, 9) root length after 7 day flooding treatment, 10) shoot length under the control condition, 11) shoot length after 7 day flooding treatment, 12) total root length under the control condition, 13) total root length after 0.5 µM ABA treatment, 14) crown root length under the control condition, 15) crown root length after 0.5 µM ABA treatment, 16) primary root length under the control condition, 17) primary root length after 0.5 µM ABA treatment, 18) crown root number under the control condition, and 19) crown root number after 0.5 µM ABA treatment. Figure 2 illustrates the phenotype histograms for the japonica and indica populations under stress conditions. Panels A and B show the root length response to 7-day flooding treatment, and C and D show the survival rate after 3-day 25% PEG treatment. The red arrows indicate the position of control varieties Tainung 67 (japonica) or Taichung Native 1 (indica). Two kinds of landraces were included: Ming-Ching and indigenous ones. The results of both modern varieties or landraces was normal distribution for most phenotypes checked. For the drought-resistant trait of indica rice accessions, the distribution was skewed toward more resistance. Even though some landraces showed higher resistance to the stress treatments, some modern varieties also provided similar protection.

Supplementary Fig. 1 panels A to AL illustrate the other histograms for the 19 phenotypes. All show a similar trend, that is, normal distribution; some landraces and a few modern varieties feature resistant phenotypes.

Changes in genetic diversity

We used phylogenetic, structure and principle component analyses to show the diversity and classification of the 500 accessions (Wu et al. 2022). The Taiwanese accessions were grouped into 9 sessions with K = 9 for the structure analysis, as shown in the classification section.

We explored the genetic diversity among these groups by checking for the existence of positive selection of 20 domestication- or adaptation-related genes (listed in Table S3 with gene locus information and references). We calculated selection parameters, including π (Tajima 1983), θw (Watterson 1975), as well as Tajima’s D (Tajima 1989), to test the neutral mutation hypothesis. The nearby region (± 1 Mb) of each gene were used for the calculation and the results are listed in Table 4 and Table S4. The genes of a specific group with significant selection are listed and the information illustrated huge differences among each group. For instance, 19 genes were under selection for Taiwanese V4 group, so only TGW6 was not under selection for the modern Taiwan temperate japonica rice accessions. For other groups, only a few genes showed significant selection; they are Gn1, qSH1 and qSW5 genes for V1; Bh4, Lg1, OsC1, Prog1 and TGW6 genes for V2; RAE2 gene for V6; Prog1 and qSH1 genes for V7; and An1, An2, Bh4, Edh1 and Waxy genes for V8. There was no significant selection for V3 and V5 among the 20 genes tested. Both groups include relatively primitive indica accessions in Taiwan. Thus, the genetic diversity has changed during cultivation and differs according to population.

Table 4

Selection swept analysis of some domestication- or adaption-related genes of Taiwan rice accessions. The gene region and the nearby ± 1 Mb were used for the calculation. Only significant traits/groups are listed.
Gene	Group classification	Number of sequences	π	θw	Tajima's D
An1	V4	73	0.07522	0.20383	-2.21879**
An1	V8	61	0.10125	0.21108	-1.86562*
An2	V4	73	0.03095	0.20471	-2.98748***
An2	V8	61	0.04019	0.21324	-2.90897***
Bh4	V2	17	0.10986	0.24517	-2.39274***
Bh4	V4	73	0.04551	0.20470	-2.73676***
Bh4	V8	61	0.08955	0.21322	-2.07901*
Cold1	V4	73	0.06909	0.20456	-2.33085**
Ehd1	V4	73	0.03862	0.20563	-2.85917***
Ehd1	V8	61	0.05927	0.21174	-2.58027***
Gn1	V1	40	0.06544	0.13948	-1.99547*
Gn1	V4	73	0.04106	0.20555	-2.81393***
Hd3a	V4	73	0.05331	0.20573	-2.60790***
IPA1	V4	73	0.08698	0.20552	-2.02404*
Lg1	V2	17	0.10085	0.23332	-2.46130***
Lg1	V4	73	0.04990	0.20573	-2.66412***
OsC1	V2	17	0.14947	0.26621	-1.90065*
OsC1	V4	73	0.05047	0.20573	-2.65666***
OsLg1	V4	73	0.07352	0.20399	-2.25074**
Phr1	V4	73	0.09719	0.20335	-1.83741*
Prog1	V2	17	0.11562	0.27046	-2.48232***
Prog1	V4	73	0.07806	0.20573	-2.17197**
Prog1	V7	16	0.14808	0.30137	-2.23620**
qSh1	V1	40	0.05931	0.23421	-2.81371***
qSh1	V4	73	0.03236	0.20561	-2.96598***
qSh1	V7	16	0.15545	0.29526	-2.08093*
qSW5	V1	40	0.10905	0.23309	-2.00318*
qSW5	V4	73	0.07536	0.2057	-2.23050**
RAE2	V4	73	0.04642	0.20541	-2.71068***
RAE2	V7	44	0.04697	0.16912	-2.68728***
Rc	V4	73	0.06071	0.20573	-2.48110**
TGW6	V2	17	0.15465	0.2954	-2.06686*
TT1	V4	73	0.03979	0.20409	-2.83281***
Wx	V4	73	0.03674	0.20573	-2.89097***
Wx	V8	61	0.09809	0.21346	-1.93567*

Heading date 1 allele analysis showed that some indigenous rice accessions came from nearby regions

Rice is a short-day plant and was domesticated in China (a temperate zone), then was brought to subtropical and tropical zones with warmer temperature and different photoperiods. The early rice plants that grew in a temperate region fitted the daylength atmosphere very well: they flowered (also known as heading) during early autumn and were ready to be harvested about 40 days later. However, when the plants were brought to the southern region with short daylength, the reduced vegetative growth period before heading would lead to decreased yield. Many reviews provided detailed information on the regulation of rice flowering and production (e.g., Itoh and Izawa 2013; Lee and An 2015; Tsuji et al. 2013). Mutations leading to the null function of sensitivity-to-photoperiod genes would increase crop yield because rice could grow in 2 or 3 seasons instead of only one each year and also reduce stress damage caused by seasonal typhoons, monsoons or drought. Thus, such a trait could be selected out in subtropical and tropical regions.

Cultivated rice varieties and landraces exhibited large variation in flowering time, so rice heading behavior was controlled by quantitative trait loci (QTL). For instance, by using the progeny derived from a single cross between one japonica (Nipponbare) and one aus (Kasalath) line, researchers identified 15 QTL for the Heading date (Hd) trait (Yano et al. 2000). Hd1 was one of the most important loci to control rice flowering time and was identified as an Arabidopsis CO ortholog (Yano et al. 2000). By using the information from many local accessions (Takahashi et al. 2009) and the rice 3K project information (Wu et al. 2020a), about 10 Hd1 loss-of-function (LOF) alleles were identified (Yano et al. 2000; Takahashi et al. 2009; Wu et al. 2020a). The rice accessions with any of these LOF hd1 alleles would not be sensitive to photoperiod and thus could flower and mature after proper vegetative growth. Many landraces and most modern varieties in subtropical and tropical regions contained these alleles because they could adapt to the environment well and have high yield. Phylogenetic and haplotype network analysis of several of these alleles revealed that type 7 hd1 LOF mutation occurred in indica rice in insular areas in SEA, followed by introgression and expansion (i.e., brought by human beings) to nearby regions including the Indochina area and Indian subcontinent (Wu et al. 2020a). With a similar strategy and dataset, the results also suggested that type 13 mutation occurred in japonica rice in insular areas in SEA, followed by introgression and expansion of both japonica and indica accessions to nearby regions. In addition, some other hd1 LOF alleles were specific to local regions: type 3 was indica-specific and mainly from China, and type 19 was japonica-specific and mainly from Taiwan (Wu et al. 2020a).

Data mining analysis of the 129 Taiwan indigenous rice accessions in the current study indicated that 6 contained type 13 hd1 alleles, including 5 japonica accessions and one indica. In addition, 5 indica accessions had type 7 alleles, 14 japonica accessions had the type 19 hd1 allele, and 3 indica accessions had the type 3 allele. Thus, the variations in the hd1 allele type revealed that some of the early rice cultivated in Taiwan came from China and mainland or insular SEA. There must have been intensive exchanges of rice accessions in Taiwan with the nearby regions a long time ago.

In the current study, we performed a detailed analysis of the population structure changes in rice in Taiwan over thousands of years. Multidiscipline strategies including archaeological, morphological, genetic and genomic approaches were used. The materials included carbonized seeds excavated from central or southern Taiwan from 5000 years ago and those cultivated in indigenous villages or plain regions recently. We discuss which rice types arrived Taiwan in ancient times, any exchange of rice lines with nearby regions a long time ago, why there were major changes in the rice population in the last century as well as how domestication- and adaptation-related genes changed in different populations. Finally, we showed factors that have shaped the population structure of Taiwan rice accessions over these years.

Traditional Taiwan cereals were present 5000 years ago

Rice, foxtail millet and proso millet grains were excavated in southern Taiwan around 5000 BP in large quantity (Tsang et al. 2017). Multidiscipline analyses on indigenous habitat, linguistic divisions, archaeological remains, etc. were applied to search the early origin of Austronesian. Many studies proposed that the Formosan peoples and culture traits came from southern China by using archaeological data (Ferrell 1966; Bellwood 1997; Chang 1989; Deng et al. 2022; Tsang 2005) or language (Bellwood 1979; Ferrell 1969).

Alternatively, our recent study provided difference hypothesis. We performed a multidisciplinary analysis from the viewpoint of archaeology, linguistics, and genome sequence as well as seed morphology to reveal early agriculture in Taiwan (Sagart et al. 2018). By using sequence information for several domestication-related genes, we found that the functional nucleotide polymorphisms of indigenous rice lines were the same as modern japonica and indica rice varieties elsewhere in the world. Thus, the early rice that arrived in Taiwan long time ago was already domesticated and was japonica (no indica rice in eastern Asia yet during that period). Indica rice arrived in Taiwan rather late, with some modern traits. With the botanically informed linguistic fieldwork of the agricultural vocabulary of indigenous villages, along with the earlier findings in archaeology, genetics and historical linguistics, early Taiwan agriculture was found to be based on foxtail millet, broomcorn millet and rice (Hsieh et al. 2011; Tsang et al. 2017). Together, we proposed the pre-Austronesians expanded south along the coast from Northern China 5000 BCE to reach northwest Taiwan in the second half of the 4th millennium BP (Sagart et al. 2018).

The archaeological studies indicated that there were continuous rice cultivations in Taiwan from about 5000 years ago to the present time. By using 100 carbonized rice seeds excavated from 4 different sites with a time span from 5000 to 500 BP in southern Taiwan and 1300 BP in central Taiwan, we showed that rice seed size had changed over thousands of years, from a relatively small and round shape to a large and oblonga shape (Fig. 1). Previously we described the seed length, width and grain length/width (l/w) ratio of carbonized rice grains excavated from 18 sites in southern Taiwan (Table 1, Hsu et al 2019. The time span ranged from 5000 BP to 300 BP, and the seed size also showed a huge change during the ~ 5000 years: seed length average 4.72 mm, width 2.52, and l/w ratio 1.88. This analysis again demonstrated that the seeds were small and round before 3000 BP and then became large and long later on. By using the excavated rice seed morphology, there must have been movement of rice between Taiwan and the nearby regions for thousands of years. Using the seed size information, the large seeds might be from SEA, such as Funan, Langkasuka, Salakanagara, Tarumanagara or Champa during that time.

Studies of excavated rice grain indicated the changes in early cultivated rice of Taiwan

In the current study, we measured the grain size of hundreds of carbonized rice seeds excavated from 4 sites in central or southern Taiwan (Fig. 1, Table S5). Most rice grains recovered from excavated sites worldwide were preserved by carbonization, a process in which the organic structure was converted into inorganic carbon by heating (Wright 2003). The high temperature could lead to warping, shrinking or fracturing, although the seeds would not be destroyed by microbe decay (Wright 2003). This carbonization process could affect the length and width of rice grains (Ahn 1993). To differentiate japonica or indica carbonized rice seeds, the l/w ratio was used in most studies. Some researchers assume a uniform 20% shrinkage rate for the l/w ratio in the studies of all archaeological carbonized seeds (e.g., Fuller et al 2008; Fuller et al 2009; Harvey 2007.

Table S5 illustrates the average, minimum and maximum values of seed length, width and l/w values for NKLE, YHF, WCT and HLL. Using the 20% shrinkage rate, the mean l/w values were 1.98, 2.29, 2.57, and 2.70 for the 4 sites. Information on many agronomic traits, including seed length and width, from the 3K rice project is available from the Rice SNP-Seek website of IRRI (https://snp-seek.irri.org/). Using the traditional accessions (1048 lines) only, the l/w ratios were 3.05, 2.61, 3.31, 2.80, 3.06, 2.90, 2.17, 2.64 and 2.64 for aromatic, aus, ind1A, ind1B, ind2, ind3, temperate japonica, tropical japonica and subtropical japonica, respectively. Thus, the very early rice lines that may have been cultivated in Taiwan before 3800 BP (NKLE and YHF) were temperate japonica type. Since about 1500 BP (WCT and HLL), there were subtropical and tropical japonica as well as indica rice types according to the higher l/w values. This hypothesis coincided with the classification of the indigenous upland rice accessions: some primitive temperate japonica (V1 group) belong to the earliest type that arrived in Taiwan a long time ago.

Complicated exchange with nearby regions before the late Ming Dynasty

Many ornaments and tools excavated from eastern Taiwan since ~ 3000 BP were made of Fengtian jade (Hung et al. 2007). More than 100 sites dating from the early Neolithic to the Iron Age revealed an intensive jade production/industry in eastern Taiwan during that period. Electron probe microanalysis revealed that Fengtian jade was also present in excavated sites in the Philippines, Indonesia, Vietnam, Cambodia, Thailand, Malaysia and southern China (Hung et al. 2007; Hung et al. 2013; Alam et al. 2021). Therefore, there was frequent cultural contact between Taiwan and the insular areas as well as mainland SEA. With the extensive sea-based trade networks in the prehistoric world, rice seeds would accompany other crops.

The route of early japonica rice dispersal to Taiwan, the Philippines and other SEA areas was reconstructed using the whole-genome resequencing of landraces (Alam et al. 2021). The japonica component of the Taiwanese indigenous rice accessions consisted of two distinct populations, including a result of admixture between temperate japonica that presumably came from northeast Asia and tropical japonica from the northern Philippines and mainland SEA (Alam et al 2021). In the current study, we also illustrated that some indigenous rice lines with different hd1 LOF alleles were brought to Taiwan from mainland and insular SEA as well as China during the early time. Thus, there was a complicated exchange in Taiwan rice accessions with nearby regions before the late Ming Dynasty (arrival of the Han people).

Rice landraces in Taiwan provide good genetic resources for future breeding

In the current study, some of the Taiwan rice landraces, including indigenous and Ming-Ching ones, are highly resistant to abiotic stresses such as drought or flooding (Fig. 2 and Figure S1), specifically the indigenous rice accessions that were cultivated in an upland practice at higher altitudes for thousands of years. In a genome-wide study of Asian japonica landraces, several selection sweeps occurred across 12 chromosomes. The one in the long arm of chromosome 1 was due to some Taiwan indigenous rice lines, and the peak coincided with genes associated with UV tolerance (Alam et al. 2021). Therefore, these lines were resistant to several stress treatments, and they all contained good traits ready for breeding.

Climate change in recent years has caused serious problems worldwide, including to agricultural production. Problems include flooding, drought, heat, chilling, high UV, etc. Taiwanese indigenous peoples and traditional farmers have kept the rice landraces for hundreds and up to thousands of years. With many old traits preserved, they are good resources for future breeding programs.

Nan-kuan-li East, NKLE; Youhsienfang, YHF; Wuchiantsuo, WCT; Huilaili, HLL; Taiwan Agricultural Research Institute, TARI; National Plant Genetic Resources Center, NPGRC; southeast Asia, SEA; Dee Geo Woo Gen, DGWG

Acknowledgements

We would like to acknowledge our gratitude to the Taiwanese indigenous peoples and traditional farmers for their stewardship of traditional rice landraces. We thank Ms. Lie-Hong Wu for maintaining greenhouse plants. We also thank Laura Smales (BioMedEditing, Toronto, Canada) for English editing.

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 THDH, SMY and YIH.

Data Availability

The data underlying this article are available in the NCBI Short Read Archive (SRA) database (project accessions nos. PRJNA485658, PRJNA373799, PRJNA623980, and PRJEB6180).

Authors’ contributions

YICH conceived and designed the study, CCW, CKL and LTH generated sequencing data. YHW, YCT, TFH and YTT analyzed the carbonized grains. YCT, NCD, JCL, DPS, CWW, MHL, DHW, SC, YPW and SJC collected rice accessions. CCW, CKL, FJW performed bioinformatics analysis. CHT, KTL, WLC provided archaeological materials. THDH, SMY and LS participated in frequent discussions. YICH wrote the manuscript with input from all authors.

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No competing interests reported.

TableS1excavatedsite.pdf
Additional file 1: Table S1. The Chinese translation of the excavated sites and cultures used in the current study.
TableS2accessions.xlsx
Additional file 2: Table S2. Information about samples used in this study, including names, accession numbers of DNA, seed subtype information, and clustering results.
TableS3genes.xlsx
Additional file 3: Table S3. The 21 domestication- or adaptation-related genes used in the study.
TableS4selectswept.xlsx
Additional file 4: Table S4. Selection swept analysis of some domestication- or adaption-related genes of Taiwan rice accessions. The gene region and the nearby ± 1 Mb were used for the calculation. All traits/groups are listed.
TableS5.pdf
Additional file 5: Table S5. The size distribution of carbonated rice grain from the 4 excavated sites
FigureS1.pdf
Additional file 6: Figure S1.Seedling phenotype histograms of a japonicaand an indica rice population under control or stress treatments. X-axis means phenotype and Y means % of the accessions used. Panels A and B, primary root length (in cm) of japonica and indica accessions under control condition, respectively; C and D, primary root length of japonica and indica accessions after 7-day flooding treatment, respectively; E and F, shoot length (in cm) of japonica and indicaaccessions under control condition, respectively; G and H, shoot length of japonica and indica accessions after 7-day flooding treatmenton, respectively; I and J, total root length (primary + all crown roots) of japonica and indicaaccessions under control condition, respectively; K and L, total root length of japonica and indica accessions after 7-day flooding treatment, respectively; M and N, total crown root length of japonicaand indica accessions under control condition, respectively; O and P, total crown root length of japonica and indica accessions after 8-day 0.5 μM ABA treatment, respectively; Q and R, primary root length of japonicaand indica accessions under control condition, respectively; S and T, primary root length of japonica and indicaaccessions after 8-day 0.5 μM ABA treatment, respectively; U and V, crown root number of japonica and indica accessions under control condition, respectively; W and X, crown root number of japonica and indicaaccessions after 8-day 0.5 μM ABA treatment, respectively; Y and Z, survival rate (as %) after 3-day 25% PEG treatment of japonica and indicaaccessions, respectively; AA and AB, the ratio of sheet length after7-day flooding treatment (compared with control) of japonica and indicaaccessions, respectively; AC and AD, the ratio (as %) of root length after 7-day flooding treatment (compared with control) of japonica and indicaaccessions, respectively; AE and AF, the ratio (as %) of total root length after 8-day 0.5 μM ABA treatment (compared with control) of japonica and indica accessions, respectively; AG and AH, the ratio (as %) of crown root length after 8-day 0.5 μM ABA treatment (compared with control) of japonica and indica accessions, respectively; AI and AJ, the ratio (as %) of primary root length after 8-day 0.5 μM ABA treatment (compared with control) of japonica and indica accessions, respectively; AK and AL, the ratio (as %) of crown root number after 8-day 0.5 μM ABA treatment (compared with control) of japonica and indica accessions, respectively. Blue means aboriginal rice accessions, green means Mingching landraces and orange modern varieties. Arrow indicate the positions of control variety Tainung67 or Taichung Native 1 for japonicaor indica accessions, respectively.

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Population structure dynamics of Taiwan rice accessions over thousands of years as revealed by archaeological, morphological and genome sequencing information

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Abstract

Background

Results

Conclusion

Figures

Introduction

Materials and Methods

Archaeological materials

Plant materials and growth condition

Whole genome sequencing and single nucleotide polymorphism (SNP) calling

Scoring of grain-related traits

Estimation of diversity of domestication- or adaptation-related genes

Results

Continuous rice cultivation in Taiwan from 5000 years ago

Total of 265 accessions were used in the current genomic study

Classification of the indigenous upland rice accessions

Changes in phenotypes related to stress tolerance

Changes in genetic diversity

Discussion

Traditional Taiwan cereals were present 5000 years ago

Studies of excavated rice grain indicated the changes in early cultivated rice of Taiwan

Complicated exchange with nearby regions before the late Ming Dynasty

Rice landraces in Taiwan provide good genetic resources for future breeding

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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