Evaluation of Genetic Diversity of Rice Landraces (Oryza Sativa L.) in Yunnan, China

Breeding Science 57 : 91–99 (2007)

Evaluation of Genetic Diversity of Rice Landraces (Oryza sativa L.) in Yunnan, China

Yawen Zeng*1,2), Hongliang Zhang§3), Zichao Li*3), Shiquan Shen2), Jianli Sun3), Meixing Wang3), Dengqun Liao3), Xia Liu3), Xiangkun Wang3), Fenghui Xiao*1) and Guosong Wen1)

1) Key Laboratory of Agricultural Biodiversity for Plant Disease Management of Ministry of Education, Yunnan Agricultural University, 650201 Kunming, China 2) Biotechnology and Genetic Resources Institute, Yunnan Academy of Agricultural Sciences, 650205 Kunming, China 3) College of Agronomy and Biotechnology, China Agricultural University, 100094 Beijing, China

Genetic diversity and geographic distribution of rice landraces in Yunnan, Southwest China were investigated based on 31 morphological traits (including Ding and Cheng’s classification traits) using 6,121 accessions, 41 morphological traits and 12 polymorphic isozyme loci within the primary core collection of 912 accessions, and 20 microsatellite markers within the core collection of 692 accessions. Yunnan is the richest center of genetic diversity of rice (O. sativa L.) germplasm in China, in which indica varieties were derived from 108 counties in 16 prefectures and japonica varieties from 99 counties in 17 prefectures. Geographic distribution and diversity of six ecogroups and classification traits displayed clear differences. The average diversity indices of six ecogroups of rice landraces in Yunnan ranked as follows: javanica (1.2319), aman (1.1738), communis (1.1726), nuda (1.1618), aus (1.1371) and boro (0.9889), and the percentages were 3.6%, 43.9%, 32.1%, 18.1%, 2.1% and 0.2%, respectively. Lincang, Simao, Xishuangbanna and Dehong prefectures form the genetic and gene diversity center of rice landraces in Yunnan, especially Lincang Prefecture is not only the main genetic diversity center of rice landraces in Yunnan but also the diversity center of Ding’s and Cheng’s classification traits. South marginal paddy-upland rice region with Myanmar is the center of the gene diversity of rice landraces in Yunnan. A core collection from Yunnan rice landraces was identified based on morphological, isozyme and DNA variations, which have confirmed that Yunnan is the center of genetic differentiation of indica and japonica subspecies of Asian cultivated rice.

Key Words: genetic diversity, core collection, evaluation, distribution, rice landraces, Yunnan.

Introduction indica and japonica subspecies are very apparent. Variation in spikelet shape of indica and japonica rice cultivars of Genetic diversity is a ubiquitous property of all species Asian origin, and indica/japonica non-random association in in nature. The ‘Green Revolution’ has remarkably increased characters and genes reflect the independent origin of the crop productivity over the past four decades (Mann 1997). two types from different wild ancestors (Sato 1991, 1996). However, this agricultural transformation has also resulted The indica line of rice varieties evolved from the annual in problems, including the loss of crop genetic diversity gene pool of the AA genome and the japonica varieties from (Tilman 1998). Narrow genetic base in current rice cultivars the perennial gene pool of the AA genome of wild rice continues to limit the productivity of rice which has been (Yamanaka et al. 2003). The O. sativa rice accessions cultivated for more than 9000 years and is a staple food for sampled show a significant differentiation into five groups: over 50% of the human population. However, there is a re- aromatic, aus, indica, temperate japonica and tropical markably high diversity in cultivated rice. Rice landraces es- japonica (Amanda et al. 2005). China is the center of ori- pecially in the core collections from the diversity center are gin and genetic diversity of japonica and O. rufipogon, and important reservoirs of useful genes and can be exploited to one of the two centers of origin of indica rice (Chang 1976, both broaden the existing narrow genetic base and enrich the Oka 1988, Li and Rutger 2000, Li 2001, Gao et al. 2000, existing varieties with important favorable agronomic traits. Zeng et al. 2001b, 2003). At all the levels of analysis, the differences between the Yunnan is the center of genetic diversity of O. sativa in China, and indica-japonica differentiation at the isozyme Communicated by Y. Sato and RFLP levels has been well documented (Nakagahra Received January 27, 2003. Accepted October 30, 2006. 1978, Nagamine 1992, Ise et al. 2000, Li 2001, Zeng et al. § These authors contributed equally to this work 2003). Ise et al. screened 581 varieties from Yunnan (n = *Corresponding author (e-mail: [email protected]) 376) and Japan (n = 205) for the endosperm amylose content, 92 Zeng, Zhang, Li, Shen, Sun, Wang, Liao, Liu, Wang, Xiao and Wen and Yunnan rice showed a 1.7 time larger variation than Jap- cultural Sciences 1986). The statistical methods (diversity anese rice, based on the Shannon-Wiener index (http:// indices, etc.) and 60 characters of 6,121 accessions had been www.jircas.affrc.go.jp/english/publication/news/2000/No.23/ previously described in detail (Zeng et al. 2000a, 2003). 06Ise.htm). Nagamine (1992) suggested that Southwest One hundred plants of each accession from the primary Yunnan is the center of isozyme genetic variation. Yunnan core collection of 912 accessions, which accounted for 98% rice landraces can be divided into 58 variety types, which of the diversity of a total of 6,121 landraces in Yunnan (Zeng correspond to most of the types found in China, and the et al. 2002, http://www.zgzzkj.org, Li et al. 2002) were cultivars account for 8.6% of the total cultivars in China grown in five rows in a field of the experimental farm locat- (Zeng et al. 2003). It was revealed that Yunnan is the richest ed in Xingping county (500 m asl). Mid-maturity rice was center of genetic and gene diversity of O. sativa among the planted in the summer of 1999. The morphological diversity 29 provinces of China, based on the analysis of the genetic and genetic differentiation index of 41 traits (18 qualitative diversity distribution of China rice cultivars at the provincial and 23 quantitative traits) among the 5 ecological zones or 17 level, using 26 phenotypic characters of 50,526 accessions prefectures were calculated by the following two formulas from China rice germplasm collections, nine polymorphic using Foxpro software and the VB system: I = −ΣPijLogPij , th th isozyme loci from the core collection of 5,181 accessions, where Pij is the frequency of the j phenotype of the i and 36 microsatellite markers from the core collection of character, and I is the genetic diversity index (King et al. 4,300 accessions (Li 2001, Zhang et al. 2007). The present 1989); Dr = (It − Ia)/It or Dr = (CVt − CVa)/CVt, where Dr is the study on the diversity of rice landraces in Yunnan will sup- differentiation coefficient of morphological characters be- ply a basis for further investigations on the genetic diversity tween populations, It is the total genetic diversity index, Ia is of rice landraces in China and the world as well as for a bet- the average diversity index, CVt is the total diversity index ter utilization, conservation and management of O. sativa. for a certain character, and CVa is the average diversity index resources. for a certain character between populations (Nei 1977). Gene diversity index and coefficient of genetic differ- Materials and Methods entiation of 912 accessions and 692 accessions from core collections among ecological zones and subspecies were es- A total of 6,121 accessions of rice landraces with 60 char- timated, respectively, using 12 isozyme loci and 20 pairs of acters defined and evaluated were collected from the Seed SSRs. The gene diversity index was expressed as follows: H 2 Bank of the Yunnan Academy of Agricultural Sciences, = 1 − ΣPij /N, where N is the number of loci, and Pij is the fre- covering 109 counties in 17 prefectures in Yunnan, China. quency of the ith allele at the jth locus (Nei 1973). The coeffi- In order to characterize and evaluate the related traits, all cient of genetic differentiation was as follows: Gst = (Ht − Ha)/ the 912 accessions of Yunnan rice landraces were sowed in Ht, where Ht corresponds to the total gene diversity indices the middle season of rice cultivated (e.g. June 29 in 1999) in within populations, Ha is the average gene diversity index Xinping county, Yunnan (550 m above the sea level). The for each population (Nei 1977). The detection methods of subspecies (indica or japonica) and ecogroups (javanica, isozyme and SSR polymorphisms were described separately nuda, communis, aus, aman and boro) were classified ac- by Li et al. (2001), Oka (1988) and Temnykh et al. (2000). cording to the method of Cheng (1993) and Zeng et al. Twelve isozyme loci included Est-1 (0, 1, 2, 3), Est-2 (0, 1, (2001a). Indica was classified into boro, aus and aman, 2), Est-10 (0, 1, 2, 3, 4), Cat-1 (1, 2), Amp-2 (1, 2), Acp-2 (0, which boro and aus were mainly distributed below the ele- x, 1), Acp-1 (y, 1, 2), Mal-1 (1, 2), Mal-3 (y, 1, 2), Mal-x (1, vation of 800 m, but aman below the elevation of 1,200 m. 2), Pgd-1 (1, 2, 3) and Pg-2 (1, 2). Twenty SSR primers with Boro heads during the period of 5–12 September, aus before a high polymorphism that were allocated to 12 chromo- September 10 and aman after September 10. Generally somes included RM5, RM81A, RM263, RM211, RM60, speaking, japonica rice were mainly distributed over the el- RM232, RM255, RM241, RM249, RM225, RM253, RM18, evation of 1,850 m, but japonica varieties distributed below RM234, RM223, RM257, RM258, RM244, RM224, the elevation of 1000 m were javanica. Among the japonica RM247 and RM235. subspecies, except for nuda and javanica, the other cultivars belonged to communis ecogroup (Zeng et al. 2001a). This Results classification was further confirmed based on the results of the photo-thermal response observed in Nanjing, Jiangsu Geographic distribution of classification traits of rice land- Province, and of the maturation period of rice in Xinping races in Yunnan county (550 m asl) in 1999–2000 (Zeng et al. 2002). Ding’s The 6,121 accessions were collected over a wide range classification system (1959) included two subspecies (indica of 109 counties in 17 prefectures in Yunnan Province, in or japonica) and six varieties (early-mid or late, paddy or which the indica accessions were derived from 108 counties upland, glutinous or non-glutinous). Other 50 traits were ad- in 16 prefectures and the japonica ones from 99 counties in justed or standardized according to ‘Characters to be studied 17 prefectures. The percentages of javanica (from 24 coun- and standards for rating of rice genetic resources’ (Institute ties), nuda (from 77 counties), communis (from 83 counties), of Crop Germplasm Research of Chinese Academy of Agri- aus (from 12 counties), aman (from 84 counties) and boro Genetic diversity of rice landraces 93

(from 5 counties) were 3.6%, 18.1%, 32.1%, 2.1%, 43.9% groups of japonica subspecies, with high diversity indices and 0.2%, respectively (Zeng et al. 2001a). Javanica culti- up to 1.2 to 1.5. Simao, Honghe, Wenshan and Zhaotong vars were widely adapted to tropical and subtropical envi- prefectures were the centers of aus groups in the indica sub- ronments (Jiang et al. 2003). Geographic distribution of species, with indices up to 0.9 to 1.1. Xishuangbanna, classification traits of rice landraces in Yunnan are listed in Dehong and Lincang prefectures were the centers of aman, Table 1, and the rich areas in terms of classification traits with indices of 1.2 to 1.4. Dehong prefecture displayed the were found in 8 counties. Mojiang county in Simao prefec- highest diversity index for boro. The center of genetic diver- ture was a very rich area in terms of Ding’s classification sity determined based on Ding’s classification traits covered traits (including early-mid rice, paddy rice, upland rice, non- 3–6 prefectures (Table 2). The genetic diversity index of glutinous and glutinous rice) and subspecies. This was partly Cheng’s and Ding’s classification traits within and among related to the terrace culture where the Hani population grows subspecies at the prefecture level revealed that the landraces various rice landraces. Menghai county in Xishuangbanna from Lincang and Dehong prefectures exhibited the highest prefecture was an enriched area for upland rice landraces. genetic diversity. Zhenkang county in Lincang prefecture was the distribution center of javanica. Nuda accsessions, mostly consisting of Diversity and its geographic distribution patterns upland rice, were grown mainly in the Simao, Xishuangbanna Geographic distribution patterns of genetic and gene and Lincang prefectures, Southwest Yunnan. Mojiang diversity at the phenotypic, isozyme and SSR levels are county in Simao prefecture was rich in communis, japonica shown in Table 3. The difference in diversity of Yunnan and aman accessions. Zhenxiong and Weixin counties in landraces among the prefectures was relatively large. The Zhaotong prefecture were enriched areas for aus. Boro acces- highest average genetic diversity indices (It 1.2930-1.238, sions were mainly distributed in Ruili county in Dehong pre- Ia 0.3985-0.3672) of rice landraces estimated using 31 fecture. characters in 6,121 accessions and 18 qualitative traits in 912 accessions were detected in Southwest Yunnan (Lincang, Genetic diversity of classification traits of rice landraces in Xishuangbanna, Dehong and Simao prefectures). However, Yunnan the landraces of 912 accessions from the core collection The genetic diversiy of 6,121 accessions at the levels of displayed a lower diversity in terms of 23 quantitative traits classification traits was calculated separately using 31 char- such as grain size. The increasing diversity of the grain size acters (Table 2). The genetic diversity indices of the land- during varietal differentiation appeared to have resulted from races for every classification trait varied considerably. The speciation or human selection in each geographical region results revealed that the average diversity indices of the six (Uga et al. 2003). Wenshan, Baoshan, Yuxi, Zhaotong, ecogroups of rice landraces in Yunnan could be ranked as Chuxiong, Honghe, Nujiang and Qujing prefectures showed follows: javanica (1.2319), aman (1.1738), communis the second highest diversity indices (It 1.2211-1.0750, Ia (1.1726), nuda (1.1618), aus (1.1371) and boro (0.9889). 0.3613-0.2707), while Lijiang, Kunming, Dongchuan and Lincang was the richest center of genetic diversity in 17 pre- Diqing prefectures, the lowest (It 1.0174-0.8114, Ia 0.2686- fectures, especially for the javanica, nuda and communis 0.1395). The average gene diversity indices of rice landraces

Table 1. Geographic distribution of classification traits of rice landraces in Yunnan Lowest Low Middle High Highest (enriched area) D% C D% C D% C D% C D% Counties Indica 0–2 93 2–4 11 4–6 2 7–8 1 8–9 Mojiang Japonica 0–1 76 1–2 9 2–4 9 4–6* 2* 6–7 Mojiang Javanica 0–5 17 5–10 5 10–15 1 15–25 0 25–30 Zhenkang Nuda 0–3 31 3–6 4 6–9 1 9–12 2 12–15 Menghai, Lancang Communis 0–1 47 1–2 23 2–4 10 4–5 3 6–7 Mojiang Aus 0–2 15 2–3 14 3–6 7 6–9 2 9–12 Zhenxiong, Weixian Aman 0–2 67 2–4 15 4–6 2 6–10 0 10–12 Mojiang Boro 10–20 1 20–30 Ruili, Yujiang Early-mid 0–1 41 1–2 24 2–3 9 3–4 3 4–5 Mojiang, Zhenxiong Late rice 0–2 67 2–4 15 4–6 8 6–10 2 10–12 Mojiang Paddy rice 0–1 40 1–2 48 2–4 12 4–6 2 6–8 Mojiang Upland rice 0–2 39 2–6 4 6–8 3 8–10 4 10–12 Menghai Nonglutinous 0–1 78 1–3 14 3–4 1 4–5 4 7–8 Mojiang Glutinous 0–2 64 2–4 12 4–6 2 6–8 1 8–10 Mojiang, Menhai Total rice 0–1 81 1–3 22 3–5 3 5–6 2 7–8 Mojiang Note: D% = (N ÷ T) × 100%, D = rice landrace density in all for counties, N = no. of rice landraces or no. of rice landraces for one classification trait from one county, T = no. of Total rice landraces or no. of rice landraces for one classification trait from Yunnan. 94 Zeng, Zhang, Li, Shen, Sun, Wang, Liao, Liu, Wang, Xiao and Wen

Table 2. Genetic diversity indices for classification traits of rice landraces in Yunnan

Classification Lowest Low Middle High Highest (center) traits IP IP IP IP IPrefectures Japonica 0–0.5 1 0.5–0.6 2 0.6–0.7 6 0.7–0.8 4* 0.8–0.9 Lincang, Dehong, Simao, Wenshan Indica 0–0.3 1 0.3–0.4 1 0.5–0.6 3 0.6–0.7 6 0.7–0.8 Lincang, Dehong, Wenshan, Xishuangbanna, Zhaotong, Honghe Javanica 0–0.3 9 0.3–0.6 1 0.6–0.9 1 0.9–1.2 4 1.3–1.5 Lincang, Dehong Nuda 0–0.6 7 0.6–0.8 1 0.8–1.0 2 1.0–1.2 6 1.2–1.4 Lincang Communis 0–0.7 1 0.7–0.9 1 0.9–1.1 5 1.1–1.3 7 1.3–1.5 Lincang Aus 0–0.2 3 0.2–0.3 4 0.3–0.6 2 0.6–0.9 4 0.9–1.1 Simao, Wenshan, Honghe, Zhaotong Aman 0–0.5 1 0.6–0.8 2 0.8–1.0 2 1.0–1.2 9 1.2–1.4 Lincang, Dehong, Xishuangbanna Boro 0.9–1.2 Dehong, Honghe, Yuxi Early-mid rice 0.6–0.7 1 0.8–0.9 3 0.9–1.0 2 1.0–1.1 5 1.1–1.2 Lincang, Baoshan, Simao, Wenshan, Nujiang, Zhaotong Late rice 0.5–0.7 1 0.7–0.9 1 0.9–1.1 2 1.1–1.3 10 1.3–1.5 Lincang, Dehong, Xishuangbanna Paddy 0.5–0.7 1 0.7–0.9 1 0.9–1.1 2 1.1–1.3 10 1.3–1.5 Lincang, Dehong, Xishuangbanna Upland 0–0.3 5 0.3–0.6 2 0.6–0.9 1 0.9–1.2 4 1.2–1.5 Lincang, Dehong, Xishuangbanna, Wenshan, Honghe Nonglutinous 0.5–0.7 1 0.7–0.9 1 0.9–1.1 2 1.1–1.3 9 1.3–1.5 Lincang, Dehong, Xishuangbanna, Wenshan Glutinous 0–0.3 4 0.3–0.6 3 0.6–0.9 1 0.9–1.2 4 1.2–1.5 Lincang, Dehong, Xishuangbanna, Simao Total rice 0.8–0.9 1 0.9–1.0 2 1.0–1.1 2 1.1–1.2 7* 1.2–1.3 Lincang, Dehong, Simao, Xishuangbanna, Wenshan Note: I = genetic diversity index for Chang’s six ecogroups or Ding’s six classification traits of rice landraces with 30 traits from one prefecture; P = No. of prefectures, *P = I of 7 counties between 1.1–1.2 of total rice landraces, and I of four counties between 0.7–0.8 of japonica, respectively. revealed based on 34 alleles at 12 polymorphic loci among diversity indices of qualitative traits in the five ecological 912 accessions, and by 20 microsatellite primers among 692 zones ranged from 0.2705 (Northeast Yunnan plateau rice accessions, varied considerably in the respective prefectures, region) to 0.4041 (South Yunnan single/double cropping ranging from 0.2396 in Dongchuan prefecture to 0.4860 in rice region). Gene diversity indices among the ecological Dehong prefecture at the isozyme level, and from 0.5778 in zones were similar to each other at the isozyme loci, while at Dali prefecture to 0.7707 in Simao prefecture at the SSR the level of the microsatellite markers, they ranged from level. The landraces from Simao, Dehong, Xishuangbanna 0.6890 (Northeast Yunnan plateau rice region) to 0.7692 and Lincang prefectures showed the highest gene diversity (South marginal paddy-upland rice region). In addition, (Hi 0.4860-0.4702, Hs 0.7706-0.7594). Lincang prefecture among the five ecological zones of Yunnan, the South mar- displayed the highest genetic diversity indices (It = 1.2930; ginal paddy-upland rice region was the richest gene diversity Ia = 0.3985) in terms of 18 qualitative traits of the accessions center of rice landraces in terms of diversity indices for mor- from the core collection) and higher gene diversity indices phological traits (1.2849, especially paddy versus upland (Hi = 0.4602, Hs = 0.7594). Simao prefectures showed higher was 0.6892), isozymes (0.4779) and DNA patterns (0.7692) genetic diversity indices (It = 1.2387; Ia = 0.3826) and the (Fig. 1). highest gene diversity index (Hs = 0.7707). Dehong prefecture also showed higher genetic diversity indices (It = Genetic differentiation of landraces from core collection 1.2582; Ia = 0.3672) and the highest gene diversity index within and among subspecies (Hi = 0.4860). Xishuangbanna prefecture displayed both Genetic differentiation (Dr) of landraces from the core higher genetic diversity indices (It = 1.2712; Ia = 0.3727) and collection at the subspecies level was detected based on the gene diversity indices (Hi = 0.4718, Hs = 0.7623). The results analysis of 41 characters (18 qualitative and 23 quantitative revealed that Lincang, Simao, Dehong and Xishuangbanna traits), 12 isozyme loci and 20 SSR markers. Table 3 shows prefectures was the genetic and gene diversity centers of that indica-japonica differentiation in O. sativa occurred at Yunnan rice landraces at the morphological, isozyme and the phenotypic level because of the adaptation to different SSR levels (see Fig. 1). environments. In 17 prefectures (Dst of 12 isozyme loci In the five ecological zones, the morphological diversi- ranged from 0.9130 to 0.1824), allelic frequencies showed a ty indices (Ia) or variation coefficients (CVa) and the gene di- differentiating between the indica and japonica subspecies. versity indices of 34 alleles at 12 polymorphic loci (Hi) and It is obvious that the low temperature of the japonica areas 20 microsatellite markers (Hs) are listed in Table 4. Genetic at elevations from 1,850 to 2,700 m asl (Dongchuan, Diqing, Genetic diversity of rice landraces 95

Table 3. Geographic pattern of genetic and gene diversity at the phenotypic, isozyme and SSR levels 912 accessions from the primary core collection It of 31 traits of 6121 Hs of 20 microsatellite

Prefectures accessions of rice Ia of 18 qualitative Ia of 23 quantitative 12 isozyme loci primers of 692 accessions landraces from Yunnan from core collections traits traits Hi Gst Lincang 1.2930 0.3985 0.2172 0.4715 0.1454 0.7594 Xishuangbanna 1.2712 0.3727 0.2077 0.4718 0.1298 0.7623 Dehong 1.2582 0.3672 0.1918 0.4860 0.0842 0.7686 Simao 1.2387 0.3826 0.2105 0.4702 0.1824 0.7707 Wenshan 1.2211 0.3557 0.2178 0.4534 0.1736 0.7361 Baoshan 1.1908 0.3613 0.2216 0.4648 0.0557 0.6844 Yuxi 1.1817 0.3610 0.2016 0.4473 0.1467 0.6875 Nujiang 1.1780 0.2707 0.2216 0.4343 0.4644 0.6735 Zhaotong 1.1702 0.3454 0.1905 0.4215 0.0599 0.7122 Honghe 1.1658 0.3441 0.2061 0.4331 0.0417 0.7405 Dali 1.1409 0.3458 0.1905 0.3570 0.0165 0.5778 Chuxiong 1.1220 0.2767 0.2119 0.3875 0.1729 0.6781 Qujing 1.0750 0.2823 0.2126 0.2827 0.1298 0.6501 Lijiang 1.0174 0.2686 0.2311 0.3856 0.1831 0.6917 Kunming 0.9736 0.2546 0.2228 0.4601 0.2091 0.5780 Dongchuan 0.9412 0.1171 0.1080 0.2396 0.9130 – Diqing 0.8114 0.1395 0.1801 0.4017 0.5000 0.6094

Note: It = total genetic diversity indices for 31 phenotypic traits of rice landraces from prefectures in Yunnan; Ia = average diversity index for 18 qualitative/23 quantitative traits in primary core collection from prefectures in Yunnan; Hi = average gene diversity index for 12 isozyme loci in primary core collection from prefectures in Yunnan; Gst = coefficient of genetic differentiation between indica and japonica subspecies in primary core collection from prefectures in Yunnan; Hs = average gene diversity index for 20 microsatellite primers in secondary core collection from prefectures in Yunnan.

species between 1,400 and 1,800 m asl, where 60 counties in the Dali, Honghe, Baoshan, Zhaotong, Dehong, Qujing, Xishuangbanna, Lincang, Yuxi and Chuxiong prefectures were located. Dst values in these 10 prefectures ranged from 0.0599 to 0.1729. In the past, indica rice accounted for 60% of the total production and japonica for 40%. However, recently the production of japonica rice has increased, and presently it accounts for 80% of the total production. In all these cases, the temperature is the major factor for the indica- japonica differentiation in O. sativa at the phenotypic and isozyme levels. The ecological zones for qualitative traits showed a higher genetic differentiation than that of quantitative traits, and indica-japonica differentiation was similar, while gene differentiation among the ecological zones was higher than that of indica-japonica. Indica-japonica differentiation among the prefectures at isozyme level ranged from 0.0165 to 0.9130. Coefficients of genetic differentiation among the 17 prefectures and five ecological zones for the quantitative traits (0.0759, 0.0158) and qualitative traits (0.2357, 0.1082) Fig. 1. Prefectures and ecological zones of rice landraces. I (Central Yunnan single cropping japonica-indica rice region), II (South at the isozyme (0.1242, 0.1256) and SSR (0.0268, 0.0427) Yunnan single/double cropping rice region), III (South mar- levels were relatively higher. However, the coefficients of ginal paddy-upland rice region), IV (Northeast Yunnan plateau genetic differentiation (Dr) between indica and japonica rice region), V (Northwest Yunnan cold highland japonica rice were lower, either at the morphological (0.0288 for quantita- region). tive traits, 0.0271 for qualitative traits), isozyme (0.0603) or DNA (0.0311) levels. It was confirmed that Yunnan is the Nujiang, Kunming and Lijiang prefectures) and the hot-wet center of genetic differentiation for indica and japonica sub- indica areas from 76 to 1,463 m asl (Simao and Wenshan species of Asian cultivated rice. In the subspecies, the Dr prefectures) led to such a differentiation at the isozyme values between paddy and upland rice in the japonica sub- level. There was a mixed belt of indica and japonica subspecies at the isozyme level (0.0054) was lower than that of 96 Zeng, Zhang, Li, Shen, Sun, Wang, Liao, Liu, Wang, Xiao and Wen

Table 4. Genetic diversity among different ecological zones and subspecies Core collections (accessions) 31 traits of 6121 accessions of Yunnan rice landraces (I) 912 692 Ecological zones Average Glutinous 23 CVa 18 Ia 12 Hi 20 Hs and subspecies Early-medium Paddy versus genetic versus Quantitative Qualitative Isoezyme Microsatellite or late upland diversity Index nonglutinous traits traitsa locus primers I 1.2147 0.6574 0.2361 0.4797 0.2258 0.3798 0.4637 0.7314 II 1.2622 0.3567 0.4375 0.5957 0.2179 0.4041 0.4675 0.7423 III 1.2849 0.2399 0.6892 0.5996 0.2082 0.3955 0.4799 0.7692 IV 1.1825 0.6909 0.0434 0.6407 0.2244 0.2705 0.4290 0.7179 V 1.1322 0.6058 0.3622 0.2397 0.1926 0.3497 0.4312 0.6890 indica 1.1454 0.1791 0.1556 0.4694 0.2061 0.3891 0.4049 0.7315 japonica 1.2081 0.6192 0.6794 0.6329 0.2172 0.4036 0.4158 0.7462 Rice landraces 1.1570 0.6365 0.4768 0.3768 0.2172 0.4036 0.4483 0.7626 Note: I, II, III, IV and V see Fig. 1; I = total genetic diversity indices for 31 phenotypic traits of rice landraces from ecological zones or indica/ japonica or rice landraces in Yunnan. CVa = average diversity index for 23 quantitative traits in primary core collection from ecological zones or indica/japonica or rice landraces in Yunnan. Ia = average diversity index for 18 qualitative traits in primary core collection from ecological zones or indica/japonica or rice landraces in Yunnan. Hi = average gene diversity index for 12 isozyme loci in primary core collection from ecological zones or indica/japonica or rice landraces Yunnan; Hs = average gene diversity index for 20 microsatellite primers in secondary core collection from ecological zones or indica/japonica or rice landraces in Yunnan. indica (0.1271), and the Dr value between paddy and upland and japonica subspecies was mainly distributed in over 60 rice at isozyme level was also low (0.0124). counties between elevations of 1,400 m and 1,800 m. Most varieties in the mixed belt displayed the characteristics of Discussion both indica and japonica subspecies, which made it difficult to classify them by elevation. The elevations of Zhenyuan, Geographic distribution of classification traits of landraces Lancang and Ximeng counties in Simao prefecture were in in Yunnan the range of 1,600–1,800 m, 1,360–1,500 m and 1,200–1,300 Yunnan is a plateau at low latitudes which extends m, respectively. Therefore, the vertical distribution of the from the Qinghai-Tibet Plateau. The province is character- indica and japonica rice varieties in Simao prefecture reflect- ized by an alternate distribution of mountains, plateaus and ed the distribution pattern of the whole Yunnan landraces. basins with diverse geographic and climatic types. The diverse climate includes all the climatic types found in China Genetic diversity of Yunnan rice landraces such as tropical, south subtropical, mid-subtropical, north There were variations in the genetic diversity of differ- subtropical and warm temperate zones. Since the Yunnan ent classification traits and the geographic distribution of ge- rice landraces are widely distributed geographically, from netic diversity in the Yunnan rice landraces. The South mar- 21°8′32″N to 29°11′18″N and 97°31′39″E to 106°11′47″E, ginal paddy-upland rice region was the richest gene diversity they grow at very different altitudes and under diverse cli- center among the five ecological zones of rice landraces in matic conditions. Such a wide geographic distribution and Yunnan, based on the isozyme and DNA gene diversity indi- diverse growing conditions contribute to the high genetic di- ces (0.4799 and 0.7692). Lincang, Simao, Xishuangbanna versity of rice landraces from Yunnan. and Dehong prefectures represented the genetic and gene di- The vertical distribution of rice landraces from Yunnan versity centers of the rice landraces in the 17 prefectures of showed considerable variations between the major indica Yunnan. In particular, Lincang prefecture was not only main and japonica zones and the mixed zone. The lowest latitude genetic diversity center of the rice landraces in Yunnan but and the highest altitude where Yunnan landraces grew corre- also the main diversity center of Dingying’s and Cheng’s sponded to about 76 m above sea level in Hekou county in classification traits. Honghe prefecture in the southeastern part of Yunnan and There was a geographic diffusion trend starting from 2,700 m in Weixi county in Diqing prefecture. It was re- the southwestern region, the center of diversity, to other re- markable that the indica and japonica subspecies in Simao gions, and there was also a close connection with the ecolog- prefecture were distributed vertically along the altitude. In ical environment. Accordingly, the following 3 geographical general, the area below 1400 m belonged to the indica belt, distribution regions could be roughly identified. 1. The di- and most varieties growing below 1000 m belonged to the versity central region lay in southwest Yunnan. The results javanica, aman, boro and late-maturity rice types. The area showed that there was a high genetic diversity and a large over 1,800 m corresponded to the japonica belt, and most va- number of accessions in Southwest Yunnan. The region not rieties grown above 1,900 m belonged to the communis, aus only contained a large number of accessions and displayed and early-mid maturity rice types. The mixed belt of indica the highest genetic diversity and gene diversity indices, but Genetic diversity of rice landraces 97 also harbored three species of wild rice (O. granulata, ular data for a large number of accessions with sufficient O. rufipogon, O. officinalis) in China (Fig. 1), for the following polymorphism to identify genetic diversity in germplasm reasons. (1) Southwest Yunnan is the distribution center of accessions. PCR-based markers are suitable for large-scale Yunnan’s granite rock and granite-formed soils, character- analysis, and microsatellites are becoming popular, both for ized by a complex holocrystalline granular structure consid- the detection of genetic diversity and breeding research. ered to have solidified from magma at a considerable depth It is generally recogenized that the indica-japonica below the surface, which affected and will continue to exert differentiation in O. sativa at the phenotypic level is largely a major influence on rice evolution and diversity, due to attributable to adaptation to very different environments, some chemical elements with irradiation rays from granite- including temperature and water conditions (Scond 1982, formed soils had been absorbed by rice and lead to gene mu- Glaszmann 1987, Oka 1988). Li and Rutger (2000) reported tation in plants during a long time. (2) The diversity of the that absolute within-county gene diversity in each of the rice landraces in this region is closely related to the ecologi- groups was correlated with ecological variability and envi- cal variability and environmental heterogeneity. The land- ronmental heterogeneity rather than with geographical con- scape and topography especially in the Hengduan Moun- ditions. It remains to be determined what caused such a tains, Gaoligong Mountains, West Yunnan Great Gorge and geographic differentiation at the DNA level. We noted that southern part of Yun-Gui Plateau, an extremely complicat- the absolute within-prefecture genetic and gene diversity of ed. The vertical distribution of rice is obvious, with eleva- classification traits was correlated with ecological variabili- tions up to 2,000 m and a high frequency of mutation in- ty (all the climate types in China), environmental heteroge- duced by the ultraviolet radiation and son on. (3) The neity (the vertical distribution ranged from 76 to 2700 m asl) climate in most regions belongs to the subtropical highland and geological characteristics (granite rock). For instance, monsoon (monthly average temperature is in the range of the prefectures with the maximum phenotypic and isozymic 10–23°C and rainfall in the range of 800–2,000 mm), while diversity (Lincang, Simao, Dehong and Xishuangbanna) in some parts, tropical, south subtropical and mid- subtropi- were most heterogeneous. Also, the highest genetic diversity cal conditions predominate and an affected by drought and corresponded to the ecogroups (javanica, aman, communis, floods, low temperatures and insect and disease damage in nuda, aus and boro) or Ding’s classification traits (early- normal years. (4) The predominant diversity of Ding and mid or late rice, paddy or upland rice, glutinous or non- Cheng’s classification traits and other traits is partly related glutinous rice) differentiation in to indica and japonica sub- to the rice culture practised by the Hani (terrace) and Dai species. In addition, genetic differentiation of the landraces (glutinous and soft rice) populations. It was reported that the among the 17 prefectures at the isozyme level was higher waxy mutation was preferentially selected by most Asian (0.1242), and the indices from the five ecological zones at peoples (Yamanaka et al. 2004). (5) Lincang prefecture is the isozyme and SSR levels were 0.1256 and 0.0427, respec- the richest center of genetic diversity of rice landraces in tively. Most varieties (indica-like and japonica-like types) Yunnan as well as the diversity center of Ding’s 5-grade in the mixed belt, i.e. verieties with a wide compatiblility va- classified characters and of javanica, nuda and communis rieties (Zeng et al. 2000b), showed the characteristics of groups, based on the morphological traits of rice landraces both indica and japonica subspecies, making it difficult to from 17 prefectures in Yunnan. Such results reveal the pres- classify them, with differences at elevations from 1,400 to ence of the richest areas of elite rice germplasm in China. 2. 1,800 m. Indica-japonica differentiation was lower, either of The diversity diffusion regions included Wenshan, Baoshan, the morphological (0.0288 for quantitative traits, 0.0271 for Yuxi, Nujiang, Honghe, Dali, Chuxiong and Qujing prefec- qualitative traits), isozyme (0.0603) or DNA (0.0311) levels. tures which surround the diversity center, and displayed on We suggested that Southwest Yunnan in China is the center obvious in vertical distribution of indica and japonica rice. of genetic differentiation of indica and japonica subspecies 3. The regions with a low genetic diversity were mainly of Asian cultivated rice. The genetic differentiation by natu- Lijiang, Kunming, Dongchuan and Diqing prefectures, possi- ral selection to facilitate reproductive isolation involved the bility due to the lower diversity in paddy-upland, glutinous- assumption of the origin of geographic races (ecogroups or nonglutinous and glume-hair characters. Additionally, the classification traits), subspecies (indica-japonica) and spe- low diversity of the landraces from Kunming prefecture was cies (O. sativa, O. granulata, O. rufipogon and O. officinalis). also related to the extensive use of improved varieties. Yunnan in Southwest China is one of the centers of genetic Genetic differentiation within and among prefectures or diversity of Asia cultivated rice ecological zones of O. sativa Southwest China, especially Yunnan, is the richest cen- It is generally recognized that phenotypic traits are con- ter of genetic and gene diversity of O. sativa among the 29 trolled by genes and are affected by the environment, but can provinces of China based on the analysis of the genetic di- adapt to environments, which results in additional polymor- versity distribution of China rice cultivars at the provincial phism in genetic diversity and reveals genetic variation in- level, using 26 phenotypic characters of 50,526 accessions directly. Although isozyme and molecular data can reveal from the China rice germplasm collections and nine poly- genetic differentiation directly, it is difficult to obtain molec- morphic isozyme loci of 2,080 accessions from the core 98 Zeng, Zhang, Li, Shen, Sun, Wang, Liao, Liu, Wang, Xiao and Wen collection (Li 2001). The genetic diversity of 4,310 acces- Acknowledgements sions from primary core collection of Chinese rice landraces from the 6 rice zones and 29 provinces of China based on 36 This research was supported by the National Basic Re- microsatellite markers, showed that a higher genetic diversity search Program Foundation of China (973 Program Founda- and low differentiation occurred in Yunnan and Guizhou, tion, no. 2006CB100205, 2004CB117201), the National and that the center of gene diversity of Chinese rice land- Natural Science Foundation of China (No. 30260060, races at the DNA level lay in Yunnan, China (Zhang et al. 30660092), Yunnan Provincial Natural Science Foundation 2007). A total of 6,121 accessions were collected over a (No. 2002C0077M), Yunnan Introduction and Foster Talent wide range of 109 counties in 17 prefectures in Yunnan (No. 2005PY01-14) and Cooperation Program between Province, in which indica accessions were drived from 108 Province and Zhejiang University from Yunnan provincial counties in 16 prefectures and japonica accessions from 99 Scientific and Technology Department (2006YX12). counties in 17 prefectures. The genetic diversity center based on the concentrations of 8 minerals in brown rice from Literature Cited Lincang, Simao, Xishuangbanna and Dehong prefectures showed a similar trend to that of both the genetic and gene Amanda, J.G., T.H. Tai, J. Coburn, S. Kresovich and S.R. McCouch diversity center of rice landraces, based on morphological (2005) Genetic Structure and Diversity in Oryza sativa L. traits and isozyme and SSR marker (unpublished data) in 17 Genetics 169: 1631–1638. Chang, T.T. (1976) The origin, evolution, cultivation, dissemination prefectures of Yunnan. The genetic diversity included and diversification of Asian and African rices. Euphytica 5: Yunnan Province (China), Bhutan, Assam (India) adn North 425–441. Thailand (Nakagahra and Hayashi 1977, 1984, Glaszmann Cheng, K.S. (1993) Classification of indica and japonica subspecies in 1987). The diversity within indica rice was evenly distribut- Oryza sativa. Yunnan Sci. Techn. Press, Kunming, China. p. 1– ed throughout tropical Asia, while variation within japonica 45. rice reveled that the hilly part of continental Southeast Asia, Ding, Y. (1959) Rice Cultivation in China. In “Classification of Chi- was the region with the highest genetic diversity and proba- nese cultivated rice” Ding, Y. (ed.), Agriculture Press, Beijing, ble origin (Glaszmann 1987). The center of origin is consid- China. p. 181–215. ered to correspond to the area with the highest diversity, and Gao, L.Z., S. Ge and D.Y. Hong (2000) Allozyme variation and popula- furthermore, the center of origin of crops harbors many wild tion genetic structure of common wild rice Oryza rufipogon relatives and is the source of dominant genes (Vavilov Griff. in China. Theor. Appl. Genet. 101: 494–502. Glaszmann, J.C. (1987) Isozymes and classification of Asian rice vari- 1926). Southwest Yunnan is probably such a center. Yunnan eties. Theor. Appl. Genet. 74: 21–30. is one of the centers of genetic diversity of Asian cultivated Ise, K., Y.Q. Sun, L.Y. Dai, C.R. Ye, Y. Sunohara and K. Tomita (2000) rice, which is documented by the abundance of indica and Genetic variation in endosperm amylose content in rice genetic japonica rice landraces (Zeng et al. 2003), and is strongly resources of Yunnan, China and artificial induction of mutants supported by morphological (Li 2001) and isozyme with low amylose content. Japan J. Tropical Agric. 44: 269– (Nakaghara 1978, Nagamine et al. 1992, Wang and Sun 275. 1997, Ise et al. 2000, Li 2001) studies as well as RFLP (Liu Jiang, N., Z. Bao, X. Zhang, H. Hirochika, S.R. Eddy, S.R. McCouch and et al. 1995) and SSR analysis (Zhang et al. 2007). Therefore, S.R. Wessler (2003) An active DNA transposon family in rice. Yunnan was previously considered as one of the original Nature 421: 163–167. centers (Chang 1976, Oka 1988, Nakaghra 1978, Nakaghra King,L.M. and B.A.Schaal (1989) Ribosomal DNA variation and dis- et al. 1984). tribution in Rudbckia missouriensis. Evolution 43: 1117–1119. Li, Z.C. (2001) Studies on sampling strategy for core collection of However, Wang and Sun (1997) suggested that Yunnan Chinese landraces and genetic diversity of phenotypes and may be a secondary center of origin due to the archeological isozymes. PhD dissertation, China Agric. Univ., Beijing. findings of ancient rice grains that may have appeared far Li, Z.C., H.L. Zhang, Y.W. Zeng, Z.Y. Yang, S.Q. Shen, C.Q. Sun and later in the region, compared with those in the middle and X.K. Wang (2002) Studies on sampling schemes for the estab- lower reaches of the Yangtze River in China. However, this lishment of core collection of rice landraces in Yunnan, China. concept is only supported by indirect archaeological data Genet. Resour. Crop Evol. 49: 67–74. and lack of biodiversity and ecosystem evidence, because Li, Z. and J.N. Rutger (2000) Geographic distribution and multilocus Yunnan, the well-known “Plant, Animal and Microbe King- organization of isozyme variation of rice (Oryza sativa L.). dom”, is one of the regions with the richest biodiversity in Theor. Appl. Genet. 101: 379–387. the world; including more than 17000 species (62.9% of the Liu,K.D., Q.F.Zhang, D.P.Zhang and Y.F.Xue (1995) Genetic varia- total in China) of vascular plants (Zeng et al. 2001b), and tion and indica-japonica differentiation in Yunnan indigenous rice. Acta Botanica Sinica 37: 718–724. lacking of the archeological evidence for most plants due to Mann, C. (1997) Reseeding the green revolution. Science 277: 1038– the ecosystem consisting of microbe decomposing and ani- 1042. mal utilization. Therefore, it has some questions that only Nagamine, T. (1992) Genetic variation in isozymes of indigenous rice based on the archeological evidence for rice origin center. varieties in Yunnan Province of China. Jpn. J. Breed. 42: 507– 513. Nakagahra, M. (1978) The differentiation, classification and center of Genetic diversity of rice landraces 99

genetic diversity of cultivated rice (Oryza sativa L.) by vars and their evolutionary significance during the domestica- isozyme analysis. Tropical Agric. Res. Ser. 11: 77–82. tion process of rice. Theor. Appl. Genet. 108: 1200–1204. Nakagahra, M. and K.I. Hayashi (1977) Origin of cultivated rice as Yamanaka, S., I. Nakamura, H. Nakai and Y.I. Sato (2003) Dual origin detected by isozyme variations. JARQ 11: 1–5. of the cultivated rice based on molecular markers of newly col- Nakagahra, M., T. Akihama and K.I. Hayashi (1984) Geographical dis- lected annual and perennial strains of wild rice species, Oryza tribution of esterase genotypes of rice in Asia. Rice Genet. nivara and O. rufipogon. Genet. Resour. Crop Evol. 50: 529– Newsl. 1: 118–120. 538. Nei, M. (1973) Analysis of gene diversity in subdivided populations. Zeng, Y.W., Z.C. Li, Z.Y. Yang, X.K. Wang, S.Q. Shen, H.L. Zhang Proc. Natl. Acad. Sci. USA 70: 3321–3323. and Y.M. Chen (2000a) Law and center of diversity of main Nei, M. (1977) F-statistics and analysis of gene diversity I subdivided characters of indigenous rice (Oryza sativa L.) in Yunnan. J. populations. Ann. Human Genet. 41: 225–233. Huazhong Agri. Univ. 19: 511–517. Oka, H.I. (1988) Origin of cultivated rice. Japn. Sci. Soc. Press, Tokyo. Zeng, Y.W., F.R. Xu, S.Q. Shen and J.Y. Deng (2000b) Correlation of Sato, Y.I. (1991) Variation in spikelet shape of indica and japonica rice indica-japonica classification and morphological character of cultivars of Asian origin. Jpn. J. Breed. 41: 121–134. Yunnan nuda rice landraces. Chinese J. Rice Sci. 14: 115–118. Sato, Y.I. (1996) Rice Culture Viewed from DNA. NHK Books, Zeng, Y.W., Z.C. Li, Z.Y. Yang, S.Q. Shen, H.L. Zhang and X.K. Wang Tokyo, 227 p. (2001a) Geographical distribution and ecogroups classification Scond, G. (1982) Origin of the genetic diversity of cultivated rice of Indica-Japonica subspecies of Yunnan local rice resources. (Orzya sativa): study of polymorphism scored at 40 isoezyme Acta Agronomica Sinica 27: 15–20. loci. Jpn. J. Genet. 57: 25–75. Zeng, Y.W., J.J. Wang, Z.Y. Yang, S.Q. Shen, L.H. Wu, X.Y. Chen and Temnykh, S., W.D. Park, N. Ayres, S. Cartinhour, N. Hauck, L. Lipovich, J.G. Men (2001b) The diversity and sustainable development of Y.G. Cho, T. Ishii and S.R. McCouch (2000) Mapping and crop genetic resources in the Lancang River Valley. Genet. genome organization of microsatellite sequences in rice (Oryza Resour. Crop Evol. 48: 297–306. sativa L.). Theor. Appl. Genet. 100: 697–712. Zeng, Y.W., S.Q. Shen, Z.C. Li, G.S. Wen, H.L. Zhang, H.J. Li and Tilman, D. (1998) The greening of the green revolution. Nature 396: X.K.Wang (2002) A study of the photo-thermal reaction of 211–212. core collection of Yunnan rice and its early-mid and late-types. Uga, Y., Y. Fukuta, R. Ohsawa and T. Fujimura (2003) Variation of flo- J. Southwest Agric. Univ. 24: 502–505. ral traits in Asian cultivated (Oryza sativa L.) and its wild rela- Zeng, Y.W., S.Q. Shen, Z.C. Li, Z.Y. Yang, X.K. Wang, H.L. Zhang and tives (O. rufipogon Griff.). Breed. Sci. 53: 345–352. G.S. Wen (2003) Ecogeographic and genetic diversity based on Vavilov, N.I. (1926) Centers of origin of cultivated plants. Trends. morphological characters of indigenous rice (Oryza sativa L.) Pract. Bot. Gener. Sel. 16: 3–24. in Yunnan, China. Genet. Resour. Crop Evol. 50: 566–577. Wang, X.K. and C.Q. Sun (1997) Origin and Differentiation of Chinese Zhang, D.L., H.L. Zhang, X.H. Wei, Y.W. Qi, M.X. Wang, J.L. Sun, Cultivated rice, China Agricultural University Press, Beijing. S.X. Tang, Z.E. Qiu, Y.S. Cao, X.K. Wang and Z.C. Li (2007) p.1–91. Genetic structure and diversity of Oryza sativa L. in Guizhou, Yamanaka, S., I. Nakamura, K.N. Watanabe and Y.I. Sato (2004) Identi- China. Chinese Sci. Bull. 52: 343–351. fication of SNPs in the waxy gene among glutinous rice culti-