Rice Science, 2012, 19(1): 21−28 Copyright © 2012, China National Rice Research Institute Published by Elsevier BV. All rights reserved

Genetic Diversity of Wild Rice Species in Yunnan Province of China CHENG Zai-quan1, YING Fu-you1, LI Ding-qing1, YU Teng-qiong1, FU Jian1, YAN Hui-jun2, ZHONG Qiao-fang1, ZHANG Dun-yu1, LI Wei-jiao1, HUANG Xing-qi1, 2 (1Biotechnology & Genetic Resource Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650223, China; 2 Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650223, China) Abstract: Yunnan Province of China is one of the important centers for origin and evolution of cultivated rice worldwide. Wild rice is the ancestor of the cultivated rice. Many elite traits of wild rice have widened the genetic basis in cultivated rice. However, many populations of wild rice species have disappeared in the past few years. Therefore, the current status of wild rice resources should be updated and the genetic diversity of wild rice species should be examined for further germplasm preservation and utilization. Our investigations showed that the number of natural wild rice populations declined sharply in Yunnan Province during the past few years due to various reasons. Fortunately, one population of Oryza rufipogon, three of O. officinalis and ten of O. granulata have been newly found in different ecological sites, which were confirmed by inter-simple sequence repeat (ISSR) marker analysis in this study. ISSR analysis and investigation of some important traits of nutritional values indicated that the genetic diversity of the currently existing wild rice resources in Yunnan is still rich. The demonstration of genetic diversity of wild rice by a combined use of geographical distribution, morphological traits, nutrition contents and ISSR markers would be helpful for the conservation and exploration of these important wild rice resources. Key words: wild rice resource; natural distribution; genetic diversity; genetic trait

Genetic resources play an important role in increasing yield potential in rice. As genetic diversity among commercial cultivars decreases, it’s getting difficult to find new genes from cultivars for futher improvement of grain yield and quality, and sufficient resistance to biotic or abiotic stress, which motivates scientists to explore wild species to meet those demands (Wang et al, 2000). Wild rice is an important gene bank harboring lots of good traits and genes to enrich the genetic basis of cultivars (He, 1998; Fan et al, 2000a, b; Qin et al, 2000; Zhong et al, 2000; Cai et al, 2001; Feng et al, 2002; Li et al, 2002). There are many successful examples of dramatic increases of rice yield with the employment of wild resources, which contributes to the solution of the food problem for 1.3 billion people in China (Zhou and Tan, 1999). During 1926–1937, by making use of the natural hybrids of Oryza rufipogon (wild rice) with cultivated rice, Ding et al bred the variety Zhongshan 1 with wild rice genes. Up to date, at least 95 varieties have been bred using Zhongshan 1 as a parent (Li et al, 2009). Another example is that YUAN Longping and his colleagues led national effort to develop hybrid rice with male sterile genes from O. rufipogon (Pang, 1998). The hybrid rice has tremendousely increased grain yield of rice and made great contribution to world food production. The gene WBB1 responsible Received: 29 November 2010; Accepted: 23 February 2011 Corresponding author: HUANG Xing-qi ([email protected])

for resistance to rice bacterial blight in the entire life of rice was derived from O. rufipogon as well (Zhang, 2002). Many other reports have demonstrated various good traits derived from wild resources to improve rice cultivars (Pang, 1998; Lu et al, 2002; Yang, 2003). Wild rice distributes in all continents except Europe, North America and Antarctic areas in the world, mostly in tropical and sub-tropical areas of Asian. China, as one of the important origin countries of wild rice, has three major wild rice species: O. rufipogon Griff. (A′A′, 2n = 24), O. granulate Baill (GG, 2n = 24) and O. Officinalis Wall (CC, 2n = 24) (Zhang, 2002). Yunnan Province is located in the southwest China at a latitude from 21°09′32′′ to 29°15′04′′ N and a longitude from 97°31′39′′ to 106°11′47′′ E, with altitude varying from 76 m above sea level to 6740 m. The mountains and rivers run from north to south across the province, which forms eight climate zones with unique topography and biological resources. Yunnan Province has been considered as one of the precious green treasures on the earth, known as the ‘plant and wildlife kingdom’, with great biodiversity (Fan et al, 2000b). Many wild rice populations have been found in the past few decades since the first O. officinalis was found in Jinghong of Yunnan in 1936 by WANG Qiyuan (Cheng et al, 2004). According to some previous reports, wild rice populations were identified in pools, river sides, bamboo fields, banana

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tree fields, rubber tree fields, shrub fields and forest fields from the altitude of 400 m to 1000 m in the southern five prefectures of Yunnan. The number of wild rice species and ecological types (populations) in Yunnan Province were the highest among all related provinces of China (Gao et al, 2000). Each wild rice species has many subtypes or ecological populations in Yunnan Province, which can be used for breeding rice cultivars with high yield and multiple resistances. Many scientists reach an agreement that Yunnan wild rice is one of the most important parts of wild rice resources in China because all the three wild species in China have been found in Yunnan, and each species has a lot of ecotypes. Due to the increasing interference of human being activities, the proper ecological environments for many plants including wild rice have been changed or damaged seriously. Many recent reports have shown that wild rice resources were consistently reduced and some natural populations even disappeared in Yunnan since the 1990s. Therefore, wild rice resources need to be protected and conserved urgently (Fan et al, 2000b; Gao et al, 2000). However, several issues have occurred to the rice community in Yunna Province, such as the possibility to find new wild rice populations, recent changes of wild rice resources, important traits in the wild rice species for cultivar improvement, the genetic diversity in the wild rice resources nowadays, and molecular analysis and comparison of morphological traits and some important nutritional contents of seeds. In order to answer these questions, we carried out the following studies in the past few years for the genetic diversity of wild rice resources in situ in Yunnan Province of China.

MATERIALS AND METHODS Field investigation Comprehensive investigation of wild rice resources in Yunnan From 2001 to 2006, investigation was conducted in Yunnan to find new populations of wild rice resources and to confirm if the wild rice plants in those populations identified previously still exist naturally. Some plants of the presently existing wild rice populations were grown in a relocation base. The conditions for growing the wild rice plants were similar to cultivated rice. Rice cultivars as control were also planted about 3 km far away from the wild rice relocation base. Important characteristics of some wild rice plants of nine ecotypes were investigated during the whole growth period for three years.

Morphological investigation of wild rice in Yunnan Important traits of 10 plants sampled from each wild rice ecotype were scored and recorded in the relocation base and in the original populations. The relocation bases of all the three wild rice species have similar climate and environmental conditions. Evaluation of agronomic traits Resistance to diseases and abiotic stress in wild rice of Yunnan The methods reported by Feng et al (2002) and Yoshimura et al (1998) were used to evaluate the resistance to blast and bacterial blight, respectively. The methods reported by Fan et al (2000a, b) were adapted to evaluate the resistances to abiotic stress of drought and chilling. Protein and amylose contents in husked wild rice seeds Eight ecotypes of wild rice species (Table 1) and six cultivated rice (O. sativa) (Table 2) were analyzed. Seeds of wild rice were collected from their original populations or the wild rice protection populations in Jinghong. The cultivated rice varieties Yunhui 290 and Dianchao 9 belong to indica rice, Hexi 35 and Yuyou 1 belong to japonica rice, Xiangyaxiannuo belongs to indica glutinous rice and X-Jingdaonuo belongs to japonica glutinous rice. Seeds of each cultivated rice were collected from the similar regions of wild rice and were husked. Total protein content in the husked grains was measured by the method of Zhen et al (1997). Amylose content was determined according to the methods of Cheng et al (2004) and Zhang et al (1996). Each assay was repeated three times and data were presented as the mean ± SD. Inter-simple sequence repeat (ISSR) analysis Plant materials Five sub-populations of Yuanjiang O. rufipogon and four sub-populations or types of Jinghong from Yunnan; four of O. officinalis (two populations from Jinghong, wide leaf type and normal type, one from Mengding, and one from Gengma); 16 of O. granulata from different ecological regions were used for ISSR analysis. Some of them were newly found populations. Total genomic DNA extraction SDS method was used to extract total genomic DNA of rice. The procedure of genomic DNA extraction of O. granulata was modified slightly from the SDS method (Sun et al, 1997). ISSR analysis A total of 22 ISSR primers were ordered from the Shanghai Sangon Company (China). After screening,

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Table 1. Summary of important characteristics of representative populations of three wild rice species in Yunnan, China. Species O. rufipogon

Type Erect type

Population Banna

Strong and board-spectrum resistance to blast, thick and erect leaf blade, relatively erect stem, high-tilling, big panicle, high seed-setting rate, good grain quality, big anther, strong growth ability

Red-awn type

Banna

Moderate resistance to blast, strong resistance to bacterial blight, thick and erect leaf blade, extremely high-tilling, big panicle, high seed-setting rate, good grain quality, big anther, strong growth ability

Yuanjiang

Moderate resistance to blast, strong resistance to bacterial blight, thick and erect leaf blade, extremely high-tilling, extreme tolerance to chilling, drought tolerance, good grain quality, big anther, strong growth ability

Gengma

Moderate resistance to blast, resistance to bacterial blight, resistance to stemborer and planthopper, exellent grain quality, big panicle, thick rhizome, strong growth ability, outward stigma, extreme tolerance to chilling

Mengding

Resistance to blast, bacterial blight, stemborer and planthopper, high seed setting rate, good grain quality, big panicle, thick rhizome, strong growth ability, extreme tolerance to chilling Similar to O. officinalis from Gengma, except for extremely broad leaf blade and chilling tolerance Extreme resistance (immunity) to bacterial blight, big and outward stigma, thich rhizome, drought tolerance, reproductive growth period is extremely long (nor light sensitiveness), wide adaptability

O. officinalis

Jinghong Banna

O. granulata

Mengding

Important characteristics

Similar to O. granulata from Banna with good grain quality

The underlined are the traits investigated newly. The traits in bold are the most valuable for improvement of rice cultivars.

Table 2. Seeds of different Yunnan wild rice species or types. Species O. rufipogon

O. officinalis

O. granulata O. sativa

Type Yuanye Yuanye Jingye Yaoye I Yaoye III Yaoye I Yaoye II Yaoye IIIA Youye Youye Yunhui 290 Diancha 9 Yuyou 1 Hexi 35 Xiangyaxiannuo X-Jingdaonuo

Collected site Yuanjiang Jinghong Jinghong Jinghong Kunming Jinghong Zhedian Mengding Gengma Jinghong Mengding Jinghong Jinghong Yuxi Yuxi Jinghong Yuxi

Collected date Nov. 2001 Nov. 2001 Nov. 2001 Nov. 2001 Nov. 2001 Nov. 2002 Nov. 2001 Nov. 2001 Nov. 2001 Nov. 2001 Sept. 2001 Sept. 2001 Sept. 2001 Sept. 2001 Sept. 2001 Sept. 2001

12 primers (Table 3) were selected for genetic diversity analysis to all samples of the three wild rice species (Chen et al, 2002). PCR conditions were described according to the method of Wu et al (2004). Data analysis including percentage of polymorphic bands and Nei’s gene diversity was conducted using POPGENE (version 1.32) software. Jaccard similarity coefficients were calculated using NTSYS-pc (version 2.0) (Numerical Taxonomy System, Rohlf, 1993). Genetic relationship among samples was analyzed using the unweighted pair group method (UPGMA) (Sneath and Sokal, 1973).

Table 3. ISSR primers used in analysis of genetic diversity. Primer ISSR1 ISSR2 ISSR3 ISSR4 ISSR5 ISSR6 ISSR7 ISSR8 ISSR9 ISSR10 ISSR11 ISSR12

Primer sequence BDBCACACACACACA VHVGTGTGTGTGTGTGT DBDGAGAGAGAGAGAGA GCACACACAC CTCTCTCTCTCTCTCTRG CTCTCTCTCTCTCTCTRC CCCGTGTGTGTGTGT GSGGTGTGTGTGTGT CSCGAGAGAGAGAGA GCWGAGAGAGAGAGAG CCAGTGGTGGTGGTG GCGACACACACACACA

Molecular analysis of genetic diversity of wild rice resources in Yunnan The materials were the representatives of different populations in Yunnan. The molecular analysis method was similar to the above description.

RESULTS New populations of wild rice identified in Yunnan Our comprehensive investigation identified several new wild rice populations. One population of O. rufipogon was found in Jinghong, belonging to Jinghong type of O. rufipogon. The plants in the new population were different from those in other ecological populations or types of O. rufipogon with

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resistance to insects or diseases. The main differences were shown in wider and longer leaves, bigger panicles and higher seed-setting rate, and the blast resistance was stronger in Jinhong type than in other types of O. rufipogon. Two new populations of O. officinalis were found at Genma and Menghai, respectively. They were the only naturally existing O. officinalis popuplations in Yunnan. Ten new populations of O. granulata were found in some banana fields, riversides and forest fields in Jinghong and Simao of Yunnan. All the newly found populations of wild rice were ecological populations differred from the other previously found populations. The ISSR analysis showed some different traits and genetic backgrounds of these newly found populations (Fig. 1). Comparison of the population number of wild rice between new and existent populations O. rufipogon Our investigation showed that 23 previously found populations of O. rufipogon had no wild rice plants in Yunnan at present. This fact indicates that in the past 20 years, wild rice in situ became extinct at a surprisingly high speed that almost one population (in one ecological site) of O. rufipogon disappeared each year in Yunnan. One population of O. rufipogon recently found at Mandiu of Jinghong has some wild

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rice plants, and another population of O. rufipogon previously found at Yuanjiang also has some wild rice plants, but they differentiated into five sub-populations. Actually, it was found that there were many plants in Yuanjiang O. rufipogon population, although the plant number was once reduced in the past 20 years. The plant number of O. rufipogon in Yuanjiang was only 15−20 in 2003, but gradually increased to 380–400 in 2006 after establishment of protection base around the Yuanjiang population. O. officinalis O. officinalis distributed over five counties of three prefectures in Yunnan. The total population number of the identified O. officinalis was 14 (Cheng et al, 2004). Our investigation showed that 12 of the 14 populations had no plants at their original sites now. It is surprising that in the past 20 years, almost one natural population of O. officinalis disappeared in every two years in Yunnan. Fortunately, we found two populations of O. officinalis that were never recorded before, one in Gengma and the other in Menghai. The plant number of O. officinalis in Gengma was only 5–6 in 2002. After construction of protection base at that site, the plant number recovered gradually, and reached 40–50 in 2006. O. granulata O. granulata distributed widely in Yunnan. So far,

Fig. 1. Genetic backgrounds revealed by ISSR-marker based analysis of different populations of three wild rice species in Yunnan, China. A, O. rufipogon. 1, Dongxiang from Jiangxi Province; 2, Mandiu, Jinghong (1); 3, Mandiu, Jinghong (2); 4, Purple-stem of Jinghong; 5, Green-stem of Jinghong; 6, Yuanjiang (1); 7, Yuanjiang (2); 8, Yuanjiang (3); 9, Yuanjiang (4); 10, Yuanjiang (5); 11, O. longistaminata; 12, Yuyou 1; 13, Hexi 35; 14, NC; 15, Dianlong 201; M, M2000 marker. B, O. officinalis. 1, Jinghong 1; 2, Jinghong 2; 3, Menghai; 4, Mengding; 5, Wide leaf type. C, O. granulata. 1, Jinghongxi; 2, Zhengkang; 3, Mengbeixin; 4, Simao; 5, Erhe; 6, Chishuiguan; 7, Mengkuang; M, M2000 marker. The underlined are newly found populations of wild rice in Yunnan Province, and others are wild rice reported before and the cultivated rice varieties. The numbers in the parenthesis present the subpopulation of sample plants from different wild rice growing fields in the same original site.

CHENG Zai-quan, et al. Genetic Diversity of Wild Rice Species in Yunnan Province of China

some O. granulata populations have quite many plants scattering naturally in large areas up to several hectares. This was very special for O. granulata in comparison to small population size naturally for O. rufipogon and O. officinalis. Data of investigation since 1950s indicated that O. granulata including 122 populations distributed in 20 counties of 7 prefectures in Yunnan. Ten populations of O. granulata have been recently found from year 2001 to 2006. Our investigation concluded that 85 of the previously found populations of O. granulata have no plants at their original sites presently. In other words, O. granulata permanently disappeared at a speed of 3–4 populations each year in the past two or three decades in Yunnan. At present, only 37 populations of O. granulata have plants at their original sites, mainly at Simao and Lincang prefectures in Yunnan (Fig. 2). Wild rice resources need urgent protection in Yunnan Our investigations suggested that the extinction of wild rice resources in Yunnan urgently needs public attention. Take O. granulata as an example, in the past three decades, the extinction speed of O. granulata populations have kept increasing. Consequently, 85 of the 122 previously identified populations have disappeared. Among the 37 existing populations of O. granulata at their original sites currently, 28 (76%) are approaching to the edge of extinction. The environmental conditions are becoming more unfavourable for the survival of these 28 populations of O. ganulata. Plant number in each of these 28 populations is gradually reducing. Unless some effective ways are taken to protect these populations, O. granulata would disappear sooner or later in Yunnan. Only 9 populations of O. granulata are relatively safe at present, because their original sites are in steep mountains that are free from the interference of human beings and animals. In general, the populations and the plant number in each population of Yunnan wild rice have decreased sharply in the past 20 years, especially for O. rufipogon

Fig. 2. Comparison of wild rice populations at different periods.

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and O. officinalis. It is surprising that the extinction rate of wild rice in Yunnan is extraordinarily high. Genetic diversity of wild rice resources assessed by phenotypic traits in Yunnan Prior to this study, no systematic investigation was conducted to phenotypically characterize the wild rice species, especially for growth-developmental characteristics during the whole life period. The important or special traits of the three wild rice species are summarized in Table 1. It was found that each wild rice species had unique or important traits that were not observed in other species and cultivars. For example, O. rufipogon from Yuanjiang displayed extremely strong tolerance to chilling. The plants of O. officinalis from Gengma had the highest seed setting rate (82.0% ± 1.2%) among all the wild rice species. O. ganulata had extremely strong tolerance to drought with a long reproduction period from April to November, indicating it is photo-period insensitive. Moreover, all the three wild rice species shared some characteristics with cultivated rice. For example, all the three wild species had tolerance to bacterial blight and long outward stigma. Most of the plants of different wild rice species maintained their phenotypic traits in the relocation base after being transplanted from their own original populations. This observation suggests that most of the traits are genetically controlled. However, some traits such as plant height changed. For example, the plant height of O. officinalis plants under shrub reached 4 m in Gengma, but only 1.5–2.0 m at the relocation base. The plant height of an O. granulata population reached 80–90 cm in Dongwo, but only 60 cm at the relocation base. The average protein contents in the husked seeds of O. rufipogon, O. granulata and O. officinalis were 14.47% ± 0.64%, 16.28% ± 2.05% and 15.30% ± 0.57%, respectively (Fig. 3). O. officinalis from Gengma had the highest protein content of 19.3%. The seed protein content was significantly higher in wild rice species than in O. sativa, suggesting greater nutritional value of wild rice. Amylose contents ranged from 8.8% to 13.0% among the samples of Yunnan wild rice, with an average of 12.0% ± 0.38%, 9.70% ± 1.40%, and 11.28% ± 0.43%, respectively for O. rufipogon, O. officinalis and O. granulata (Fig. 4). All the three wild rice species had significantly lower amylose content than O. sativa. Generally speaking, rice with low amylose content is favored for steamly cooked rice because of softness and taste feeling. This observation indicates that wild rice may have genes controlling lower amylose content, which would be useful to

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Protein content (%)

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Fig. 3. Average protein content in the husked seeds of three wild rice species and O. sativa. AA, O. sativa; A′A′, O. rufipogon; CC, O. officinalis; GG, O. granulate. 20 15.96±1.16

Amylose content (%) Percentage (%)

16 12.0±0.38

12

11.28±0.43 9.70±1.4

8 4 0

3.88±1.03

AA(g)

AA

A'A'

CC

GG

Fig. 4. Average amylose content in the husked seeds of three wild rice species and O. sativa. AA, O. sativa; A′A′, O. rufipogon; CC, O. officinalis; GG, O. granulate. AA(g) means glutinous rice of O. sativa.

improve rice quality. In general, most of the wild rice species found in Yunnan have many special and important traits, such as strong resistance and good quality which are very useful for rice cultivar improvement. Genetic diversity of wild rice resources assessed by ISSR analysis in Yunnan Genetic diversity of O. rufipogon in Yunnan A total of 12 ISSR primers were identified polymorphic among the sampled entries of O. rufipogon (Table 3). There were two populations of O. rufipogon naturally existing in Yunnan, and three

populations of O. rufipogon conserved in the relocation base. The five populations of O. rufipogon were highly diverse, expressed by Nei’s value of 0.26 among different populations and 0.13 among the plants inside the populations (Table 4). The ISSR markers generated 80 polymorphic bands with a percentage of polymorphic bands of 64.52%. The genetic distance (I) was 0.37 among different populations and 0.19 among the plants inside the populations. The genetic distance of O. rufipogon was high between Yuanjiang and Jinghong type. The differences of these types of O. rufipogon were similar in the morphological and molecular analysis. Genetic diversity of O. officinalis in Yunnan The genetic variation among different populations was higher than the variation among different plants inside a population of O. officinalis. The genetic variation coefficient (h = 0.22) among populations of O. officinalis was lower than that of O. rufipogon. Genetic diversity of O. granulata in Yunnan For 16 populations, 135 bands were resulted from the PCR with 12 ISSR primers, with an average of 11.2 bands per primer. Among 135 bands, 93 were polymorphic with the percentage of polymorphic bands of 68.8%. The lengths of amplified bands ranged from 100 to 2 000 bp. In addition, the observed average number of alleles (Na) was 1.81, the mean Nei’s gene diversity (h) was 0.33 and the mean Shannon information index (I) was 0.43. Within the population Banna, 105 bands were resulted from the PCR with 10 ISSR primers (ISSR1– ISSR10). However, only 27 bands were found to be polymorphic with the percentage of polymorphic bands of 25.7%. Similarly, in the population Mending, 78 bands were produced after PCR with 8 ISSR primers (ISSR1, ISSR2, ISSR4, ISSR5, ISSR6, ISSR9, ISSR10, ISSR12). Among 78 bands, only 17 were polymorphic, showing the percentage of polymorphic bands of 21.8%. A cluster analysis was used to generate two dendrograms based on the Jaccard coefficients of ISSR markers among 14 populations of O. granutula

Table 4. Genetic diversity of two wild rice species revealed by the ISSR marker analysis. Species O. rufipogon O. granulata

Range Among 5 populations Within population plants Among 16 populations Within Population Banna Within Population Mending

No. of ISSR No. of ISSR bands polymorphic bands 124 80 75 25 135 93 105 27 78 17

PPB of ISSR

Na

Ne

h

I

64.52 33.33 68.84 25.71 21.79

1.66 1.34 1.81 1.25 1.22

1.45 1.23 1.52 1.15 1.11

0.26 0.13 0.33 0.09 0.07

0.37 0.19 0.43 0.14 0.10

PPB, Percentage of polymorphic bands; Na, Observed number of alleles; Ne, Effective number of alleles; h, Nei’s gene diversity; I, Shannon information index.

CHENG Zai-quan, et al. Genetic Diversity of Wild Rice Species in Yunnan Province of China

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(Fig. 5), and a cluster analysis of ISSR markers within population Banna (Fig. 6). The genetic diversity was high among 16 populations, but low within any population of O. granulata in Yunnan. The population number was larger in O. granulata than in O. rufipogon. The genetic diversity (h) and distance (I) of O. granulata populations were higher than those of O. rufipogon populations. However, the genetic diversity (h) and distance (I) within an O. granulata population were lower than those within an O. rufipogon population. In general, O. granulata was genetically more diversified than either O. rufipogon or O. officinalis.

DISCUSSION Currently, wild rice O. rufipogon only naturally exists in Yuanjiang and Jinghong in Yunnan province and most of the plants within a population are genetically diversified. Fortunately, we had collected some plants from three populations that have naturally disappeared and preserved them in the relocation base. Large genetic distance has been found among five populations of O. rufipogon. It is surprising to discover a very large genetic distance between Yuanjiang and Jinghong types of O. rufipogon, but these two types have a close genetic relationship with Dongxiang type of O. rufipogon from Jiangxi Province, China. In addition, there is a rich genetic variation among different plants in the currently existing populations ex situ. It is a pity that some populations of O. rufipogon in Yunnan have disappeared and the genetic diversity in those populations have lost forever. Fortunately, the genetic diversity among the currently existing O. rufipogon is still very high among the five populations and the plants of each population. This conclusion receives a support from the substantial variation for morphological characteristics and growth-development traits among the currently existing populations of O. rufipogon in Yunnan (unpblished data). It is noticed that the genetic diversity of O. officinalis has dramatically decreased because many naturally ecological populations have disappeared during last two or three decades (Chen et al, 2002). There are two naturally ecological populations of O. officinalis, and other two ecological populations collected and preserved ex situ in the relocation base. Great amount of variation in plant height, leaf size, stem size and seed-setting rate have been found among the four populations. Results from molecular marker analysis of ISSR showed a great amount of genetic diversity. Relatively speaking, the genetic diversity in O. officinalis is lower than in O. rufipogon.

Ind1 Ind2 Ind3 Ind4 Ind5 Ind17 Ind18 Ind19 Ind20 Ind7 Ind6 Ind8 Ind9 Ind10 Ind16 Ind21 Ind22 Ind23 Ind24 Ind11 Ind12 Ind13 Ind14 Ind15

0.85

0.90

0.95

1.00

Coefficient (Jaccard)

Fig. 5. Dendrogram of 14 populations of O. granutula based on the results of ISSR marker analysis by UPGMA method.

Fig. 6. Dendrogram of population Banna of O. granutula based on ISSR marker analysis by using UPGMA method.

Luckily, there is a certain degree of genetic diversity among different populations and different plants inside each ex situ population of O. officinalis preserved and protected in the relocation base and in situ population in original sites (Cheng et al, 2004). Even though the number of natural populations for O. granulata resource have seriously decreased in the past (Qian et al, 2001), there are 37 naturally ecological populations at present in Yunnan. In addition, there are other three ex situ populations collected and preserved in the relocation base. O. granulata has more populations than either O. rufipogon or O. officinalis. The results from molecular marker analysis showed that O. granulata had the most genetic diversity among the three wild rice species in Yunnan.

ACKNOWLEDGEMENTS This work was supported by the Yunnan Natural

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Science Foundation Project (Grant No. 2008C004); the Natural Science Foundation of China (Grant No. 31160067), and Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201003021). We also thank Dr. YAN Wengui from Rice Center of United States Department of Agriculture (USDA) for his proofreading of this paper.

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