Genetic Resources and Crop Evolution (2006) 53: 245–252 DOI 10.1007/s10722-004-6132-y
# Springer 2006
Genetic erosion from modern varieties into traditional upland rice cultivars (Oryza sativa L.) in northern Thailand R. Ishikawa1,*, S. Yamanaka2, Y. Fukuta3, S. Chitrakon4, C. Bounphanousay5, K. Kanyavong5, L.-H. Tang6, I. Nakamura7, T. Sato8 and Y.-I. Sato9 1
Faculty of Agriculture and Life Science, Laboratory of Plant Breeding, Hirosaki University, Hirosaki 036-8561, Japan; 2Gene Research Center, University of Tsukuba, Tsukuba 305-8572, Japan; 3Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, DAPO Box 7777 Metro Manila, Philippines; 4Rice Research Center, Thanyabury, Pathum Thani 12110, Thailand; 5National Agriculture Research Center-Vientiane, P.O. Box 811, Napok, Vientiane, Lao PDR; 6Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, China; 7Graduate School of Science and Technology, Chiba University, Matsudo 271-0092, Japan; 8Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan; 9 Research Institute for Humanity and Nature, 335 Takashima-cho, Marutamachi-dori Kawaramachi nishi-iru, Kamigyo-ku, Kyoto 602-0878; *Author for correspondence (e-mail:
[email protected]; phone: +81-172-39-3778; fax: +81-172-39-3750) Received 4 September 2003; accepted in revised form 20 February 2004
Key words: Genetic erosion, Isozyme, Northern Thailand, Plastid type, Upland rice (Oryza sativa L.)
Abstract The purpose of this study was to assess the extent of genetic erosion of traditional upland germplasm in northern Thailand as a result of gene-flow from distinct strains carrying different genotypes. Even modern variety specific markers have not been developed, there is a comparative population in Laos. Thus, both populations were compared with various characters to evaluate gene-flow from modern variety to landraces. Glutinous and glabrous strains are predominated in Laos. However, such strains were drastically decreased in north–east Thailand. Gene diversity is higher in Thailand, compared to Laos at seven isozyme loci. This was a result of the higher frequencies of Indica strains and heterozygotes in Thailand. Plastid type was also determined by using an INDEL marker. Nearly half of Indica strains carried the Japonica plastid. Heterozygotes also tended to carry Japonica cytoplasm. Such nuclear–cytoplasm substituted strains and heterozygotes were probably generated by natural hybridization. Japonica strains tended to be a maternal donor rather than Indica ones. Or Indica strains would easily release pollens, which grow outside of upland fields.
Introduction Rice (Oryza sativa L.) landraces are an important gene resource for the resistance of pests, fungus, and abiotic stress, which have not always been incorporated into modern varieties in recent rice breeding programs yet. Collection and evaluation of landraces are now part of the fundamental work
of rice geneticists, to supply new tools to rice breeders (Vaughan 1991). Primitive upland cultivars are one of these useful landraces, and have been traditionally cultivated by minority people mainly in mountain areas of Southeast Asia (Sato 1987, 1991; Oka 1988). Upland rice fields are maintained by the slash and burn farming system, in which forests are first
246 slashed and then burned to create upland fields. Generally, upland cultivars are grown during the rainy season without irrigation, depending only on rainfall. Glaszmann (1987) and Sato T. (1994) reported that these cultivars were characterized by the waxy (glutinous) endosperm, non-apiculus hair (glabrous), and Japonica specific alleles for particular isozyme loci. Detail for isozyme polymorphism suggested that primitive upland landraces were classified into Japonica type. According to IRRI, farmers tend to abandon landraces for modern varieties in Southeast Asia, where the economic situation is rapidly changing. Recent spread of modern varieties over local area and rapid change of the economic situation in developing countries have made it difficult to collect landraces as genetic resources, especially in Thailand (Sato Y.-I. 1994). Nan province locates in the northern region of Thailand faced the border of Laos, upland rice cultivation is still predominant. Recently, however, modern varieties are being introduced into paddy fields around upland fields. Thus, environmental condition surrounding landraces are rapidly changing. As soon, traditional cultivation style and landraces may be lost. It is very severe situation to maintain natural bio-diversity which will be necessary for rice breeding conquering many kinds of obstacles for cultivation at various environmental conditions world wide. Modern varieties cultivated nearby landraces result in outcrossing with upland landraces. This may generate weedy rices and make the progeny population polymorphic (Ishikawa et al. 2003). But that type of bio-diversity will not be useful for plant breeding, but contaminate natural diversity. However, this type of gene-flow is unknown well. In this report, we examined how much pseudo bio-diversity existed in northern Thailand when compared with Laotian population where primitive upland cultivation is still managed. Easily handling genetic markers such as isozymes and PCR mediated molecular markers were performed to evaluate these population structures. Isozymes have been used as well-known genetic markers to evaluate genetic resources in rice (Glaszmann 1987; Sano and Morishima 1992). Plastid DNA markers used in this report gave us critical clues to know the direction of gene-flow and maternal genetic background in particular
populations (Nakamura et al. 1997; Ishikawa et al. 2002a, b).
Materials and methods Upland landrace Thirty-nine upland cultivars as landraces were collected from 10 sites where upland fields were maintained by the slash and burn system at Nan province, northern Thailand in 1999 (Figure 1). In total, 10 sites were visited, NN71A (Table 1). Seeds were collected on a single plant basis. Such seed samples from single plants were treated as single strains. Strains representing different phenotypes were collected as different ones even in single fields. In order to assess of these genetic resources, we compared respective characters described as below with another kinds of upland cultivars collected from Laos. The collection sites in Laos were near Nan province, Thailand. Strain designation was same as described in the above. One hundred and thirty-one strains collected in Laos were examined as comparative primitive population. Isozyme genotypes of Laotian population were mentioned in our previous report (Ishikawa et al. 2002c). In total, 131 strains were examined for morphological characters. Among them, 123 strains were next examined for plastid type to compare with Thailand population.
Field observation and character investigation Apiculus hair (Aph) and spikelet size were recorded for all strains. Presence of Aph was recorded as Aph + and absence as glabrous or . Based of field observation, plants with no hairs on spikelets were also glabrous for leaf surface. Thus, the variation was due to the particular allele for Gl locus. Endosperm characteristics governed by Wx locus was recorded as non-glutinous (+) or glutinous (). The precise collection sites were documented with global positioning system (GPS) from GERMIN (EMPEX) Co. Ltd. as shown in Figure 1 and Table 1.
247
Figure 1. A map showing the observation sites (NN71–80) in Nan province, Thailand. A. Geographical distribution of observation sites. B. Altitude of each site (meters above the sea).
248 Table 1. Locations of visited fields in northern Thailand.
Site NN71a NN71b NN72 NN73 NN74 NN76 NN77 NN78 NN79 NN80 Total
Altitude (m)
Latitude
Longitude
240 290 520 440 220 450 740 550 320 250
N18 510 2600 N18 510 3200 N18 510 4700 N18 510 3300 N18 590 21 N19 340 1900 N19 340 5600 N19 350 4900 N18 450 5100 N18 440 1800
E100 380 5700 E100 390 0700 E100 360 3900 E100 320 5900 E100 460 0800 E100 560 1100 E101 000 4100 E101 040 3300 E100 590 3700 E101 000 4200
No. of strains 3 3 1 4 3 5 6 2 10 2 39
Isozyme analysis Nuclear genotypes of collected strains were examined for seven genes, Acp1, Amp2, Cat1, Est2, Pgi1, Pgi2, and Pox2. Seeds were germinated at 30 C in darkness. Crude extracts of three-day-old seedlings were used for isozyme analysis as described by Ishikawa et al. (1991). Because of existence of heterozygotes, more than two seeds were used to extract isozymes to detect heterozygotes.
tissues (100 mg). PCR amplification was performed with Taq DNA polymerase (Takara Co.) to examine the ORF100 region of rice chloroplast DNA (cpDNA) that was characterized with a primer set, ORF/ORF2 (Kanno et al. 1993). The INDEL marker can discriminate plastid types into Indica and Japonica. The company’s recommended buffer, 0.2 mM dNTPs, and 2 mM MgCl2 in final concentration, and the primers was used. After 3 min heat treatment at 94 C, 45 cycles (98 C for 10 s, 60 C for 30 s and 72 C for 1 min) were used in the amplification followed by 72 C for 5 min as post-treatment. PCR products were then electrophoresed in 2% agarose gel and classified into deletion (D) and non-deletion (ND) types. D type plastids have 69 bp deletion in ORF100, which is consistent with Indica nuclear markers generally. ND type is consistent with Japonica nuclear markers. This plastid marker is effective in evaluation of plastid origin (Chen et al. 1993). Plastid subtype ID (PS-ID) sequences based on the polymorphic DNA sequences of the linker sequences between rpl14 and rpl16 (Nakamura et al. 1997) were used to confirm plastid origin in detail.
Discrimination score Plants examined for isozyme genotypes were next classified into Indica or Japonica types by using the discrimination scores (Ishikawa et al. 1991; Sano and Morishima 1992). Genotypic diversity was calculated for seven isozyme loci, Acp1, Amp2, Cat1, Est2, Pgi1, Pgi2, and Pox2. We used the scores given to alleles at the above seven loci. Each allele was known to be specific to Indica or Japonica type. Therefore, discrimination scores were given to each allele in our previous study (Ishikawa et al. 1991). Based on genotypes, their discrimination scores were averaged over the above seven loci. When the scores were 0.0–0.4 and 0.6–1.0, these cultivars were regarded as Japonica and Indica type, respectively. This classification gives us clear differences of their scores. Heterozygotes were excluded from calculation of discrimination scores. Polymorphism in chloroplast DNA Total genomic DNA was isolated by the CTAB method (Murray and Thompson 1980) from leaf
Results Morphological and physiological characters In Thailand, a total of 39 strains collected from 10 sites were examined. In the investigation of endosperm, 31 out of 39 strains (79%) were glutinous (waxy) and eight were non-glutinous (Table 1). Glutinous endosperm was predominated in Laotian mountain areas (79%). There was no difference for the frequency of glutinous rice strains. It was due to the preference of farmers in both countries. In general, glabrous strains were a unique character in addition to the glutinous trait. Ninety-eight strains (74%) in Laos were glabrous. Significant reduction of glabrous strains was found in Thailand. Only 17 out of 22 strains (44%) were glabrous. Glabrous and glutinous characters were not correlated with each other in Thailand. There were no obvious differences for any spikelet size characters. Even relative larger grain size was
249 Table 2. Spikelet size variation and other characters of collected strains. Spikelet size (mean ± SD)
Thailand Laos
Wx
Aph
No. of strains
Length (mm)
Width (mm)
Length/width ratio
+
+
39 131
9.38 ± 0.77 9.56 ± 0.92
3.78 ± 0.54 3.70 ± 0.51
2.5 ± 0.4 2.6 ± 0.4
8 27
31 103*
22 33
17 98
* One strain was sterile. Thus, the endosperm character could not be counted.
Table 3. Allelic frequencies at seven isozyme loci and the genotypic diversity. Allele Locus
Country
0
1
2
1/2
Total
Diversity
Acp1
Thailand Laos Thailand Laos Thailand Laos Thailand Laos Thailand Laos Thailand Laos Thailand Laos
– – – – – – 10 70 – – – – 5 121
15 21 21 111 15 102 22 49 16 19 18 109 34 10
23 110 15 19 22 27 7 11 20 108 17 21 – –
1 0 3 1 1 2 0 1 3 4 4 1 – –
39 131 39 131 38* 131 39 131 39 131 39 131 39 131
0.503 0.284 0.520 0.262 0.525 0.350 0.584 0.568 0.563 0.299 0.586 0.282 0.223 0.140
Amp2 Cat1 Est2 Pgi1 Pgi2 Pox2
* One strain could not be scored.
generally found in Japonica type strains in tropical area. Characterization of nuclear genes by isozyme genotypes Isozyme polymorphism was much higher in the Thailand population. Genotypic diversity was calculated at seven loci (Table 2). The scores ranged from 0.223 for Pox2 to 0.586 for Pgi2. In Laos, these scores ranged from 0.140 for Pox2 to 0.568 for Est2. Except for Est2, the scores were much less than those in the Thailand population. At the Est2 locus, multiple alleles made the score relatively higher (Table 3). Except for Pox2, the examined isozyme genes carry co-dominant alleles. Thus, they easily represented heterozygotes. Such heterozygotes were frequently found in Thailand population. At NN79 site, we collected much more samples at this site. It
was due to the extraordinary variation found in the site. Four of the 10 strains were recognized as heterozygotes (data not shown). Discrimination scores as multi-locus analysis based on the isozyme genotypes, showed that 16 Indica, 16 Japonica, 1 intermediate, and 6 heterozygotes. The frequency of Indica strains (41%) in Thailand was much higher than that in Laos (13%). It was also notable that the frequency (15%) of heterozygotes in Thailand was considerably higher than in Laos (5%) (Table 4). Cytoplasmic genotype and the relationship with nuclear genotype Maternal genotypes were determined by the polymorphism of ORF100 region in cpDNA (Figure 2). Twenty-seven out of the 39 Thailand strains were ND type and 12 remainders were D type. These types were reconfirmed with the linked PS-ID
250 Table 4. Comparison of plastid–isozyme discrimination between two countries. Frequency of isozyme discrimination2 (%) Country
Plastid type1
J
M
I
Heterozygote3
Total
Thailand
ND D ND D
15(38) 1 (3) 98(80) 0 (0)
1(3) 0(0) 1(1) 1(1)
7(18) 9(23) 4 (3) 12(10)
4(10) 2 (5) 6 (5) 1 (1)
27(69) 12(31) 109(89) 14(11)
Laos 1
ND: non-deletion type, D: deletion type. J, M, and I indicating Japonica, intermediate, and Indica types, respectively. These classifications were based on the (D score). 3 Heterozygotes were detemined by isozyme genotypes. D scores of heterozgotes were not calculated. 2
Discussion Genetic erosion in Nan province Figure 2. Cytoplasmic origin of strains detected by INDEL marker, ORF100. D/ND; mixed loading of two kinds of PCR products amplified with template DNA of Indica and Japonica types plastids. Size of D and ND are 1000 and 900 bp, respectively. J and M; Japonica strains and an intermediate strain carried ND type plastid. I and H; Indica and heterozygotes carried both types of plastid types.
sequence. ND type was consistent to 6C7A and D type to 8C8A, but not other combination was found. Fifteen of 16 Japonica strains carried ND type of cytoplasm, but the rest one carried D type (Table 4). Intermediate type for multi-locus discrimination (isozyme discrimination) carried ND type. Among Indica type strains, seven carried ND type and nine carried D type. Heterozygotes revealed four ND type strains and two D type ones. Among 131 upland strains in Laos, 123 strains were next examined plastid type. A 109 strains carried ND type and 14 carried D type. All Japonica type strains carried ND type. Among 16 Indica type strains four of them carried ND type and the rest carried D type. Six out of seven heterozygotes carried ND type. The frequency of the Indica strains with ND type was 18% in Thailand, which was much higher in Laos (3%). In both countries, heterozygotes tend to carry ND type. There were 10% or 5% Indica strains carrying ND type.
We found that traditional upland cultivars are still grown in northern Laos with the slash and burn system (Ishikawa et al. 2002c; Yamanaka et al. 2002). In such traditional upland fields, farmers generally cultivate Japonica landraces with glutinous endosperms and glabrous hulls. This is partly due to the preference for glutinous endosperm among native people in these mountain areas. The low frequency of modern varieties may also be due to the traditional cultivation style performed in the upland fields; chemical fertilizer has never been used in traditional slash and burn fields in Laos. It would result in low productivity of modern varieties under the condition. Geographical and ecological conditions in the Thailand–Laos border areas were quite similar. We could not see any difference for utilization and preference of glutinous rice by local people. They prefer to eat glutinous rice grains. In Thailand, however, Indica modern varieties are introduced and cultivated near or in upland fields, where they are not cultivated traditionally (interviewed from native farmers done in the trip by us). These modern varieties were also cultivated in paddy fields near upland fields in middle part of Laos (Ishikawa et al. 2002c). In Nan of Thailand, Indica strains (41%) and non-glabrous strains (51%) which have never been there in general, were found in high frequency. This situation is now being accelerated based on our field
251 observation and molecular studies done in this study. Because of out-crossing, introgression of the germplasm from the modern varieties into landrace will increase not only in northern Thailand but also Laos soon. We have not developed the molecular markers specific to modern varieties. Instead of such markers, Indica specific markers helped us to estimate how much frequency such out-crossing happened in these countries. Of course, Indica type landraces should be also found in upland fields. However, such particular Indica landraces are really rare. Thus, we speculate lowland landraces would be the source of pollens resulted in outcrossing. Hybridization Spontaneous crop-wild relative hybridization has been reported in grass and other species (Ellstrand and Hoffman 1990). As for the case of maize, geneflow was molecularly examined among Zea species (Doebley 1990). Gene-flow was found in both directions, wild to cultivars and vise versa. In contrast to cross-pollinated plants, rice plants are generally regarded as self-pollinated plants. About 1% cross-pollination could happen naturally (Morishima et al. 1992). Our results, however, suggest that Indica–Japonica out-crossings have occurred frequently in Nan province and the direction of gene-flow was mainly from Indica to Japonica estimated by the frequency of the cytoplasm donor. Higher frequency of Japonica maternal origins in heterozygotes suggested that outcrossing events in Thailand, would usually happen with Japonica cultivars as cytoplasm donor. It would be due to that the primitive upland cultivars are generally Japonica type occupied most upland fields. As upland cultivars carried the ND type cytoplasm, we easily distinguished from Indica modern varieties carrying D type. Nuclear and plastid data were summarized with the site information. Indica strains and heterozygotes were frequently found near airport. It suggests that earlier introduction of modern varieties into urban areas made them easily penetrate to landraces near the fields. Such gene-flow was much lower in traditional upland fields like in Laos. It would be due to the much wide introduction of modern varieties
around upland fields in Thailand. Gene-flow between different types of cultivars is easily happened where those cultivars are closely cultivated with each other (Ishikawa et al. 2002c, 2003). In the case, such introgression generated weedy strains. In fact, we observed strains with lower fertility in Thai fields. Some of them would turn to be weedy type and others would to be nuclear–cytoplasm substituted strains. Rarely they would be intermediate type as seen in this report. This phenomenon may explain by our previous data referred as non-random association of Indica–Japonica traits (Sato et al. 1990). Wild relatives also face the risk of hybridization with modern varieties. They have been confirmed by several traits (Suh et al. 1997; Tang and Morishima 1998). In some cases, such hybridization may generate new cultivar types. Our previous report described that spontaneous out-crossing events might take part in generation of new ecospecies (Ishikawa et al. 2002b). Even in Japanese lowland rice strains, we can detect nuclear–cytoplasmic substituted strains. As seen in this report, there are frequent out-crossing events in upland fields, evaluated by the frequency of heterozygotes and nuclear– cytoplasm substituted strains. Thus, there is a relatively high risk of gene-flow from modern varieties carrying genes for high productivity into primitive landraces where both strains are in sympatric (Ishikawa et al. 2003). It is probable that same situations arise in many developing countries (Sato 1994). Current improvement of genetic engineering technology will also create other problems. Genetically modified (GM) rice plants will soon appear as popular varieties carrying high potential productivity and stress resistance. If such GM plants take part in introgression with such new genetic characters, hybridized progeny will disturb natural populations so rapidly. We are now preparing rice Bio-resource database to assess any genetic resources. The database will enable us to know on-going genetic erosion when compared the genetic resources in Asian countries with de novo collections. These resources have been accumulated with the help of native scientists in each country in the past. We hope the data-base will help all of us to access genetic resources on demand.
252 Acknowledgements This study was supported in part by Grant-in-Aid (No. 10041162) for scientific research and in part by Bio-resource project from the Ministry of Education, Science and Culture, Japan.
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