RFLP tagging of a salt tolerance gene in rice

A salt tolerant rice mutant (M-20) was obtained through selection in vitro. Its tolerance was stably inherited over eight generations and most traints between M-20 and its sensitive original 77–170 (Oryza sativa) were very similar. By deriving an F₂ population of M-20 × 77–170 and splitting every F₂ individual into two parts, with one part planted in normal conditions and another part in saline conditions, the inheritance of salt tolerance in rice was studied. Under normal conditions, there was no apparent segregation among F₂ individuals. Under saline conditions, however, the segregation of traits was obvious. According to our standards, the ratio of salt sensitive:moderately-tolerant:tolerant plants was 25:42:18, in accordance with a 1:2:1 ratio. It suggested that the improvement of salt tolerance in our materials was induced by the mutation of a major tolerant gene which showed incomplete dominance. By use of 130 RFLP probes distributed throughout the rice genome, the gene was tagged by a single copy DNA probe, RG4, which was located on chromosome 7. The genetic distance between the salt tolerant gene and RG4 was 7.0 ± 2.9 cM. Based on the split method, a method which could be currently used to evaluate the damage of salt stress in rice was proposed.     

Comparison of Some Characteristics Between Phragmites communis and Its Salt Tolerant Variant

R5002-12, a salt tolerant line of Phragmites communis Trin., which was obtained from ethyl methane sulfonate (EMS) treated callus selected under saline stress, was compared with its wild line in respect to their molecular biological, physiological and biochemical characterizations. Five arbitrary primers were screened which showed differences in DNA amplified polymorphism between the variant and its wild line. Some new proteins appeared in the salt tolerant plant under salt stress. Electrophoresis of peroxidase and esterase also showed some differences in isozyme expression between them. The chlorophyll content of the variant was higher than that of the original variety, whether the plants were under salt stress or not.     

芦苇耐盐变异体与野生型植株某些特性的比较

地通过细胞工程方法得到的耐盐芦苇（Ｐｈｒａｇｍｉｔｅｓ ｃｏｍｍｕｎｉｓ Ｔｒｉｎ．）变异系Ｒ５００２－１２与野生型植株进行了分子生物学和生理生化特性比较分析。筛选到５个ＲＡＰＤ引物对变异体和野生型植株的ＤＮＡＦ的随机扩增表现出不同的多态性,表明该变异体在分子水平上发生了变化。生理生化分析的结果表明,平行生长状态下的两种株系,其可溶性蛋白和同工酶的表达水平和种类不一样,变异体在ＮａＣｌ胁迫下,表达     

水稻零等位RFLP标记的遗传学研究

采用8种限制性内切酶与247个已定位到水稻12条染色体上的分子探针组合,对典型籼稻(Oryza sativa L.ssp.indica)“南京11”和粳稻(O.sativa L.ssp.japonica)“金南凤M”以及中间型品种“Bellemont”之间存在的零等位位点进行了分析,9个探针揭示出零等位位点的存在。其中,RG573、G122、G93、G402和G1318特异揭示了“南京11”的零等位,G261特异揭示了“金南凤M”的零等位,RG229特异揭示了“Bellemont”的零等位,C397揭示了“南京11”和“Bellemont”的零等位,而RG131揭示了“金南凤M”和“Bellemont”的零等位。利用“南京11”/“金南凤M”的杂交F_2群体,对其中58个单株进行了7个零等位RFLP标记及程氏指数法中的6个形态性状的测验,据此计算出各植株的零等位基因型及籼、粳分类指数,结果表明:零等位标记在F_2群体中呈显性遗传。在F_2群体中进一步对G93、G122、G261、G402、G1318、C397、RG573零等位标记与程氏指数之间进行了成组数据t测验,发现G93、G122、G402、G1318、G397差异达显著或极显著水平,G261、RG573的t值也较大,显示出零等位标记在籼、粳交后代,可特异鉴定籼、粳差异。     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

Genetic Analysis of Null Alleles in Rice Doubled Haploid Plants from Anther Culture

In the research of constructing a rice(Oryza sativa) molecular map, 4 RFLP markers, i.e. RG 229, RG 419, RG 424 and RG 353, detected the null alleles in the indica and japonica parental rice. RG 229 indicated two null alleles in indica rice Gui 630, and each of the other markers revealed a null allele in japonica rice 02428. Genetic analysis in the doubled haploid (DH) population consisting of 81 plants showed that the linkage relationships between these null allele loci and the neighboring molecular markers shown in McCouch' s rice molecular map were changed. In addition, the marker RG 684 could detect its null alleles in some DH plants, though the RG 684 sequence did exist in the genomes of both parents. Appearence of null alleles might be induced by transpositional changes on chromosomes.     

水稻苗期耐盐突变体的遗传分析及基因定位

在籼稻品种R401辐射诱变的M2群体中筛选到一个苗期耐盐突变体, 在150 mmol/L的NaCl溶液处理下对照植株枯萎死亡, 而突变体植株依然存活。以粳稻品种Nipponbare(不耐盐)和耐盐突变体作亲本, 构建了一个F2群体, 调查该群体在150 mmol/L的NaCl溶液胁迫下的表现, 发现Nipponbare和耐盐突变体苗期耐盐性的差异受单个主基因控制, 耐盐为隐性, 将该基因暂时命名为SST(t)。利用该F2群体, 采用集团分离分析(Bulked segregant analysis, BSA)法将SST(t)定位在第6染色体上, 进一步对F2群体中137个典型的耐盐单株的分子标记进行分析, 将该基因定位在InDel标记ID26847和ID27253之间, 约2.3 cM (或406 kb)的区间内, 与两标记分别相距1.2 cM和1.1 cM。     

Rice allergenic proteins, 14-16 kDa albumin and alpha-globulin, remain insoluble in rice grains recovered from rice miso (rice-containing fermented soybean paste)

The rice grains (RG) and rice seed proteins remaining in rice miso were investigated with a view point to the potential allergenicity of rice miso. RG ranging from 36 to 180 mg dry weight per g dry miso were separated from several samples of commercially available rice miso. Scanning electron microscopy of the recovered RG indicated that starch granules disappeared almost completely while protein bodies remained intact in RG. Most of the major seed proteins were extracted from RG by heating with 1% SDS/2% 2-mercaptoethanol and detected by SDS-polyacrylamide gel electrophoresis. Major rice allergenic proteins, 14-16 kDa albumin (Alb14-16) and alpha-globulin (alpha-Glb) were also detected by immunoblotting using the specific antisera, and their contents were estimated to be 1.7 to 9.0 and 1 to 7 mg protein per g dry RG respectively. However, the major rice proteins, including glutelin and prolamin, in RG were insoluble in salt, alcohol, and urea solutions, but soluble in 6 M guanidine hydrochloride (Gu-HCl). By immunoblotting and ELISA, no Alb14-16 and only a slight amount of alpha-Glb were detected even in the 6 M Gu-HCl fraction, indicating that these major allergenic proteins are denatured and are present in an insoluble form in rice miso.     

小麦耐盐细胞系及后代耐盐稳定性的生化分析

以离体培养筛选出的耐盐小麦变异系及其后代为材料,对其耐盐稳定性进行有关生理生化特性分析。结果表明：（１）随着耐盐愈伤组织耐盐能力的提高,过氧化物同工酶酶带谱加,与对照相比,三个区域出现了明显差异;（２）从耐盐愈伤组织酯酶同工酶酶谱发现,耐盐愈伤组织具有Ｃ２,Ｃ５,Ｃ９,Ｃ１１新增加酶带;（３）耐盐系后代幼苗能维持较高的Ｋ＾＋／Ｎａ＾＋比值,在盐浓度为０．９％时,其Ｋ＾＋／Ｎａ＾＋为０．７８８,而对     

Construction of a BAC contig containing the xa5 locus in rice

 The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F₂ population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Received: 12 January 1998 / Accepted: 27 May 1998     

Non-allelic interaction conditioning spikelet sterility in an F2 population of indica/japonica cross in rice

Significant segregation of spikelet fertility occurred in an F₂ population derived from a spikelet fertility-normal F₁ hybrid produced by a cross between Palawan, a japonica variety, and IR42, an indica variety. To identify factors controlling the fertility segregation, we used 104 RFLP markers covering all 12 rice chromosomes to investigate the association of spikelet fertility and marker segregation. We found that the segregation of two sets of gene pairs was significantly (P < 0.001) associated with fertility segregation. The first pair of genes was linked to RFLP marker RG778 on chromosome 12 and RFLP markers RG690/RG369 on chromosome 1. A significant reduction in fertility was observed when the plants were homozygote at RG778 with the indica allele as well as homozygote at RG690/RG369 with the japonica allele. The second pair of genes was linked to RG218 on chromosome 12 and RG650 on chromosome 7, respectively. The recombinant homozygote at these two loci showed a significant reduction on spikelet fertility. The non-allelic interaction effect was further modified by a gene linked to RG778, resulting in even lower fertility. The results of this study provides the first evidence of chromosomal localization of sporophytic sterility genes whose interaction can result in a reduction of spikelet fertility in the F₂ derived from fertility-normal F₁.     

Mapping of quantitative trait loci for salt tolerance at the seedling stage in rice

Salt tolerance was evaluated at the young seedling stage of rice (Oryza sativa L.) using recombinant inbred lines (MG RILs) from a cross between Milyang 23 (japonica/indica) and Gihobyeo (japonica). 22 of 164 MG RILs were classified as tolerant with visual scores of 3.5-5.0 in 0.7% NaCl. Interval mapping of QTLs related to salt tolerance was conducted on the basis of the visual scores at the young seedling stage. Two QTLs, qST1 and qST3, conferring salt tolerance, were detected on chromosome 1 and 3, respectively, and the total phenotypic variance explained by the two QTLs was 36.9% in the MG RIL population. qST1 was the major QTL explaining 27.8% of the total phenotypic variation. qST1 was flanked by Est12-RZ569A, and qST3 was flanked by RG179-RZ596. The detection of new QTLs associated with salt tolerance will provide important information for the functional analysis of rice salt tolerance.     

耐盐水稻种质资源的筛选

用0.8% NaCl溶液和国际水稻所水稻耐盐性9级评价方法对前人选留的38份水稻耐盐种质资源(用0.5%NaCl盐土筛选)进行了重复筛选,以期获得极端耐盐水稻种质资源.同时研究了不同盐浓度对水稻发芽率的影响.结果表明:高浓度盐溶液显著抑制水稻种子的发芽率,但品种间有较大差异;筛选出苗期极端耐盐品种6份,其中1级1份,2级4份,3级1份;极端耐盐水稻品种苗高的增长受高浓度盐溶液抑制,但品种间差异不显著;而盐溶液对极端耐盐水稻品种出叶速率的影响因品种而异.     

Proteomics Reveals New Salt Responsive Proteins Associated with Rice Plasma Membrane

The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.     

Proteomics reveals new salt responsive proteins associated with rice plasma membrane

The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.     

Mapping QTLs for phosphorus deficiency tolerance in rice (Oryza sativa L.)

 The amplified fragment length polymorphism (AFLP) technique combined with selective genotyping was used to map quantitative trait loci (QTLs) associated with tolerance for phosphorus (P) deficiency in rice. P deficiency tolerant cultivar IR20 was crossed to IR55178-3B-9-3 (sensitive to P-deficiency) and 285 recombinant inbred lines (RILs) were produced by single-seed descent. The RILs were phenotyped for the trait by growing them in P-sufficient (10.0 mg/l) and P-deficient (0.5 mg/l) nutrient solution and determining their relative tillering ability at 28 days after seeding, and relative shoot dry weight and relative root dry weight at 42 days after seeding. Forty two of each of the extreme RILs (sensitive and tolerant) and the parents were subjected to AFLP analysis. A map consisting of 217 AFLP markers was constructed. Its length was 1371.8 cM with an average interval size of 7.62 cM. To assign linkage groups to chromosomes, 30 AFLP and 26 RFLP markers distributed over the 12 chromosomes were employed as anchor markers. Based on the constructed map, a major QTL for P-deficiency tolerance, designated PHO, was located on chromosome 12 and confirmed by RFLP markers RG9 and RG241 on the same chromosome. Several minor QTLs were mapped on chromosomes 1, 6, and 9. Received: 21 April 1998 / Accepted: 9 June 1998     

Primary Study of Rice AFLP Analysis-Optimization of Reaction Conditions and Analysis of Thermo sensitive Genic Male Sterile Rice Allelic Mutant Lines

AFLP analysis was performed between a pair of thermo-sensitive genic male sterile (TGMS) rice allelic mutant lines (5460S and 5460F). The reaction conditions for rice AFLP assay were optimized. The relative efficiencies for polymorphism detection of RFLP, RAPD and AFLP were compared. The results indicated that the efficiency for polymorphism detection in rice was in the order of AFLP > RAPD > RFLP, and also indicated that AFLP was a powerful DNA molecular marker technique for polymorphism detection, especially in the case of extremely low polymorphism, such as isogenic lines and allehc mutant hnes. Some of the AFLP products between the TGMS rice allehc mutant lines were cloned. Three of them were used as mixed probes to screen BAC library of rice line 5460S. 12 positive clones were screened out. In addition, the advantages and disadvantages of these three molecular marker systems were discussed.     

Effects of genetic background and environment on QTLs and epistasis for rice (Oryza sativa L.) panicle number

A double-haploid (DH) population and a recombinant inbred (RI) line population, derived from a cross between a tropical japonica variety, Azucena, as male parent and two indica varieties, IR64 and IR1552, as female parents respectively, were used in both field and pot experiments for detecting QTLs and epistasis for rice panicle number in different genetic backgrounds and different environments. Panicle number (PN) was measured at maturity. A molecular map with 192 RFLP markers for the DH population and a molecular map with 104 AFLP markers and 103 RFLP markers for the RI population were constructed, in which 70 RFLP markers were the same. Six QTLs were identified in the DH population, including two detected from field experiments and four from pot experiments. The two QTLs, mapped on chromosomes 1 and 12, were identical in both field and pot experiments. In the RI population, nine QTLs were detected, five QTLs from field conditions and four from the pot experiments. Three of these QTLs were identical in both experimental conditions. Only one QTL, linked to CDO344 on chromosome 12, was detected across the populations and experiments. Different epistasitic interaction loci on PN were found under different populations and in different experimental conditions. One locus, flanked by RG323 and RZ801 on chromosome 1, had an additive effect in the DH population, but epistatic effects in the RI population. These results indicate that the effect of genetic background on QTLs is greater than that of environments, and epistasis is more sensitive to genetic background and environments than main-effect QTLs. QTL and epistatic loci could be interchangeable depending on the genetic backgrounds and probably on the environments where they are identified. Received: 26 May 2000 / Accepted: 19 October 2000     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998