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10 September 2007, Volume 21 Issue 5 Previous Issue    Next Issue
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研究报告
实验技术
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研究报告

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InDel and SNP Markers and Their Application in Map-Based Cloning of Rice Genes PAN Cun-hong ,WANG Zi-bin ,MA Yu-yin ,YIN Yue-jun ,ZHANG Ya-fang ,ZUO Shi-min ,CHEN Zong-xiang ,PAN Xue-biao 2007, 21(5): 447-453 .  Abstract ( )   PDF (1095KB) ( )   The high-density markers are necessary for map-based cloning of rice genes,but the available marker could not reach the satisfying level.InDel(insertion-deletion length polymorphism) and SNP(single nucleotide polymorphism) are new molecular markers,which could basically meet the need of the fine mapping.InDel and SNP markers could be developed through bioinformatics approaches.InDel and SNP markers are valuable markers with the characteristics of low cost,high specificity and stability.The methods for designing InDel and SNP markers were introduced and the example of mapping of the rice rolled leaf gene was used to describe the design procedures in practice.In addition,some key factors for improving the design efficiency were discussed.

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Fine Mapping of C(Chromogen for Anthocyanin) Gene in Rice FAN Fang-jun ,FAN Ye-yang ,DU Jing-hong ,ZHUANG Jie-yun 2007, 21(5): 454-458 .  Abstract ( )   PDF (1445KB) ( )   Seven residual heterozygous lines(RHLs) displaying different genotype compositions in the genomic region covering probable locations of C(chromogen for anthocyanin) gene on the short arm of rice chromosome 6 were selected from the progenies of the indica cross Zhenshan 97B/Milyang 46.Seeds were harvested from each of the seven plants,and the resultant F2∶3 populations were used for fine mapping C gene.It was shown in the populations that the apiculus coloration matched to basal leaf sheath coloration in each plant.By relating the coloration performances of the populations with the genotype compositions of the RHLs,the C locus was located between rice SSR markers RM314 and RM253.By using a total of 1279 F2∶3 individuals from two populations showing coloration segregation,the C locus was then located between RM111 and RM253,with genetic distances of 0.7 cM to RM111 and 0.4 cM to RM253.Twenty-two recombinants found in the two populations were assayed with seven more markers located between RM111 and RM253,including six SSR markers and one marker for the C gene candidate,OsC1.The C locus was delimited to a 59.3-kb region in which OsC1 was located.

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MITE-TRAP:A Marker Technique Based on Miniature Inverted Repeat Transposable Element and Target Region Ampilifcation Polymorphism for Rice and Other Plants MA Zhong-you ,SU Jing-ping ,SUN Lin-jing ,LIU Xue-jun ,WANG Chun-min ,WANG Sheng-jun ,ZENG Bin ,LIANG Yong-shu ,YAN Shuang-yong 2007, 21(5): 459-463 .  Abstract ( )   PDF (1028KB) ( )   A MITE-TRAP marker method was developed,in which the fixed primer,designed from the miniature inverted repeat transposable element(MITE) sequence,was instead of EST or gene sequence in target region amplification polymorphism(TRAP) technique,and the arbitrary primer was the same as that in TRAP.As different rice materials were analyzed by using the fixed primer designed from the MITE sequence in rice,each PCR reaction could amplify one or several clear bands with sizes ranging from 100-1500 bp on a 1.5% agarose gel,which possessed better reproducibility.Some polymorphic bands in these rice materials were also obtained.Moreover,this method could be applied in other plant species directly,such as cotton,tomato and Arabidopsis.Some advantages of the method and its application were discussed

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Establishment of Sequence Related Amplification Polymorphism and Segregation of the Polymorphic Loci in F2 Population for Rice JIANG Shu-kun ,ZHONG Ming ,XU Zheng-jin ,ZHANG Li ,ZHANG Xi-juan ,MA Hui ,CHEN Li-jing ,GUO Zhi-fu 2007, 21(5): 464-468 .  Abstract ( )   PDF (1358KB) ( )   An effective PCR protocol for detecting sequence related amplified polymorphism(SRAP) in rice was developed.SRAP markers were used for segregation analysis in an F2 population derived from a cross between Shennong 606 and Lijiangxintuanheigu.Among 110 primer pairs,35 pairs generated 143 polymorphic bands with an average of 4.09 polymorphic bands per primer pair and 24 markers(16.78%) showed the genetic distortion(P<0.05).Of them,11 markers deviated towards pollen parent Shennong 606 and 12 markers towards ovule parent Lijiangxintuanheigu,only one towards heterozygote.It was found that the segregation distortion might be caused by gamete and zygote jointly.

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Recombination Structure of bar Gene Expression Cassette in Rice Transformed by Particle Bombardment ZHAO Yan ,QIAN Qian ,WANG Hui-zhong ,GAO Zhen-yu ,YAN Mei-xian ,HUANG Da-nian 2007, 21(5): 469-474 .  Abstract ( )   PDF (1117KB) ( )   The recombination structure of bar gene cassette(containing the promoter,coding region and terminator) in rice(Oryza sativa L.) genome transformed by particle bombardment was studied by Southern blotting and primer combination PCR technology.Multiple copies of bar gene cassette were recombined and linked to 1-3 transgene arrays in rice genome.Different transgene arrays were insulated by rice genome DNA and tended to integrate in a large area of rice chromosome,lying in nearby loci and forming transgene clusters.There were three kinds of link forms among bar gene fragments including head to head,head to tail and tail to tail when forming transgene arrays,always accompanied with truncation.

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Rapidly Obtaining the Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture ZHU Li ,FU Ya-ping ,LIU Wen-zhen ,HU Guo-cheng ,SI Hua-min ,TANG Ke-xuan ,SUN Zong-xiu 2007, 21(5): 475-481 .  Abstract ( )   PDF (1304KB) ( )   The "double T-DNA" binary vector p13HSR which carried two independent T-DNAs,containing hygromycin phosphotransferase gene(hpt) in one T-DNA region and three target genes(hLF,SB401,RZ10) in the other T-DNA region,was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation.T0 plants with the three target genes and hpt gene were selected for anther culture.It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes through co-transformation followed by anther culture,with an efficiency of 9.87%.RT-PCR analysis indicated that the target genes were inserted into rice genomic DNA and successfully transcripted.It was noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

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Transformation of ICE1 Gene Mediated by Agrobacterium Improves Cold Tolerance in Transgenic Rice XIANG Dian-jun ,ZHANG Yu ,YIN Kui-de 2007, 21(5): 482-486 .  Abstract ( )   PDF (1563KB) ( )   An Arabidopsis thaliana transcriptional factor gene ICE1 cloned by RT-PCR was successfully transferred into rice variety Kenjiandao 10 by Agrobacterium-mediated transgenic technique.PCR amplification and Southern blot analysis indicated that ICE1 had been integrated into rice genome.Compared with non-transgenic plants,transgenic lines exhibited high resistance to hygromycin B and inherited in Mendelian single-copy.Under the same low temperature stress conditions,transgenic plants had lower mortality rate and higher increasing range of proline contents compared with non-transgenic plants,suggesting that cold tolerance of transgenic rice plants with ICE1 gene was improved.

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Identification and Genetic Analysis of Fertility Restoration in Dongxiang Wild Rice YANG Kong-song ,CHEN Xiao-rong ,FU Jun-ru ,ZHU Chang-lan ,PENG Xiao-song ,HE Xiao-peng ,HE Hao-hua 2007, 21(5): 487-492 .  Abstract ( )   PDF (1510KB) ( )   Two populations of Dongxiang wild rice from Shuitaoshu and Dongtangshang were crossed with five male sterile rice lines with different cytoplasmic backgrounds,and the seed setting rate of the F1 was used to judge the existence or not of the fertility restoration in the Dongxiang wild rice.With P1,F1,P2,F2 populations,the Akaike′s information criterion(AIC) was used to identify the existence of major genes affecting quantitative traits,and when the major genes existed,the genetic effects of the major genes and polygenes and their genetic variance were estimated through segregation analysis.The seed setting rates of the F1 generations varied from 45.98% to 76.57%,suggesting that the Dongxiang wild rice had the ability of fertility restoration.One major gene and polygene mixed model was the most fitted genetic model for this trait in all the F2 populations.The heritability values of the major genes varied from 56.63% to 88.29% and those of the polygenes varied from 2.74% to 30.97%,and the total heritability values were from 63.17% to 94.01%.The major gene inheritance of the combination Zhong 9A/Dongtangshang was controlled by the additive effect without dominant effect,and the other nine combinations were complete dominant inheritance.

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QTL Analysis for Morphological and Physiological Characteristics of Flag Leaf at the Late Developmental Stage in Rice TONG Han-hua ,MEI Han-wei ,XING Yong-zhong ,CAO Yi-ping ,YU Xin-qiao ,ZHANG Shan-qing ,LUO Li-jun 2007, 21(5): 493-499 .  Abstract ( )   PDF (983KB) ( )   A recombinant inbred line(RIL) population derived from a cross between two elite indica varieties,Zhenshan 97 and HR5,was used to investigate the morphological and physiological characteristics of the functional leaves(flag leaves) at the late developmental stage in rice.A total of 26 QTLs referring the flag leaf characteristics,including morphological characteristics(length,width,and the ratio of length to width),metabolism characteristics(fresh weight,dry weight,water content),and chlorophyll content(chlorophyll a,chlorophyll b,and total chlorophyll contents) were detected,and the phenotypic variation of each QTL ranged from 4.77%-31.67%.Among them,the QTLs for the flag leaf morphology were mainly located on chromosomes 1 and 4,and the QTLs underlying the flag leaf metabolite were on chromosomes 3,7 and 10,whereas those affecting the chlorophyll contents of flag leaf were on chromosomes 3,4 and 9.Interestingly,a main QTL at marker interval RM321-MR6444 on chromosome 9 was identified, which would be beneficial for breeding late-stage vigor super-high-yielding rice.

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Influence of Genetic Drift of Restoring Gene on Seed Purity of Yuetai A,a Honglian-Type Cytoplasmic Male Sterile Line WANG Ji-feng ,LU Zuo-mei 2007, 21(5): 500-506 .  Abstract ( )   PDF (48KB) ( )   Seed samples of Yuetai A,a Honglian-type cytoplasmic male sterile(CMS) line were investigated to assess the seed purity and to analyze the cause of off-types by imitating the biological contaminant to Yuetai A in Nanjing,Jiangsu Province and Lingshui,Hainan Province in 2004-2006.The results showed that male sterility of Yuetai A was stable and seed impurity of Yuetai A resulted mainly from genetic drift of restoring-gene(Rf-gene) after biological contaminant.All of maintainer lines,restorer lines and thermo-sensitive genic male sterile(TGMS) lines used in the study could pollinate Yuetai A by directly penetrating Rf-gene into Yuetai A or pollinate Yuetai B led to Rf-gene into Yuetai A indirectly,and then produce F1 plants and ’iso-cytoplasm restoring-generations’.Furthermore,Yuetai A was easily outcrossing and simultaneous heading with many materials used in hybrid rice seed production,which resulted in high biological contaminant rate.After picking out all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene from drifting into Yuetai A,the seed purity of Yuetai A and Yuetai B had been raised to 100%.

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Changes of Some Agronomic Traits in japonica Rice Varieties During Forty-Seven Years of Genetic Improvement in Jilin Province,China WU Zhi-hai ,XU Ke-zhang ,ZHAO Ying-jun ,HE Xiao-liang ,WANG Xiao-ling ,LING Feng-lou 2007, 21(5): 507-512 .  Abstract ( )   PDF (2641KB) ( )   To identify changes in agronomic traits associated with yield increase,25 typical japonica rice varieties that were widely planted during the 47 years(1958-2005) were used.The grain yield was linearly increased from 6 198.68 kg/hm2 in 1958 to 12 682.8 kg/hm2 in 2005,with an average annual increase of 137.96 kg/hm2 and an annual increase rate of 2.33% according to the regression equation.Number of spikelets per panicle,grain weight per panicle,seed setting rate and total panicle number increased over released time,which were significantly and positively correlated with yield,with the highest coefficient of correlation(r=0.85**) between number of spikelets per panicle and yield.Moreover,the plant height and stem-leaf angle decreased,but leaf area index at the filling stage increased and heading date postponed over released time.The result indicated that the increase of grain yield was mainly attributed to magnifying of total sink capacity with the increase of number of spikelets per panicle as the key factor.

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Germination of Pollen and Growth Dynamic of Pollen Tube in Rice CHEN Shi-qiang ,WANG Zhong ,LIU Man-xi ,XIE Zhao-wei 2007, 21(5): 513-517 .  Abstract ( )   PDF (1350KB) ( )   The germination of rice pollen grain in vitro and the growth of pollen tube in pistil were observed with a microscope.The stigma was removed at different time after pollination to investigate seed setting rate of floret.Results showed that rice pollen grain germinated 2 min after pollination and pollen tube would penetrate stigma into style in 5-10 min,30 min later the end of pollen tube reached the bottom of ovary,then some pollen tubes arrived at the embryo sac 40 min after pollination and meanwhile a small amount of callose formed in the pollen tubes,50 min later a great deal of callose was found,and the pollen grain began to shrink.The growing speed of pollen tube in rice stigma,style and ovary was 1500,5000,5400 μm/h,respectively.The seed setting rate was quite low when the stigma was removed at 10-15 min after pollination,and then it increased gradually from 20 min to 50 min,finally it was over 60% at 50 min after pollination and tended to be stable.

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Effects of High Temperature Stress on Super Hybrid Rice Guodao 6 During Flowering and Filling Phases TAO Long-xing ,TAN Hui-juan ,WANG Xi ,CAO Li-yong ,CHENG Shi-hua 2007, 21(5): 518-524 .  Abstract ( )   PDF (2576KB) ( )   Super hybrid rice Guodao 6 was used to analyze its tolerance against natural high temperature stress with different sowing times in field and a greenhouse treatment with controlled high temperature(40-42℃) during flowering to filling was conducted in 2005 and 2006.Differences in seed setting rate under different treatments were observed and heat injury index,grain yield and yield components were analyzed.Compared with control hybrid rice Xieyou 46,Guodao 6 showed stable yield and spikelet fertility and lower heat injury index under high temperature stress,indicating higher tolerance to high temperature.The spikelet sterility was positively correlated with daily average temperature,especially with daily maximum temperature and the coefficients of correlation for Guodao 6 were 0.8604 and 0.9850(P<0.05),respectively.Meanwhile,daily maximum temperature showed less effect on seed setting rate than daily average temperature during filling,their coefficients of correlation for Guodao 6 were 0.7352 and 0.9317(P<0.05),respectively.

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Protective Roles of Brassinolide in Rice Seedlings under Heat Stress CAO Yun-ying ,ZHAO Hua 2007, 21(5): 525-529 .  Abstract ( )   PDF (3079KB) ( )   Two indica rice(Oryza sativa L.) materials,Xieqingzao B(heat-sensitive) and 082(heat-tolerant),were used to study the role of brassinolide(BR) in protection of rice seedlings from heat stress.Young seedlings were subjected to higher temperature and sprayed with 0.005 mg/L of BR.Analysis was conducted on the contents of chlorophyll,protein,malondialdehyde(MDA),electrolyte leakage,the activities of peroxidase(POD) and superoxide dismutase(SOD) and their isozymes expression levels in leaves.Under the heat stress treatment,application of BR significantly increased the contents of chlorophyll and protein,the activities of POD and SOD,and reduced the level of MDA and the leakage of electrolyte in the leaves of the heat-sensitive material Xieqingzao B,whereas BR had less effect on those of the heat-tolerant material 082,relatively.The BR treatment enhanced the expression level of POD isozymes in the leaves of both materials.Under the heat stress and BR treatment,the expression of four SOD isozymes were reduced in 082,but the expression of two SOD isozymes A and B were increased in Xieqingzao B.This suggested that BR plays an important role in protection rice seedlings from heat stress through enhancing the activities or expression levels of protective enzymes in the leaves.The materials with various heat-tolerance might differ in the mechanism of response to heat stress with BR application.

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Effects of Row-Spacing on Morphological and Eco-Physiological Characteristics in Rice YAN Chuan ,DING Yan-feng ,WANG Qiang-sheng ,LI Gang-hua ,HUANG Pi-sheng ,WANG Shao-hua 2007, 21(5): 530-536 .  Abstract ( )   PDF (3169KB) ( )   With japonica rice Wuyunjing 7 and indica rice Fengyouxiangzhan as test materials,effects of row-spacing on morphological and eco-physiological characteristics of rice were studied.The yield of medium row-spacing treatment(30 cm×13.3 cm) was the highest,while that of narrow row-spacing(20 cm×20 cm) was the lowest,with a significant difference at 0.05 level.Medium row-spacing treatment significantly diminished internode length and enhanced internode thickness,wall thickness,the number of vascular bundles of internode,and the lodging resistance compared to wide row-spacing(40 cm×10 cm) and narrow row-spacing treatments.The content of dry matter in basal internode at the heading stage and the 20th day after heading,and the amount of dry matter transferred to grain at the filling stage were the highest at the medium row-spacing treatment.Moreover,there were appropriate leaf area index and higher photosynthetic rate of flag leaf at medium row-spacing treatment.Furthermore,the distinctive differences were found in the content of CO2,light intensity,air temperature and humidity in the population among different row-spacing treatments,and medium row-spacing treatment showed ideal microclimate within the population of rice.It clearly suggested that medium row-spacing treatment is necessary for high-yielding cultivation of rice.

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Elongation of the Uppermost Internode for Changxuan 3S,a Thermo-Sensitive Genic Male Sterile Line with Elongated Uppermost Internode Gene in Rice XIAO Hui-hai ,WANG Wen-long 2007, 21(5): 537-542 .  Abstract ( )   PDF (42KB) ( )   Changxuan 3S,a thermo-sensitive genic male sterile(TGMS) line with eui gene,is derived from Pei′ai 64S by irradiation of 350 Gy 60Co γ-ray.In order to elucidate the uppermost internode elongation of TGMS line with eui gene,Changxuan 3S and its parent Pei′ai 64S were used to study the effect of temperature on panicle exsertion.At 24℃,the uppermost internode of Changxuan 3S elongated fastest from the 4th day before flowering to 0 day(flowering),being 2.1-fold as that of Pei′ai 64S,whereas it elongated slowly during the 12th to 4th day before flowering and the 1st to 3rd day after flowering.The uppermost internode of Changxuan 3S exserted from flag leaf sheath at 22℃,24℃ and 26℃,and the length of elongated uppermost internode increased with the decreasing temperature.At 28℃,the panicles of Changxuan 35 were enclosed in the leaf sheath,but the degree of panicle enclosure was obviously lower than that in Pei′ai 64S.Cytological studies on Changxuan 3S showed that the uppermost internodes elongation was attributed to the increase of cell number and cell elongation,and the latter was more significant.Moreover,the number of outermost and innermost parenchyma cells and the cell length in the uppermost internode reduced with the increasing temperature.

实验技术

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Accurate Measurement of Pasting Temperature of Rice Flour by a Rapid Visco-Analyser BAO Jin-song 2007, 21(5): 543-546 .  Abstract ( )   PDF (488KB) ( )   Pasting properties are among the most important characteristics of starch,determining its applications in food processing and other industries.Pasting temperature derived from the Rapid Visco-analyser(RVA),in most cases,is overestimated by the Thermocline for Windows software program.Here,two methods facilitating accurate measurement of pasting temperature by RVA were described.One was to change parameter setting to ’screen’ the true point where the pasting viscosity began to increase,the other was to manually record the time when the pasting viscosity began to increase and calculate the pasting temperature with the formula of(45/3.8)×(T1-1)+50 for rice flour.The latter method gave a manually-determined pasting temperature,which correlated significantly with the gelatinization temperatures measured by differential scanning calorimetry.

研究简报

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Mapping of Rice Fertility-Restoring Gene for Yinshui Cytoplasmic Male Sterility in a Restorer Line R68 LI Liang-jie ,ZHOU Hai-peng ,ZHAN Xiao-deng ,ZHUANG Jie-yun ,CHENG Shi-hua ,CAO Li-yong 2007, 21(5): 547-549 .  Abstract ( )   PDF (1589KB) ( )   An F2 population derived from the cross of Zhong 9A/R68 was used to map the fertility-restoring(Rf) gene for Yinshui cytoplasmic male sterility(CMS).A fertile bulk was constructed by pooling equal amount of 10 highly fertile lines and a sterile bulk was obtained by pooling equal amount of 10 highly sterile lines.Four hundred and thirteen pairs of SSR primers,which distributed on 12 chromosomes,were screened for polymorphism between the parents and between the two bulks.RM283 on chromosome 1 and RM5756,RM258,RM6100,RM171 on chromosome 10 were found to be polymorphic between the parents and between the two gene bulks.Ten highly fertile and ten highly sterile lines were selected from F2 population of Zhong 9A/R68 to screen the gene bulks.The results showed that the five SSR markers were linked to fertility-restoring genes.A total of eighty two-excessive sterile lines were selected from Zhong 9A/R68 F2 population to estimate the genetic distance between the five SSR markers and fertility-restoring genes respectively.A Rf gene was located on chromosome 1 with a distance of 6.7 cM to RM283,and the other Rf gene was mapped on the long arm of chromosome 10 flanked by RM5756,RM258,RM6100 and RM171 at distances of 10.4,8.0,2.4 and 4.2 cM,respectively.

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A Strain Isolated from Brown Planthopper and Its Molecular Identification ZHANG Jue-feng ,WU Hong ,CHEN Jian-ming ,ZHENG Xu-song ,CHEN Lie-zhong ,YU Xiao-ping 2007, 21(5): 551-554 .  Abstract ( )   PDF (2007KB) ( )   A strain of yeast-like-symbiotes(YLS) was isolated from the eggs of brown planthopper(Nilaparvata lugens St°al,BPH),and named as strain 34.The 26S rDNA sequence of strain 34 was highly homologous with that of the YLS directly isolated from BPH,and the strains both belonged to the Yarrowia lipolytica of subphylum Ascomycotina class Pyrenomycetes.The results showed that the strain 34 had the genetic relationship with the yeast-like-symbiotes of BPH.Moreover,it was concluded that the YLS of BPH could be cultured in vitro

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Resistance and Susceptibility to Several Groups of Insecticides in the Small Brown Planthopper,Laodelphax striatellus(Homoptera: Delphacidae) MA Chong-yong ,GAO Cong-fen ,WEI Hua-jie ,SHEN Jin-liang 2007, 21(5): 555-558 .  Abstract ( )   PDF (80KB) ( )   The resistance to nine insecticides and the sensitivity to seventeen insecticides were evaluated in two field populations of the small brown planthopper(SBPH),Laodelphax striatellus Fallén,collected from Wuxi City,Jiangsu Province and Huzhou City,Zhejiang Province in April 2006.The results detected by topical application showed that Wuxi and Huzhou populations developed high level resistance to imidacloprid(79.6-fold and 44.6-fold),and propoxur(76.6-fold and 40.1-fold),mid-high level resistance to carbaryl(29.8-fold and 45.3-fold),respectively,and remained susceptible or low level resistance to diazinon,fenitrothion,fenobucarb,carbosulfan,etofenprox and fenvalerate(1.4-to 8.1-fold).The assay on toxicities of the 17 insecticides to the third instar nymphs of Wuxi and Huzhou populations by using rice stem dipping method showed that the toxicity of fipronil and butylene-fipronil to SBPH was the highest,with the LC50 values being 0.21-0.41 mg/larva,and the recommended insecticides of field efficacy trial were thiamethoxam,nitenpyram,chlorpyrifos,pymetrozine,isoprocarb and dichlorvos.Furthermore,the resistance management strategies of the small browm planthopper to insecticides were also discussed.