Introgression of Gene for Non-Pollen Type Thermo-Sensitive Genic Male Sterility to Thai Rice Cultivars

For the two-line hybrid rice system, pol en sterility is regulated by recessive gene that responds to temperature. The recessive gene controlling thermo-sensitive genetic male sterility （TGMS） is expressed when the plants are grown in conditions with higher or lower critical temperatures. To transfer tgms gene（s） control ing TGMS to Thai rice cultivars by backcross breeding method, a male sterile line was used as a donor parent while Thai rice cultivars ChaiNat 1, PathumThani 1, and SuphanBuri 1 were used as recurrent parents. The BC2F2 lines were developed from backcrossing and selfing. Moreover, the simple sequence repeat （SSR） markers were developed for identifying tgms gene and the linked marker was used for assisting selection in backcrossing. The identification lines were confirmed by pol en observation. The results showed the success of introgression of the tgms gene into Thai rice cultivars. These lines will be tested for combining ability and used as female parent in hybrid rice production in Thailand.     

SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene （ben） in Rice

In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.     

SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene (ben) in Rice

In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.     

Genetic analysis of blast resistance On rice cultivars with wide—spectrum resistance

In recent years,indirect selection based ontightly linked genetic markers has been sug-gested to pyramid the resistance genes.How-ever,resistance parents used in the linkagestudies were usually near isogenic lines possessing a single resistance gene derived fromthe original resistance donor.Little was knownon the interaction among the genes at differentloci.In the present study,the genetic controlof blast resistance in two wide—spectrum re-vistance rice cultivars was analyzed.A susceptible indica varieties Xianfenglwas used as the female parent,and the blastresistance cultivars Tetep and Hongjiaozhanwere used as the male parents.The threeparental lines were inoculated with each of 20races of Pyricularia oryzae Cav at tilleringstage.The race ZC_(13) was selected to inoculatethe 161 F_3 lines of Xianfeng1/Tetep and 175 F,lines of Xianfengl/Hongjiaozhan.Eighteen in-dividuals of each line were inoculated.In both populations,all the individualsdemonstrated extreme reaction to the blast dis-ease.In the F_3 po     

Quantitative Trait Loci for Resistance to Stripe Disease in Rice （Oryza sativa）

In order to map the quantitative trait loci for rice stripe resistance, a molecular linkage map was constructed based on the F2 population derived from a cross between Zhaiyeqing 8 and Wuyujing 3. Reactions of the two parents, F1 individual and 129 F2:3 lines to rice stripe were investigated by both artificial inoculation at laboratory and natural infection in the field, and the ratios of disease rating index were scored. The distribution of the ratios of disease rating index in Zhaiyeqing 8/Wuyujing 3 F2:3 population ranged from 0 to 134.08 and from 6.25 to 133.6 under artificial inoculation at laboratory and natural infection in the field, respectively, and showed a marked bias towards resistant parent (Zhaiyeqing 8), indicating that the resistance to rice stripe was controlled by quantitative trait loci (QTL). QTL analysis showed that the QTLs detected by the two inoculation methods were completely different. Only one QTL, qSTV7, was detected under artificial inoculation, at which the Zhaiyeqing 8 allele increased the resistance to rice stripe, while two QTLs, qSTV5 and qSTV1, were detected under natural infection, in which resistant alleles came from Zhaiyeqing 8 and Wuyujing 3, respectively. These results showed that resistant parent Zhaiyeqing 8 carried the alleles associated with the resistance to rice stripe virus and the small brown planthopper, and susceptible parent Wuyujing 3 also carried the resistant allele to rice stripe virus. In comparison with the results previously reported, QTLs detected in the study were new resistant genes to rice stripe disease. This will provide a new resistant resource for avoiding genetic vulnerability for single utilization of the resistant gene Stvb-i.     

Bacterial Community in Different Populations of Rice Brown Planthopper Nilaparvata lugens （St~l）

The structures of bacterial communities in the brown planthopper （BPH） Nilaparvata lugens （St~l） from different geographic and resistant virulent populations were analyzed by using denatured gradient gel electrophoresis （DGGE）. Results showed that the bacterial communities in BPH nymph from the first to the fifth instars varied with nymphal growth and development. The bacterial communities in the first-instar BPH nymph were similar to those in adults. Nine geographic BPH populations were divided into three groups based on the cluster analysis of DGGE fingerprint. The first group was from the Philippines; the second group was from Thailand and Hainan, Yunnan and Zhejiang provinces of China; and the third group was from Vietnam and Guangxi, Hunan and Jiangxi provinces of China. BPH populations adapted to different resistant rice varieties. The BPH populations from Mudgo （with resistant gene Bphl） and ASD7 （with resistant gene bph2） differed with those of the susceptible rice variety TNI.     

Improvement of Resistance to Bacterial Blight by Marker-Assisted Selection in a Wide Compatibility Restorer Line of Hybrid Rice

4183 is a promising wide compatibility restorer line with good grain quality. Its hybrid rice Shuangyou 4183 (Shuangjiu A/ 4183) was registered in Anhui Province in 2003. However, the line and its hybrid rice are susceptible to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). R4183 carrying Xa21 was developed to improve bacterial blight resistance of 4183 through introducing the broad-spectrum resistance gene Xa21 by marker-aided selection. R4183 had similar level of bacterial blight resistance to IRBB21, the resistant donor, while maintained the wide compatibility, restoring ability and other good economic traits of the recurrent parent 4183. Critical issues on improvement of bacterial blight resistance of hybrid rice and breeding strategies were also discussed.     

Genetic analysis and QTL mapping of stalk cell wall components and digestibility in maize recombinant inbred lines from B73 × By804

The cell wall composition and structure of the maize stalk directly affects its digestibility and in turn its feed value.Previous studies of stem quality have focused mostly on common maize germplasm,and few studies have focused on high-oil cultivars with high grain and straw quality.Investigation of the genetic basis of cell wall composition and digestibility of maize stalk using high-oil maize is desirable for improving maize forage quality.In the present study,a high-oil inbred line(By804)was crossed as male parent with the maize inbred line B73 to construct a population of 188 recombinant inbred lines(RILs).The phenotypes of six cell-wall-related traits were recorded,and QTL analysis was performed with a genetic map constructed with SNP markers.All traits were significantly correlated with one another and showed high broad-sense heritability.Of 20 QTLs mapped,the QTL associated with each trait explained 10.0%–41.1%of phenotypic variation.Approximately half of the QTL each explained over 10%of the phenotypic variation.These results provide a theoretical basis for improving maize forage quality by marker-assisted selection.     

Marker-assisted Selection of ZmC4Ppc in Rice Breeding and Yield Trait Performances of Advanced Lines

The full-length of intact Zea mays gene for phosphoenolpyruvate carboxylase gene (ZmC4Ppc) is 6 781 bp. The products of PCR for this gene were not clear with poor repeatability, resulting in that it was difficult for marker-assisted selection (MAS) both in rice and maize. For selecting the markers for MAS, sequences presented only in maize rather than in rice were identified by BLAST, and used for primer design using Primer Premier 5.0. A pair of specific primer termed MRpc (Forward: 5' AAGCAGGGAAGCGAGACG 3', Reverse: 5' GATTGCCGCCAGCAGTAG 3') was used for selection of transformed rice, and ZmC4Ppc could be highly and constitutively expressed at each tested developmental stages in the transformed rice selected by using MRpc. Thus, MRpc was used for MAS of progenies carrying ZmC4Ppc gene in rice and some restorer lines with ZmC4Ppc (e.g. FPM881) derived from ZmC4Ppc-transformed Kitaake backcrossed with a restorer line Shuhui 881 were obtained. The analyses on genetic background, PEPCase activity, net photosynthetic rate, general combining ability (GCA) and specific combining ability (SCA) of FPM881 showed that similarity of genetic background reached above 95%, the PEPCase and net photosynthetic rate were higher than those of the control, and some of the progenies carrying ZmC4Ppc gene had better GCA and SCA for grain yield per plant, number of panicles per plant, and 1000-grain weight than those of the control. This suggested that the introduction of maize ZmC4Ppc gene via MAS and its stable expression could increase grain yield of rice and would likely provide a pathway for rice varietal improvement.     

Osa-mi R439 Negatively Regulates Rice Immunity Against Magnaporthe oryzae

Osa-miR439 is a rice-specific microRNA family. Here we showed that Osa-miR439 acted as anegative regulator in rice immunity against blast fungus Magnaporthe oryzae. Osa-miR439 differentiallyresponded to M. oryzae between susceptible and resistant rice accessions. The accumulation ofOsa-miR439 was constitutively more in the susceptible accession than in the resistant one. Transgeniclines overexpressing Osa-miR439a (OX439a) showed higher susceptibility associating with lower inductionof defense-related genes and less hydrogen peroxide (H2O2) accumulation at the infection sites than thecontrol plants. In contrast, transgenic lines expressing a target mimic of Osa-miR439 (MIM439) displayedcompromised susceptibility associating with increased H2O2 accumulation. Furthermore, we found thatthe expression of three predicted target genes was decreased in OX439a but increased in MIM439 incomparison to control plants, and this expression was differential in susceptible and resistant accessionsupon M. oryzae infection, indicating that Osa-miR439a may regulate rice blast resistance via these genes.Our results unveiled the role of Osa-miR439a in rice blast resistance and provided the potentiality toimprove the blast resistance via miRNA.     

玉米株高主效QTL qPH2.4的定位分析

课题组前期以玉米自交系郑58为轮回亲本,以昌7-2为供体亲本,通过分子标记辅助选择获得染色体单片段代换系Z12和W16。Z12和W16在bin2.07区域均含有1个来源于昌7-2的染色体片段,株高均显著高于轮回亲本郑58。利用郑58和Z12为材料构建F2分离群体,基于重测序和混合分离分析(BSA)策略,将株高主效QTL qPH2.4定位于第2染色体13.95 Mb(201 457 953~215 022 157 bp)的区域内。利用在目标区间内筛选出的20对多态性分子标记对包含743个单株的郑58×Z12 F2分离群体和包含1 720个单株的郑58×W16 F2分离群体进行基因型分析,结合田间株高数据进行QTL定位,将株高主效QTL qPH2.4定位在InDel分子标记ph-18和ph-19之间,区间的遗传距离为0.57 cM,物理距离为626 kb。参考B73基因组(RefGen_v4)注释信息,该区间内存在17个注释基因,其中,包含可以调控油菜素内酯信号的基因Zm00001d006677。     

小麦抗条锈基因Yr17的新SCAR标记的建立与应用

为深入了解四川小麦新品种(系)中抗条锈病基因的组成,并为开展分子标记辅助育种提供依据,以小麦抗条锈病基因Yr17的近等基因系以及抗条锈病品系L15与感条锈病材料SY95-71的杂交F2代群体为材料,采用集群分离分析法(BSA)结合SSR分子标记进行分析,发现1条由引物Xgwm415扩增的与抗条锈基因Yr17连锁的特异带。对该特异带克隆测序发现,该序列长390 bp,与栽培大麦中的一个序列表达标签(EST)有90%的相似性,与水稻基因组中的一个"核苷酸结合位点(NBS)-富亮氨酸重复(LRR)"型抗病基因的编码区有70%的相似性。根据该序列设计特异引物,建立SCAR标记,命名为SC-372。利用SC-372对54个四川省近年育成的小麦品种(系)进行检测,发现在15个抗病品种(系)中能特异扩增。进一步利用SC-372及前人报道的Yr17连锁标记VENTRUP/LN2对Yr17近等基因系及偏凸山羊草进行扩增比较发现,本实验建立的SCAR标记对小麦背景中的Yr17基因有更好的检测效率。因此可将SC-372用于分子标记聚合小麦抗条锈病基因的育种实践中。     

小麦BNS雄性不育中国春恢复基因的连锁群检测和QTL初步定位

BNS是一个新发现的温敏小麦雄性不育系,有良好的不育性和自身转换性,在杂交小麦利用和不育资源研究中有重要价值。为定位BNS的恢复基因,首先以BNS的高恢复系中国春为材料,创建BNS×中国春F2作图群体,建立自交结实率和花粉可育率两个表型BSA池;然后用中国春缺体-四体系检测恢复相关连锁群;最后用这些连锁群上的SSR分子标记筛选BSA池,用检测的连锁标记筛选F2作图群体,进一步定位恢复基因的QTL位点。结果表明,用BNS与中国春缺四体杂交,根据F1不育性检测到4个相关连锁群,分别是1A、1B、2B和7B;利用4个相关连锁群和4个非相关连锁群共8个染色体上的222对SSR分子标记筛选2对共4个BSA池,结果在3个相关连锁群上检测到8对连锁标记;用这8对连锁标记筛选F2群体210个个体植株,检测到5个QTL位点,位于1A、1B和2B染色体上。这些位点中,1个与自交结实率相关,2个与两个表型均相关,是主效QTL位点,另2个与花粉可育率相关,是微效QTL位点。这些结果为BNS恢复基因分子标记选择和精细定位奠定了基础。     

Identification of RAPD Markers and Development of SCAR Markers Linked to a Powdery Mildew Resistance Gene,and their Location on Chromosome in Wheat Cultivar Brock

Abstract

Wheat cultivar Jing 411 which is susceptible to powdery mildew, and wheat cultivar Brock and NILs of Jing 411, which are resistant to powdery mildew were analysized for polymophisms using 213 random amplified polymorphic DNA primers. Only one primer (S2092) stably produced a polymorphic band between the resistant and susceptible plants. Linkage analysis of this marker (S2092₉₇₂) revealed that the polymorphism existed in a 131 F₂ segregating population. S2092₉₇₂ was closely linked to a powdery mildew resistance gene in wheat cultivar Brock, at a linkage distance was 4.9 cM. S2092₉₇₂ was converted to sequence characterized amplified region (SCAR) markers SCAR₈₆₀ and SCAR₂₀₀. The two SCAR markers were used for detecting F₂ segregating population. SCAR₈₆₀ and SCAR₂₀₀ existed in resistant plants but were absent in the susceptible plants. We concluded that S2092₉₇₂ was located on the chromosome 3BL. These markers will be useful for marker-assisted selection and gene pyramiding in wheat resistance breeding.     

利用RAPD技术寻找大豆抗孢囊线虫4号小种标记初报

以高抗大豆孢囊线虫４号生理小种的２个大豆品系１２５９系（黄色）和１２５９系（双色）及其抗感亲本灰皮支黑豆和晋遗９号，以及另外两个高抗品种ＰＩ４３７６５４和元钵黑豆，两个高感品种鲁豆１号和鲁豆７号为试验材料，进行大豆抗孢囊线虫的ＰＡＰＤ分析。     

一个与马铃薯青枯病抗性连锁的SRAP标记筛选

以两个马铃薯二倍体分离群体ED和CE共105个基因型为材料,利用群分法(BulkedSegregantAnalysis,BSA)筛选与马铃薯青枯病抗性基因连锁的分子标记。在8个正向引物和11个反向引物组成的88对SRAP引物组合中,筛选获得了一个与马铃薯青枯病抗性紧密连锁的SRAP标记M32,遗传连锁分析表明,该标记在ED群体和CE群体中与抗性位点之间的遗传距离分别为10.2cM和17.3cM。     

Rice Germin-Like Proteins: Allelic Diversity and Relationships to Early Stress Responses

Germin-like protein (GLP) markers were associated with quantitative trait loci (QTL) for resistance to the rice blast pathogen, Magnaporthe oryzae in multiple rice (Oryza sativa) mapping populations. Twelve paralogous OsGLP gene family members are located within the physical QTL region on chromosome 8, and gene silencing studies suggest that they contribute collectively to the resistance phenotype. We compared sequence and expression profiles of OsGLP alleles in two resistant and two susceptible parental rice lines to find functional polymorphisms that correlated with the resistant phenotype. Based on coding and promoter sequences, the genes belong to two germin subfamily groups (GER3 and GER4). OsGLP members from both subfamilies were constitutively expressed and developmentally regulated in all cultivars. Transient induction above constitutive levels was observed for some OsGLPs, especially GER4 subfamily members, at early time points after M. oryzae infection and mechanical wounding. Varying 5′ regulatory regions and differential expression of some family members between resistant and susceptible cultivars corresponded with differential hydrogen peroxide (H₂O₂) accumulation after the same stimuli. OsGLP of both GER subfamilies localized to the plant cell wall. The protein location and early gene induction suggest that OsGLPs protect rice leaves at early stages of infection before fungal penetration and subsequent ingress. Our data suggest that regulation of OsGLP genes defines resistant versus susceptible phenotypes.     

Changing allele frequencies associated with specific resistance genes to leaf blast in backcross introgression lines of Khao Dawk Mali 105 developed from a conventional selection program

Key genomic regions associated with blast resistance against a broad spectrum of isolates could be identified in backcross introgression lines developed by conventional breeding program. In this article, eighty three BC₃F₂ backcross introgression lines (BILs) derived from a cross between IR68835-98-2-B-2-1-1 (a broad spectrum blast resistance variety) and KDML105 (a susceptible variety) were developed by phenotypic selection against a mixture of six virulent blast isolates (MXL) that are widely spread out in the rainfed lowland of the North and Northeast of Thailand. The resistance spectrum of the BILs was assessed by inoculating with 12 different Magnaporthe oryzae isolates that showed differential responses on the parental cultivars and the MXL that was used for phenotypic selection. All BILs showed highly resistant reactions (low disease score) to the MXL and to two blast isolates THL48 and THL149. Four markers, i.e., RM246, RM241, RM303 and RM164 completely favoring the IR68835 allele (shifting of allele frequencies from KDML105 to IR68835) were identified on chromosomes 1, 4 and 5, respectively. Therefore, these markers could be linked to the resistance genes functioning against the MXL, THL48 and THL149. Furthermore, significant shifting of alleles was identified at six markers located on chromosomes 2, 4, 8, 9 and 12. Seven DNA markers linked to specific resistance genes were identified, in which allele from IR68835 at 6 markers, i.e., RM6, RM205, RM211, RM252, RM273 and RM342 reduced disease score (DS), against blast isolates THL16, THL329, THL458, THL831, THL868 and THL96036 while allele from KDML105 at RM208 (Pi-kd on chromosomes 2) reduce DS against THL84, THL191, THL557 and THL1108. In this study, the use of DNA markers enabled the identification of specific resistance genes in the backcross breeding materials developed from routine rice breeding program through the conventional phenotypic selection. In this experiment, the usefulness of breeding materials from conventional breeding program in identifying genes corresponding to the selection was illustrated. Linked markers and their genomic location provide necessary information for further use of marker-assisted selection to improve blast resistance in rice breeding program.     

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-b by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-bⁱ and a translucent endosperm mutant gene Wx-mq. From F₂ generations, a sequence characterized amplified region (SCAR) marker tightly linked with Stv-bⁱ and a cleaved amplified polymorphic sequence (CAPS) marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.