Identification and Purity Test of Super Hybrid Rice with SSR Molecular Markers

Five super hybrid rice combinations, i.e. HYS-1/R105, Pei'ai 64S/E32, Liangyoupeijiu (Pei'ai 64S/9311), 88S/0293, and J23A/Q611, and their parental lines were tested by means of SSR analysis. A total of 144 SSR primer pairs distributed on 12 rice chromosomes were used, out of which 47 detected polymorphism among the tested rice lines. Among all these primers, RM337 and RM154 produced polymorphic patterns in four or more of the tested experimental materials respectively, and they could distinguish among most rice genotypes tested. Twenty-four primer pairs, two on each rice chromosome, were selected to make a reference SSR marker-based fingerprinting for the rice lines. For most of the primer pairs, F1 hybrids mainly showed complementary pattern of both parents, which could be very useful to distinguish the F1 from its parental lines. In addition, 5 primer pairs were selected as special primer pairs for five hybrid rice combinations respectively. By combining the rapid, simple method on DNA extraction, it is suggested that SSR technique has wide prospective in variety authentication and purity identification.

Physiological Character and Gene Mapping in a New Green-revertible Albino Mutant in Rice

A green-revertible albino mutant-Qiufeng M was found from the japonica rice （Oryza sativa L. ssp. japonica） Qiufeng in the field. The first three leaves of the mutant were albino with some green. The leaf color became pale green since the fourth leaf and the glume had the same phenomenon as the first three leaves. The measuring data of the pigment content confirmed the visually observed results. It truly had a remarkable changing process in the leaf color in Qiufeng M. Comparison of the main agronomic characters between Qiufeng and Qiufeng M indicated that the neck length and grain weight showed significant difference at the 1% level, and other characters were not different. Genetic analysis showed that the green-revertible albino trait was controlled by a single recessive nucleic gene. Using 209 recessive mutant individuals in the F2 population derived from the cross Pei＇ai 64S × Qiufeng M, a gene, tentatively named gra（t）, was located between the SSR markers of RM475 and RM2-22 on the long arm of chromosome 2. The genetic distance were 17.3 cM and 2.9 cM respectively.

InDel and SNP Markers and Their Applications in Map-based Cloning of Rice Genes

High-density markers are necessary for map-based cloning of rice genes, but the currently available markers are not satisfactory enough. InDel (insertion-deletion length polymorphism) and SNP (single nucleotide polymorphism) are the new generation of molecular markers and can basically meet the need of fine mapping. InDel and SNP markers can be developed through bioinformatics. These markers are valuable markers with the characters of low cost, high specificity and stability. This article introduced the methods for designing InDel and SNP markers, taking the mapping of a rice rolled leaf gene as an example. In addition, some key factors in improving the design efficiency were also discussed.

Development of a Highly Informative Microsatellite (SSR) Marker Framework for Rice (Oryza sativa L.) Genotyping

To select highly informative microsatellite markers （SSRs） and establish a useful genetic SSR framework for rice genotyping, 15 rice （Oryza sativa L.） cultivars including six indica varieties and nine japonica varieties were used to analyze the polymorphism information content （PIC） value of 489 SSR markers. A total of 1 296 alleles were detected by 405 polymorphic markers with an average of 3.2 per locus. The PIC value of each chromosome was ranged from 0.4039 （chromosome 2） to 0.5840 （chromosome 11）. Among the two rice subspecies, indica （0.3685-0.4952） gave a higher PIC value than japonica （0.1326-0.3164） and displayed a higher genetic diversity. Genetic diversity of indica was high on chromosome 12 （0.4952） and low on chromosome 8 （0.3685）, while that for japonica was high on chromosome 11 （0.3164） and low on chromosome 2 （0.1326）. A SSR framework including 141 highly informative markers for genotyping was selected from 199 SSR markers （PIC〉0.50）. Ninety-three SSR markers distributed on 12 chromosomes were found to be related to indica-japonica differentiation. Of these 93 pairs of SSR primers, 17 pairs were considered as core primers （all the japonica varieties have the same specific alleles, while the indica varieties have another specific alleles）, 48 pairs as the second classic primers （all the japonica or indica varieties have the same specific alleles, while the indica or japanica varieties have two or more other specific alleles ） and 28 pairs as the third classic primers （all the japonica and indica varieties have two or more alleles, but the specific alleles are different between japonica and indica）. Thirty-two SSR markers were selected to be highly informative and useful for genetic diversity analysis of japonica varieties. This work provides a lot of useful information of SSR markers for rice breeding programs, especially for genotyping, diversity analysis and genetic mapping.

Construction of DNA Fingerprint Using SSR Marker for Hybrid Rice Cultivars Approved by Hunan Province

[Objective] This study aimed to construct DNA fingerprint for hybrid rice cultivars those have been approved by Hunan Province. [Method] The primers which produced polymorphic and bright DNA bands were selected to construct the DNA molecular fingerprint map for 77 major hybrid rice cultivars approved by Hunan Province. [ Result] A total of 48 SSR primers were selected. Every cultivar had its unique fingerprint map so that the obtained data could identify differ- ent hybrid rice cultivars. [ Conclusion] This study made great contributions to the perfection of hybrid rice germplasm identification.

Genetic Analysis and Gene Mapping of Short Root Mutant Rice ksr1

A short root mutant ksrl with the Kasalath background was isolated from an EMS-mutagenized population in rice. The root length of 6-day-old ksr1 seedlings was only about 20% of the wild type. Genetic analysis indicated that the short root phenotype of ksrl was controlled by a recessive mutation in a single nuclear-encoded gene. To map the ksrl mutation, an F2 population was generated by crossing the ksrl mutant with Nipponbare. The KSR1 locus was linked to the SSR marker RM1223 on rice chromosome 4. Eight new SSR markers and two InDel markers were developed around this marker. KSR1 gene was further mapped to a 155 kb region, flanked by the InDel marker 4-24725K and the SSR marker RM17182.

Genetic analysis and molecular mapping of a presenescing leaf gene psll in rice （Oryza sativa L.）

A rice psl1 (presenescing leaf) mutant was obtained from a japonica variety Zhonghua 11 via radiation of 60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the big-gest length,while the leaves of the wild variety could keep green for 25―35 d. In this study,genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene (psl1) located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nippon-bare and 93-11. The psl1 was further mapped be-tween two STS markers,STS2-19 and STS2-26,with genetic distances of 0.43 and 0.11 cM,respectively,while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus,the region being delim-ited to 48 kb. This result was very useful for cloning of the psl1 gene.

Identification of QTLs for Yield and Associated Traits in F_(2) Population of Rice

Identification of quantitative trait loci(QTLs)controlling yield and yield-related traits in rice was performed in the F_(2) mapping population derived from parental rice genotypes DHMAS and K343.A total of 30 QTLs governing nine different traits were identified using the composite interval mapping(CIM)method.Four QTLs were mapped for number of tillers per plant on chromosomes 1(2 QTLs),2 and 3;three QTLs for panicle number per plant on chromosomes 1(2 QTLs)and 3;four QTLs for plant height on chromosomes 2,4,5 and 6;one QTL for spikelet density on chromosome 5;four QTLs for spikelet fertility percentage(SFP)on chromosomes 2,3 and 5(2 QTLs);two QTLs for grain length on chromosomes 1 and 8;three QTLs for grain width on chromosomes1,3 and 8;three QTLs for 1000-grain weight(TGW)on chromosomes 1,4 and 8 and six QTLs for yield per plant(YPP)on chromosomes 2(3 QTLs),4,6 and 8.Most of the QTLs were detected on chromosome 2,so further studies on chromosome 2 could help unlock some new chapters of QTL for this cross of rice variety.Identified QTLs elucidating high phenotypic variance can be used for marker-assisted selection(MAS)breeding.Further,the exploitation of information regarding molecular markers tightly linked to QTLs governing these traits will facilitate future crop improvement strategies in rice.

Characterization and Genetic Analysis of Rumpled and Twisted Leaf Mutant(rtl1) in Rice

A rumpled and twisted leaf 1(rtl1) mutant was generated from a japonica cultivar Nipponbare by ethyl methanesulfonate treatment,which was characterized as rumpled and twisted leaf at the seedling stage.The F2 populations were constructed by crossing with indica cultivars TN1 and Zhefu 802,respectively.Genetic analysis demonstrated that the phenotype was controlled by a single recessive nuclear gene.The closely linked simple sequence repeat(SSR) marker RM1155 was obtained from bulked segregant analysis.Subsequently,sequence tagged site(STS) markers were developed using the published rice genome sequence.Finally,RTL1 was located between an STS marker T1591 and an SSR marker RM1359,at the distances of 0.48 cM and 0.96 cM,respectively.These results will facilitate the cloning of the target gene in further studies.

基于SSR分子标记吉安地区的水稻亲缘关系鉴定分析

稻种亲缘关系分析是生产优质杂交水稻的必要条件。本研究选取均匀分布于水稻基因组12条染色体的SSR分子标记,对8份水稻样品进行SSR分子标记分析,通过聚类分析法将其归类并计算得出遗传相似系数后,对8份水稻样品的亲缘关系进行了鉴定。结果表明:所选取的8份水稻样品中,其相似系数为0.65~1.00,在遗传相似系数0.65处,8份水稻样品聚为两大类群,其中5号‘赣迟A04’单独为一类,其他水稻样品聚为一类。在遗传相似系数0.80处,可划分为三大类群:第一类囊括了6种样品,表明这6个品种亲缘关系较近,其中6号‘赣早A02’与7号‘吉安早A07’疑似同一样品,3号‘井冈山迟A03’与4号‘井冈山早A05’的亲缘关系最近,而6号、7号同时与8号‘吉安迟A09’保持有最近的亲缘关系;第二类只含有2号‘迟-2’样品株;第三类则仅有5号稻株‘赣迟A04’,说明其与其他7份水稻样品的亲缘关系最远。本研究结果为杂交水稻的应用提供重要信息。

Molecular mapping of two semidwarf genes in an indica rice variety Aitaiyin3(Oryza sativa L.)

Genetic analysis established that Aitaiyin3,a dwarf rice variety derived from a semidwarf cultivar Taiyin1,carries two recessive semidwarf genes.By using simple sequence repeat(SSR)markers,we mapped the two semidwarf genes,sd-1 and sd-t2 on chromosomes 1 and 4,respectively.Sd-t2 was thus named because the semidrawf gene sd-t has already been identified from Aitaiyin 2 whose origin could be traced back to Taiyin1.The result of the molecular mapping of sd-1 gene revealed it is linked to four SSR markers found on chromosome 1.These markers are:RM297,RM302,RM212,and OSR3 spaced at 4.7 cM,0 cM,0.8cM and 0 cM,respectively.Sd-t2 was found to be located on chromosome 4 using five SSR markers:two markers,SSR332 and RM1305 located proximal to sd-t2 are spaced 11.6 cM,3.8 cM,respectively,while the three distally located primers,RM5633,RM307,and RM401 are separated by distances of 0.4 cM,0.0 cM,and 0.4 cM,respectively.

Genetic analysis and gene mapping of a new rolled-leaf mutant in rice (Oryza sativa L.)

To understand the development of rice leaf blades,we identified a new rolled-leaf mutant,w32,from indica cultivar IR64 through EMS mutagenesis. The mutant showed a stable rolled-leaf phenotype throughout the life cycle. Two F2 populations were developed by crossing w32 to cultivar IR24 and PA64. Genetic analysis showed that the rolled-leaf phenotype was controlled by a single recessive gene. To determine the location of the gene,bulked segregant analysis was carried out using mutant and wild-type DNA pools and 1846 mutant-type F2 individuals derived from the cross w32/PA64 were genotyped to locate the gene on the short arm of chromosome 7. The rolled-leaf gene,tentatively named rl11(t),is likely a new gene as no other rolled-leaf genes have been identified near the region. By developing new SSR and InDel markers,the gene was delimited to a 52 kb region near the end of the short chromosome arm. Further fine mapping and cloning of the gene are currently underway.

Fine Mapping of Spr3, a Locus for Spreading Panicle from African Cultivated Rice （Oryza glaberrima Steud.）

A CSSL （chromosome segment substitution line）, SG-64, carrying a segment of chromosome 4 from African cultivated rice （CG-14） in the genetic background of var. Wuyujing-7 （japonica）, showed a spreading panicle, which was different significantly from that of Wuyujing-7 with an erect compact panicle. The gene controlling a spreading panicle is referred to as Spr3, and is mapped on chromosome 4. To uncover the genetic basis of Spr3, a large F2 population derived from cross between SG-64 and Wuyujing-7 was constructed for fine mapping of the Spr3 locus. The high-resolution linkage analysis revealed that the Spr3 locus was narrowed down to a 4.6-kb region. The delimited genomic DNA regions of Wuyujing-7 and CG-14 were sequenced and compared. Sequence mutations between Wuyujing-7 and CG-14 were evident and the candidate genes for the locus were predicted. Publicly available databases were searched for homologous cDNA sequences. However, any coding regions or other meaningful sequences for the Spr3 locus were not found within this delimited region. This result suggested that Spr3 is an unknown genetic factor in controlling the outspreading of the primary branches in rice inflorescence. In addition, NIL（Spr3） exhibited seed shattering. The formation of spreading panicle was accompanied by a few undesirable traits and the spreading panicle links with seed shattering suggest that the spreading panicle was likely lost during the domestication and selection for high seed productivity of cultivated rice.

Characterization and mapping of a novel mutant sms1 (senescence and male sterility 1) in rice

Plant senescence plays diverse important roles in development and environmental responses.However,the molecular basis of plant senescence is remained largely unknown.A rice spontaneous mutant with the character of early senescence and male sterility （sms） was found in the breeding line NT10-748.In order to identify the gene SMS1 and the underlying mechanism,we preliminarily analyzed physiological and biochemical phenotypes of the mutant.The mutant contained lower chlorophyll content compared with the wild type control and was severe male sterile with lower pollen viability.Genetic analysis showed that the mutant was controlled by a single recessive gene.By the map-based cloning approach,we fine-mapped SMS1 to a 67 kb region between the markers Z3-4 and Z1-1 on chromosome 8 using 1,074 F2 recessive plants derived from the cross between the mutant sms1 （japonica） × Zhenshan 97 （indica）,where no known gene involved in senescence or male sterility has been identified.Therefore the SMS1 gene will be a novel gene that regulates the two developmental processes.The further cloning and functional analysis of the SMS1 gene is under way.

Fine mapping of the rice bacterial blight resistance gene Xa-4 and its co-segregation marker

An F2 population developed from the Xa-4 near isogenic lines, IR24 and IRBB4, was used for fine mapping of the rice bacterial blight resistance gene, Xa-4. Some restriction fragment length polymorphism （RFLP） markers on the high-density map constructed by Harushima et al. and the amplified DMA fragments homologous to the conserved domains of plant disease resistance （R） genes were used to construct the genetic linkage map around the gene Xa-4 by scoring susceptible individuals in the population. Xa-4 was mapped between the RFLP marker G181 and the polymerase chain reaction （PCR） marker M55. The R gene homologous fragment marker RS13 was found co-segregating with Xa-4 by analyzing all the plants in the population. This result opened an approach to map-based cloning of this gene, and marker RS13 can be applied to molecular marker-assisted selection of Xa-4 in rice breeding programs.

Molecular tagging of a genic male-sterile gene in rice

The sterility of Pingxiang male-sterile rice (Pins), possibly derided from a spontaneous mutation in Pingxiang fertile rice (Pmf), was previously reported to be controlled by a single dominant nuclear gene. It can be restored to fertility either by a dominant epistatic gene or by higher temperature treatment at the early stage of inflorescence development. In order to tag the genie male-sterile gene, Pms, Pmf and Ce 64, a cytoplasmic male-sterile restoring line without the epistatic gene for Pms, were used to construct mapping populations. Two segregation populations, "(Pms/Ce 64) F1s (sterile plant)//Pmf" F1 and "Pms//(Pmf/Ce 64) F1" F1, were simultaneously developed. Subsequently, the genie male- sterile gene was mapped between a simple sequence length polymorphism marker, RM228, and a restriction fragment length polymorphism marker, G2155, with distances of 14.9 and 2.6 cM, respectively. The tagged dominant genie male-sterile gene is temporarily designated Ms-p.

Fine mapping of a semidwarf gene sd-g in indica rice (Oryza sativa L.)

The semidwarf gene sd-g which has been used in indica rice breeding in southern China is a new one, non-allelic to sd-1. To map sd-g, an F2 population derived from the cross between Xinguiaishuangai and 02428 was con-structed. The sd-g was roughly mapped between two mi-crosatellite markers RM440 and RM163, with genetic dis-tances of 0.5 and 2.5 cM, respectively. Then nine new poly-morphic microsatellite markers were developed in this region. The sd-g was further mapped between two microsatellite markers SSR5-1 and SSR5-51, with genetic distances of 0.1 and 0.3 cM, respectively, while cosegregated with SSR418. A BAC contig was found to span the sd-g locus, the region be-ing delimited to 85 kb. This result was very useful for cloning of the sd-g gene.

Fine mapping of locus S-b for F_1 pollen sterility in rice (Oryza sativa L.)

Hybrid sterility is the main barrier in util-izing the heterosis of subspecies in rice. A knowledge of the underlying molecular mechanism of the hybrid sterility will be useful for overcoming the barrier. In this research, the F1 pollen sterility locus, S-b, was mapped between SSR markers PSM8 and PSM202. To fine map the locus, one F2 mapping population of 3910 plants was developed using the near-isogenic lines of the locus. Ninety-seven recombinants be-tween two markers were selected. Moreover, a series of markers, including two SSR markers, two InDel markers and four CAPS markers, were developed on the region. Linkage analysis showed that marker W4 was co-segregated with locus S-b, while makers A8 and A14 were located on the two sides of the locus with a distance of 0.026 and 0.038 cM, respectively. The markers were then integrated with the se-quences of the clones of the region. Results showed that all the polymorphic markers were anchored on the three end-to-end jointed clones AC093089, AC079021 and AC134931. According to the physical information of the markers, locus S-b was finally de-limited to a region of 27 kb between A8 and A14. Seven ORFs were identified on the region based on the annotation results of RiceGAAS system. These results laid the foundation for further cloning the gene.

Genetic analysis and gene mapping of a narrow leaf mutant in rice (Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (un-der saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. More-over, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F2 mapping population de-rived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped be-tween the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93-11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8.

Genetic analysis and gene fine mapping for a rice novel mutant(rl_(9(t)))with rolling leaf character

Leaf shape is an important parameter for ideotype breeding in rice, and the rolling of leaf is also beneficial to efficient ripening of grains. This encourages the explorations of new genes that regulate leaf shape. In this study, genetic analysis and gene mapping were carried out for a novel rolling leaf mutant identified from japonica variety Zhonghua 11. The SSR marker analysis showed that the mutant was controlled by a single recessive gene (rl9(t)) lo- cated on chromosome 9. Fine mapping of the Rl9(t) locus was conducted with 30 new STS markers de- veloped around Rl9(t) anchored region based on the sequence diversity between Nipponbare and 93-11. The fine mapping necessitated the contruction of a PAC contig encompassing the Rl9(t) locus, which was delimited to a 42 kb region. This could therefore en- hance the cloning of the target gene in further stud- ies.