Natural Homologous Triploidization and DNA Methylation in SARII-628, a Twin-seedling Line of Rice （Oryza sativa）

A total of five pairs of diploid-triploid twin-seedlings (a diploid seedling and a triploid seedling emerged from a grain) were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ‘MSAP (methylation-sensitive AFLP)’ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on M0 generation after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.     

Genetic Analysis and Mapping of Dominant Minute Grain Gene Mi3（t） in Rice

Grain size, determined chiefly by grain length, is one of the main factors affecting the grain yield in rice production. To study the trait of rice grain size, F1 and F2 populations were developed from crosses Shuhui 881/Y34 and Shuhui 527/Y34, and genetic analysis for minute grain was performed. The F1 populations showed minute grains, and grain size segregations in the two F2 populations were both in accordance with the ratio of 3：1, indicating that minute grain in Y34 was controlled by a completely dominant gene. By using the F2 population from Shuhui 881/Y34, this dominant gene, tentatively designated as Mi3（t）, was mapped based on SSR markers in the interval between RM282 （genetic distance of 5.1 cM） and RM6283 （genetic distance of 0.9 cM） on the short arm of chromosome 3.     

Optimization of Electroporation Parameters for Immature Embryos of indica Rice （ Oryza sativa）

To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950 μF capacitance, 200 μL electroporation buffer with 70 mmol/L sodium glutamate, 100 μg/mL plasmid, 50 μg/mL carrier DNA, 20 embryos per cuvette, 0°C treatment and CC medium were the best parameters, which not only improved the transformation efficiency to 30.89%, but also ameliorated the embryo survival ratio to 95.92%. To further verify the practicability of this condition, the embryos from another indica rice variety and a rice type II metallothionein-like gene (OsMT2bL) promoter::mgfp5::gusA construct were tested, and specific GUS expression on the embryos was visualized by histochemical staining. The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues. These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.     

Effects of Different Nitrogen Fertilizer Levels on Fe, Mn, Cu and Zn Concentrations in Shoot and Grain Quality in Rice （Oryza sativa）

In a pot experiment, effects of N fertilizer application on the concentrations of Fe, Mn, Cu and Zn in shoot of rice and the quality of brown rice were studied. In the treatments with N fertilizer application, the concentrations of Fe, Mn, Cu and Zn in most parts of rice shoot increased compared with control (no N fertilizer application). This indicated that the transportation ability of microelements from root to shoot in rice was improved with N fertilizer application. Effect of N fertilizer on IR68144 was similar to that of on IR64, but the concentrations of the microelements in plant differed, suggesting that the characteristic expression of the two rice genotypes was not controlled by the amount of N fertilizer supplied. The concentrations of those microelements in brown rice increased at first and then decreased with increasing N fertilizer application, reaching the highest at 160 kg/ha, at which the Fe, Mn, Cu and Zn concentrations in brown rice increased by 28.96%, 41.34%, 58.31% and 16.0% for IR64, and by 22.16%, 13.75%, 8.75% and 20.21% for IR68144 compared with control, respectively. Moreover, N fertilizer promoted the accumulation of protein, decreased the accumulation of amylose in grain, and enhanced gel consistency of brown rice. These results indicate that appropriate N fertilizer management could increase micronutrient contents in grain and improve nutrition quality of rice.     

Grain Quality and Genetic Analysis of Hybrids Derived from Different Ecological Types in Japonica Rice （Oryza sativa）

The performance and inheritance of 7 quality traits were studied using F2 rice grain derived from 8×8 diallel crossing made by employing 8 parents of different ecological japonica rice types. Differences in each trait among 8 parents were not obvious, but in F2 generation, transgressive phenomena were found in all the traits studied, indicating that the genes controlling these traits among parents were segregated. The inheritance of grain width, grain weight, chalkiness score (CS), gelatinization temperature (GT) and gel consistency (GC) were suitable to additive-dominant model, and dominant effect contributed mainly for the 5 traits. The inheritance of grain length (GL) and amylose content (AC) did not fit into additive-dominant model, existing epistatic interactions. Dominant genes for grain width and grain weight had the efficiency of decreasing effect, and dominant genes for CS, GT and GC had the efficiency of enhancing effect. Koshihikari contained more recessive genes for gelatinization temperature than other varieties. Zhendao 88 had more dominant genes in grain width and grain weight than other varieties. Xiushui 04 possessed more dominant genes for GL and GC, and more recessive genes for CS than other varieties.     

Molecular Cloning and Characterization of Citrate Synthase Gene in Rice （ Oryza sativa）

The full-length OsCS encoding citrate synthase was isolated from rice （Oryza sativa L. subsp, japonica）, OsCS is 1477-bp long and encodes a 474 amino acid polypeptide, Its putative protein sequence is highly identical to Daucus carota, Nicotiana tabacum Beta vulgaris subsp., Arabidopsis thaliana, and Citrus junos （〉70%）. The deduced amino-terminal sequence of OsCS showes characteristics of mitochondrial targeting signal. Southern blot analysis using ORF of the OsCS as the probe indicated that this gene exists in multiple copies in rice genome. The band with predicated size of 82 kD was detected by Western blot after being induced by 0,4 mmol/L IPTG.     

Analysis of Introgressed Segments in Near-isogenic Lines for F1 Pollen Sterility in Rice （Oryza sativa）

One hundred and fifty-eight microsatellite markers showing polymorphism among parents were used to survey the introgressed segments in the 50 near-isogenic lines of F1 pollen sterility. Two hundred and sixty introgressed segments were detected in 50 near-isogenic lines, each carrying 5.2 introgressed segments on an average. Among the 260 segments, one hundred carrying F1 pollen sterility loci concentrated on the region of F1 pollen sterility genes, and the remaining one hundred and sixty without F1 pollen sterility loci distributed randomly over 12 chromosomes.Both the average number and length of the introgressed segments decreased along with the increase of backcross generations. The number of introgressed segments was less than four and the length was less than 20 cM in the near-isogenic lines after backcrossing for four or more times.     

Correlation Between Parents and F1 and Combining Ability of Parents on Seed Dormancy in indica Rice （Oryza sativa）

Dormancy indices of hulled and dehulled seeds were investigated by using 19 cytoplasmic male sterile (CMS) lines, 9 restorer lines and their 109 F1 hybrids of indica hybrid rice. The seeds of each F1 and the parents were harvested on 35 days after flowering. Combining ability was analyzed in 25 combinations made by 5 CMS lines and 5 restorer lines (North Carolina II mating design). The seed dormancy index of F1 was positively and highly significantly correlated with those of their parents and mid-parent value. Out of the 109 combinations, 82 combinations showed mid-parent heterosis, and 43 heterobeltiosis. Seed dormancy indices of F1s and their parents declined dramatically in dehulled seeds compared with hulled seeds, indicating that the hull played an important role in seed dormancy. However, the trends were similar in hulled seeds and dehulled seeds in terms of relationships between the seed dormancy indicices in F1 and their parents. The influence of hull on seed dormancy mainly depended on F1 genotype, not on the hull from maternal parent. The variances of general combining ability (GCA) in female and male parents occupied 59.2% and 31.1% of total variance, respectively. The variance of specific combining ability (SCA) in combinations occupied 9.7% of total variance, indicating that gene additive effects were principal. Among the 5 CMS lines, II112A had the highest GCA effect for seed dormancy, followed by D62A. Among the 5 restorer lines, IR112 had the highest GCA effect for seed dormancy, followed by 2786. These lines are elite parental materials for breeding F1 hybrid rice with stronger seed dormancy.     

Genetic Identification of a New Small Grain Dwarf Gene in Rice

The plant material used in the study was rice line 162d, a new small grain dwarf mutant. Polymorphic analysis of 221 SSR loci demonstrated that 162d derived from a semidwarf variety Shuhui 162 through mutation, and 162d and Shuhui 162 were just a pair of near isogenic lines. Genetic analysis of Fl and F2 populations suggested that dwarfism in 162d was controlled by a single recessive gene. Phenotypic characteristics of the mutant gene were that plant height was about a quarter of normal height, grain size about a quarter of normal size, leaf was short and broad, and seed setting rate was very low,compared with the near isogenic line Shuhui 162. The mutant gene was sensitive to gibberellin (GA3) treatment and did not located on the region near the centromere of rice chromosome 5, where dl gene located. Therefore, it was concluded that the mutant gene of 162d was a new small grain dwarf gene in rice.     

Identification of QTLs for Plant Height and Its Components by Using Single Segment Substitution Lines in Rice （Oryza sativa）

QTLs for plant height and its components on the substituted segments of fifty-two single segment substitution lines （SSSLs） in rice were identified through t-test （P〈0.001） for comparison between each SSSL and recipient parent Huajingxian 74. On the 14 substituted segments, 24 QTLs were detected, 10 for plant height, 2 for panicle length, 4 for length of the first internode from the top, 5 for length of the second internode from the top and 3 for length of the third internode from the top, respectively. All these QTLs were distributed on nine rice chromosomes except chromosomes 5, 9 and 11. The additive effect ranged from -4.08 to 3.98 cm, and the additive effect percentages varied from -19.35% to 10.43%.     

Identification and Genetic Analysis of Fertility Restoration Ability in Dongxiang Wild Rice(Oryza rufipogon)

Two populations of Dongxiang wild rice(Oryza rufipogon),Shuitaoshu and Dongtangshang,were crossed with five male sterile rice lines with different cytoplasmic backgrounds(B06S,Zhenshan 97A,Xieqingzao A,Zhong 9A,and Yuetai A),and the seed setting rate of the F1 was used to judge the fertility restoration ability in the Dongxiang wild rice.With P1.F1,P2,and F2 populations as materials,the Akaike's Information Criterion(AIC)was used to identify the major genes affecting quantitative traits,and when the major genes existed,the genetic effects of the major gene and the polygene and their genetic variance were estimated through segregation analysis.The seed setting rates of the F1 generation varied from 45.98%to 76.57%,suggesting that the Dongxiang wild rice had the fertility restoration ability.One major gone plus polygene mixed inheritance 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 from 2.74%to 30.97%,and the total heritability values were from 63.1 7%to 94.01%.The major gone inheritance of the combination Zhong 9A/Dongtangshang was controlled by the additive effect without dominant effect,and the other nine combinations were by the completely dominant inheritance.     

东乡野生稻育性恢复性的鉴定与遗传分析

以东乡野生稻水桃树、东塘上2个居群与不同细胞质来源的5个水稻不育系(B06S、珍汕97A、协青早A、中97A和粤泰A）配组,根据F1结实率的高低对东乡野生稻育性恢复性进行鉴定。建立了10个组合的P1、F1、P2、F2群体,利用混合模型理论的Akaike信息准则(AIC),在F2代中鉴定影响数量性状的主基因存在与否,主基因存在时通过分离分析估计主基因和微效基因的遗传效应以及所占总变异的分量。F1代的结实率变化范围为45.98%~76.57%,表明东乡野生稻具有一定的育性恢复性。F2代中该性状符合1对主基因+多基因的遗传模式,主基因遗传率为56.63%~88.29%,多基因遗传率为2.74%~30.97%,总基因型遗传率为63.17%~94.01%。中9A /东塘上居群杂交组合的F2代中,主基因是加性遗传,无显性效应,其他9个组合主基因是完全显性遗传。     

Development of Novel Cytoplasmic Male Sterile Source from Dongxiang Wild Rice (Oryza rufipogon)

This study was conducted to develop and characterize a novel cytoplasmic male sterile(CMS)source which was identified from Dongxiang wild rice(Oryza rufipogon)by crossing Dongxiang wild rice as female with Zhongzao 35,an indica inbred variety,as male and continuous backcrossing with Zhongzao 35.Observation under optical microscope manifested that this novel CMS belonged to typical abortion type with less pollen compared with wild abortive type cytoplasm(CMS-WA).Sequential planting showed that this novel CMS has complete and stable male sterility.Testcross experiment showed that all the 24 tested materials including maintainer and restorer lines of CMS-WA and Honglian type cytoplasm(CMS-HL)and other indica inbred varieties are the maintainers with complete maintaining ability,suggesting that this novel CMS has fertility restoration totally different from CMS-WA and CMS-HL and belongs to a novel type of CMS.So far,we only discovered a unique fertility restoration source for this novel CMS.Inheritance analysis showed that the fertility restoration of this CMS was governed by three pairs of independent dominant genes.Prospect for application of this novel CMS system in hybrid rice breeding was also discussed.     

QTL Analysis for Yield Traits Related to Low Nitrogen Tolerance Using Backcrossing Recombinant Inbred Lines Derived from Dongxiang Wild Rice (Oryza rufipogon Griff.)

【Objective】Dongxiang wild rice (Oryza rufipogon Griff.) has strong low nitrogen tolerance and is an important germplasm for low nitrogen tolerance improvement. Identification of genes responsible for low nitrogen tolerance in Dongxiang wild rice is of great importance to understand molecular mechanisms of low nitrogen tolerance and develop rice varieties with low nitrogen tolerance. 【Method】Quantitative trait loci (QTLs) for plant height and yield traits under low and normal nitrogen conditions was identified using backcrossing recombinant inbred lines (BC1F12) derived from an interspecific cross Xieqingzao B // Dongxiang wild rice/Xieqingzao B and its genetic linkage. 【Result】A total of 57 QTLs were detected in 33 regions on all chromosome, except chromosome 4 and 8. They explained individually 3.17%~63.40% phenotypic variation, and 32 QTLs of them had favorable alleles derived from Dongxiang wild rice. Nineteen QTLs were simultaneously detected under both nitrogen treatments, and 38 QTLs were only identified under single nitrogen treatment, suggesting various genetic mechanisms in rice growth and yield formation under low and normal nitrogen conditions. 【Conclusion】Fourteen QTL clusters, 43 QTLs included, scattered on seven chromosomes, indicating the common genetic-physiological mechanisms behind different traits, and the QTL pyramiding for low nitrogen tolerance can be achieved by molecular marker-assisted selection.     

应用东乡野生稻回交重组自交系群体分析糙米矿质含量QTL

【目的】 强化粮食作物的必需矿物质有利于缓解人们矿质营养缺乏症。【方法】 从协青早B//协青早B/东乡野生稻BC₁F₅群体中挑选到1个单株A58,与协青早B回交,构建了BC₂F_4:5群体。采用电感耦合等离子体原子发射光谱仪ICP-AES测定132个BC₂F_4:5株系的糙米Mg、Ca、Zn、Fe、Mn和Cu含量,应用Windows QTL Cartographer 2.5进行糙米矿质QTL分析。【结果】 共检测到17个糙米矿质含量QTL,分别位于第1、4、6、8、9和11等6条染色体上,包括Mg含量1个、Ca含量4个、Zn含量4个、Fe含量2个、Mn含量2个和Cu含量4个。这些QTL解释表型变异的5.0%~47.2%,其中8个QTL的增效等位基因来自东乡野生稻。【结论】 12个QTL聚集于5条染色体上的5个QTL簇,表明不同矿质营养元素涉及到共同遗传生理机制,可通过分子标记辅助选择方法将有利等位基因应用于稻米营养品质改良。     

Dissecting Genetic Basis of Deep Rooting in Dongxiang Wild Rice

Deep rooting is an important trait in rice drought resistance. Genetic resources of deep-rooting varieties are valuable in breeding of water-saving and drought-resistant rice. In the present study, 234 BC₂F₇ backcross introgression lines were derived from a cross of Dongye 80 (an accession of Dongxiang wild rice as the donor parent) and R974 (an indica restorer line as the recurrent parent). A genetic linkage map containing 1 977 bin markers was constructed by ddRADSeq for QTL analysis. Thirty-one QTLs for four root traits (the number of deep roots, the number of shallow roots, the total number of deep roots and the ratio of deep roots) were assessed on six rice chromosomes in two environments (2020 Shanghai and 2021 Hainan). Two of the QTLs, qDR5.1 and qTR5.2, were located on chromosome 5 in a 70-kb interval. They were detected in both environments. qDR5.1 explained 13.35% of the phenotypic variance in 2020 Shanghai and 12.01% of the phenotypic variance in 2021 Hainan. qTR5.2 accounted for 10.88% and 10.93% of the phenotypic variance, respectively. One QTL (qRDR2.2) for the ratio of deep roots was detected on chromosome 2 in a 210-kb interval and accounted for 6.72% of the phenotypic variance in 2020. The positive effects of these three QTLs were all from Dongxiang wild rice. Furthermore, nine and four putative candidate genes were identified in qRDR2.2 and qDR5.1/qTR5.2, respectively. These findings added to our knowledge of the genetic control of root traits in rice. In addition, this study will facilitate the future isolation of candidate genes of the deep-rooting trait and the utilization of Dongxiang wild rice in the improvement of rice drought resistance.     

Identification and Characterization of Drought Stress- Responsive Novel microRNAs in Dongxiang Wild Rice

MicroRNAs(miRNAs) are non-coding small RNAs, which play important regulatory roles in response to biotic and abiotic stresses. Dongxiang wild rice(Oryza rufipogon, DXWR) can survive in extreme drought environment, but its molecular mechanism of drought resistance is still largely unknown. To further explore miRNA regulatory mechanisms involved in drought resistance, we identified 138 novel miRNAs in DXWR using small RNA sequencing and bioinformatics approaches, and found that the expression levels of 67 novel miRNAs were significantly affected by drought stress. In total, 200 candidate target genes were predicted and annotated for the drought stress-responsive novel miRNAs. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways suggested that most of the target genes were related to metabolism. Stem-loop quantitative real-time PCR(qRT-PCR) results exhibited high concordance with sequencing data, which confirmed that miRNA expression patterns based on small RNA sequencing in the present study were reliable. Meanwhile, qRT-PCR validated the inverse expression patterns between several miRNAs and their target genes. These results will enhance our understanding of miRNA regulatory mechanisms in response to drought stress in DXWR, and can serve as an important reference for the protection and utilization of this valuable genetic resource.     

应用东乡野生稻回交重组自交系分析水稻耐低氮产量相关性状QTL

目的 东乡野生稻低氮耐性强,是水稻耐低氮育种的重要资源。鉴定东乡野生稻耐低氮基因对研究耐低氮遗传机制、培育耐低氮水稻品种具有重要意义。方法 利用协青早B//东乡野生稻/协BBC₁F₁₂回交重组自交系及其遗传图谱,应用Windows QTL Cartographer 2.5分析施氮肥和不施氮肥下的株高和产量相关性状QTL。结果 共检测到57个控制株高和产量性状的QTL,分布于10条染色体上的33个区域,单个QTL表型贡献率为3.17%~63.40%,其中32个QTL的增效等位基因来自东乡野生稻。19个QTL在施氮和未施氮条件下均检测到,38个QTL仅在单一环境下检测到显著效应,表明不同施氮水平下水稻性状的遗传机制不同。结论 43个QTL分别聚集于7条染色体上的14个QTL簇,表明不同性状涉及到共同遗传机制,并可通过分子标记辅助选择方法进行耐低氮有利等位基因的聚合育种。     

东乡野生稻研究进展及育种利用策略

东乡野生稻是目前世界上已发现的分布最北（N 28°14′、E 116°36′）的野生稻特异种质资源,其基因组中蕴含着抗病、抗虫、耐逆及其他诸多优良基因/QTL,前人已对其开展了大量的研究。本文从抗病、抗虫、抗逆、育性、产量及其他优良特性等方面总结了东乡野生稻的研究进展,并提出了其育种利用策略。     

栽培稻和普通野生稻基因组的随机扩增多态性DNA（RAPD）初步分析

 用35个随机引物对5个籼稻品种、5个粳稻品种和27份中国普通野生稻进行了RAPD分析。60%以上的引物能在籼稻和粳稻基因组间显示差异；中国普通野生稻与栽培稻的差异主要表现在与籼稻的不同，绝大部分供试野生稻的RAPD带型与粳稻的相同。这说明多数中国普通野生稻偏粳，但也存在偏籼的普通野生稻。