Identification and fine mapping of a fertility restorer gene for wild abortive cytoplasmic male sterility in the elite indica rice non-restorer line 9311

The wild abortive(WA)-type cytoplasmic male sterility(CMS)derived from the wild rice species Oryza rufipogon Griff.is used widely in three-line indica hybrids.The identification and mapping of restorer of fertility(Rf)genes aided in the development of WA-type hybrids.Here we report that testcross F1 plants from the WA-type CMS line and 9311 exhibited stainable pollen grains with no seed set,indicating that 9311 carries minor-effect Rfs for WA-type CMS.We developed an advanced backcross population consisting of plants harboring small regions of donor chromosomal segments from 9311 in the WATianfeng A genetic background with moderate seed setting rates.Genetic analysis showed that the pollen fertility levels of the backcross individuals are governed by a single gene from 9311 that we named Rf19(t).By use of the RICE 40 K gene chip,three introduced segments were identified in the fertile lines,and a candidate region spanning 4.37–8.29 Mb on chromosome 1 was identified for Rf19(t).Finally,Rf19(t)was fine-mapped to a region of 90 kb between the DNA marker loci STS1-163 and STS1-183,in which eight ORFs were predicted.Also,using relative expression analyses,comparative sequence analyses and functional domain analyses,we identified LOC_Os01g10530 as the most likely candidate gene for Rf19(t).Furthermore,Rf19(t)was found to function in fertility restoration,most probably by regulating the degradation of m RNA transcribed from the mitochondrial gene WA352.These results increase our knowledge of fertility restoration in WA-type CMS lines and will facilitate the development of high-quality pairs of WAtype CMS and maintainer lines.

Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene

Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluated using PCR amplification, test paper evaluation, insect resistance evaluation in both the laboratory and paddy fields, nursery evaluation of rice blast resistance and pedigree selection of agronomic traits. Larval mortalities on Bt5198 and Bt Minghui 63 were 100% when rice culms were inoculated with the eggs of the striped stem borer （SSB） in the laboratory. Bt5198 was highly resistant against SSB and the yellow stem borer （YSB） under field conditions. The F1 hybrids derived from Bt5198 and four cytoplasmic male sterile （CMS） lines were also highly resistant to SSB and YSB and had a significant heterosis. Two-year evaluation of rice blast resistance confirmed that the resistance levels of Bt5198 to leaf blast and neck blast were similar to those of Chenghui 177 and significantly better than those of Bt Minghui 63. Seed germination ability and pollen yield of Bt5198 were similar with Chenghui 177, suggesting that the introduction of the Bt gene into the new restorer line had no significant effects on seed vitality or the yield of seed production. To identify the presence of the Bt gene, it was effective to combine test paper examination with the evaluation of insect-resistance, both in the laboratory and under field conditions.

Genetic Analysis of Stripe Disease Resistance in Rice Restorer Line C224 Using Major Gene plus Polygene Mixed Effect Model

The inheritance of stripe disease resistance in a rice restorer line C224 was analyzed using the mixed effect model of major gene plus polygene for quantitative traits.In addition,the resistance was investigated in seven crosses of C224 with maintainer lines.The results showed that the stripe resistance of C224 was controlled by two major genes with additive-dominance-epistasis effects plus polygenes with additive-dominance effects （E-1 model）.These two genes had additive effects of-12.47% and-24.75%,respectively,showing negative dominance effects.There were significant epistasis and interaction effects between the two major genes.The heritability of the two major genes was 92.12%,while that of polygenes was 2.74%,indicating that the stripe resistance had dominant major gene effect.Of the seven crosses,five displayed high or medium resistance to the stripe disease.

The Application of ISSR Markers to Identify the Fertility Restorer Gene Rf6 in T. timopheevii Cytoplasmic MaleSterile Wheat(Triticum aestivum)

Inter-simple sequence repeat (ISSR) analysis was carried out on a F2 population of 147 plants derived from a cross between a wheat male fertility restorer line 2114 and a male sterile line ND44A. Out of 43 primers examined, 18 primers produced distinguishable, polymorphic bands between the two parents. Linkage analysis in the mapping population showed that two markers UBC-808 and UBC-848 were closely linked with the restorer gene Rf6 of the Triticum timopheevii CMS system. The distance between the two markers and the restorer gene was 7.9 cM and 4.9 cM, respectively. Also two parents were screened with 181 pairs of SSR primers, of which, 34.3% showed polymorphisms. But no locus was found linked with the restorer gene. Compared with the SSR technique, the ISSR approach used in the experiment provided more information and proved to be a valuable method to identify alien fragments.

Mapping of Rice Fertility-Restoring Genes for ID-Type Cytoplasmic Male Sterility in a Restorer Line R68

An F2 population derived from the cross Zhong 9A/R68 was used to map the fertility-restoring （Rf） gene for ID-type cytoplasmic male sterility （CMS）. Two bulks （a fertile bulk and a sterile bulk） were constructed by pooling equal amount of ten highly fertile lines and ten highly sterile lines, respectively. Four hundred and thirteen pairs of simple sequence repeat （SSR） primers, which evenly distributed on 12 chromosomes of rice, were selected for analyzing polymorphisms between the parents and between the two bulks. The primer RM283 on chromosome 1 and the primers RM5756, RM258, RM6100 and RM171 on chromosome 10 were found to be polymorphic between the parents and between the two bulks. These five SSR markers were linked to fertility-restoring genes. A total of 82 excessive sterile lines were selected from Zhong 9A/R68 F2 population to estimate the genetic distance between five SSR markers and fertility-restoring genes respectively. The results indicated that one Rf gene was linked to RM283 located on chromosome 1 at a distance of 6.7 cM, and the other Rfgene was mapped to the long arm of chromosome 10 flanked by RM258 and RM6100 at the distances of 8.0 cM and 2.4 cM, respectively.

Breeding of Rice Restorer Line Haihui 818 of Multi-resistant Gene Pyramiding

Haihui 818 is a new restorer line by cross breeding with Hua 23 S and Huahui 8131.Good plant type,high tillering ability,fine grain quality,strong restoring ability and high yield were shown in hybrid production.After artificial inoculation and field stress and molecular market-assisted selection,the results showed that rice-blast resistance genes Pi1,BB resistance genes Xa7,and brown planthopper resistance genes Bph3,Bph14 and Bph15 were pyramided together.Among its hybrids,Bo II You 818(Bo II A/Haihui 818)was approved for commercial production by Hainan Provincial Crop Variety Approval Committee in 2015.

Mapping of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat Using SSR Markers

LK783 was found to be a good fertility restorer for K-type male sterility of wheat. Microsatel-lite markers were employed to map the major restoring gene in LK783. Maintainer and restorer DNA pools were established using the extreme sterile and fertile plants among (KJ5418A//911289/LK783)F1 population, respectively. Seventy-nine sets of SSR primers were screened for polymorphism between the two pools, 6 of which were found polymorphic. Linkage analysis showed that Xgwm11, Xgwm18, Xgwm264a and Xgwm273 were linked to the restoring gene in LK783, while Xgwm11, Xgwm18 and Xgwm273 were co-segregated. The distance between the Rf gene in LK783 and the three co-segregated markers was 6.54 ± 4.37 cM, the distance between Rf gene and Xgwm264a was 5. 71 ± 4.10 cM. The four SSR markers were located on chromosome IBS by amplifying the DNA of nulli-tetrasomics and ditelosomics of CS with the 4 sets of primers, indicating that the major restoring gene in LK783 was located on IBS, but the relative location of the gene was different from Rfv1, allelism of the two genes should be further investigated. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility in wheat would be facilitated by using the four polymorphic markers.

Microsatellite-Aided Screening for Fertility Restoration Genes (Rf) Facilitates Hybrid Improvement

DNA markers enabled to determine the chromosomal locations of the two Rf genes(Rf3 and Rf4) in the wild-abortive cytoplasmic male sterility(WA-CMS) system. Four simple sequence repeats(SSRs) RM171, RM258, RM315 and RM443 were used to detect the allelic status with respect to the fertility restoration genes(Rf3 and Rf4) in 300 rice cultivars or breeding lines. The results revealed that out of 300 lines, 90 lines screened had Rf3, 65 lines had Rf4, and 45 lines had Rf3 and Rf4 alleles. Furthermore, 45 lines selected using SSR markers were mated with a CMS line(IR58025A) to analyze their restoring ability. Offspring of all the test lines except HHZ8-SAL9DT1-Y1, HHZ5-SAL9-Y3-1 and IDSA77 exhibited higher pollen and spikelet fertility(> 80%), thus confirming they bear the Rf alleles. The hybrid offspring of ARH12-6-1-1-B-3-1, IR32307-10-3-2-1 and Sahel 329 had the highest pollen fertility(97.39%, 98.30% and 97.10%, respectively) and spikelet fertility(95.10%, 97.07% and 96.10%, respectively).

Mapping Major Restore Genes for C-type Cytoplasmic Male Sterility in Maize with SSR Marker

Through observation about the restoration of male fertility of F2 and BC, progeny, we found that the restoring line Fengkel had two duplicating restorer genes. The restorer gene R/5 in Fengkel background was located on chromosome 5L by SSR method; it linked with bnlg1711, bnlg1346 and phi058, the genetic distances with bnlg1711, bnlg1346, and phi058 were 7.51cM, 1.68cM, and 9.87cM respectively; the restorer gene Rf4 was mapped on chromosome 8S linked with bnlg2307.

Breeding Rice Restorer Lines with High Resistance to Bacterial Blight by Using Molecular Marker-Assisted Selection

Two bacterial blight （BB） resistance genes, Xa21 and Xa4, from IRBB24 were introduced into hybrid rice restorer line Mianhui 725, which is highly susceptible to BB, by using hybridization and molecular marker-assisted selection technology. Four homologous restorer lines were obtained through testing the R target genes with molecular markers and analyzing parental genetic background. Inoculation of the four lines and their hybrids with the specific strains of Xanthomonas oryzae pv. oryzae, P1, P6 and seven representative strains of Chinese pathotype, C Ⅰ -CⅦ, showed that all of the four lines and their hybrids were highly resistant and presented broad resistance-spectrum to BB. The hybrids of G46A / R207-2 displayed good agronomic characters and high yield potential, and R207-2 was named Shuhui 207.

Inheritance of Fertility Restoration for Cytoplasmic Male Sterility in a New Gossypium barbadense Restorer

In order to clarify inheritance mechanism of fertility restoration for cytoplasmic male sterility （CMS） in a new Gossypium barbadense restorer line Hai R which was found in the fertility test crossing of G. hirsutum CMS lines with G. barbadense germplasms. 23 fertility segregation populations of F2 and backcross were used to analyze the inheritance of fertility restoring gene（s） of Hai R. The result showed that Hai R had one major dominant gene （RfB） to control the CMS fertility restoration and this fertility restoration gene functioned at the sporophytic level. The sterile cytoplasm background might not only influence the transmission rate of male gamete but also that of female gamete when the restorer gene was recessive. It could be deduced that this fertility restoration gene might come from G. harknessii cotton, Hai R is of value in the application of cotton interspecific hybrid breeding.

Genetic analysis of fertility restoring genes for AL-type male sterility in wheat

In order to screen molecular markers linked to fertility restoring genes and further improve the breeding efficiency of restorer lines,in this study,wheat varieties 18A,18B and 99AR144-1 were used as experimental materials to establish F2 fertility-segregating population. Plant quantitative trait"major gene + polygene mixed model"separation analysis method and simple sequence repeat(SSR) molecular markers were adopted for genetic analysis of four generations,including the parents(P1 and P2),and hybrid(Fl and F2) populations. The results show that AL-type fertility restoring gene is controlled by two pairs of additive-dominant-epistatic genes and additive-dominant polygene;two primers linked to fertility restoring genes were selected by SSR molecular markers, including Xgwm95 on chromosome 2A and Barc61 on chromosome 1B,with the linkage distance of 15.0 cM and 18.0 cM,respectively. Based on verification,these two markers are reliable for distinguishing AL-type wheat sterile lines and restorer lines.

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight （BB） diseases, the two most prevalent diseases jeopardizing rice production. Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food. An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions. Only one pair of parents, DH146 ×TM487, showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes, Xa21, xa13 and xa5, thus it was used in the marker-assisted selection （MAS）. F2 individuals of DH146× TM487 were genotyped using flanking markers of RM3330 and sequence tagged site （STS） marker SA7 for Pi25. The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21, xa13 and xa5 identified by the markers pTA248, RM264 and RM153, respectively in subsequent generations. Finally, after selection for agronomic traits and restoration ability among 12 pyramided lines, we acquired an elite restorer line, R8012 including all four target genes （Pi25＋Xa21＋xa13＋xa5）. Hybrid Zhong 9NR8012 derived from the selected line showed stronger resistance to blast and BB, and higher grain yield than the commercial checks uniformally in experimental plots, 2007 state-wide yield trial and 2008 nation-wide yield trial. This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes. The resultant restoring line and its hybrid would play an important role in securing rice production in China.

Influence of Genetic Drift of Restoring Gene (Rf) on Seed Purity of Yuetai A, a Honglian-Type Cytoplasmic Male Sterile Line in Hybrid Rice

The seed samples of Yuetai A, a Honglian （HL） type cytoplasmic male sterile （CMS） line in hybrid rice were investigated to assess the seed purity and to analyze the cause of off-type plants by imitating the biological contaminant to Yuetai A in Nanjing, Jiangsu Province and Lingshui, Hainan Province during 2004-2006. The seed impurity of Yuetai A mainly resulted from the genetic drift of restoring fertility gene （Rt） after biological contamination but not from its sterility unstability. All of the ten maintainer lines, five restorer lines and three thermo-sensitive genic male sterile lines used in the study could pollinate Yuetai A and Yuetai B to produce F1 plants, directly or indirectly resulting in Rf-gene drifting into Yuetai A and generating ＇iso-cytoplasm restoring-generations＇. Furthermore, high outcrossing rate and similar heading date of Yuetai A with many varieties used in rice production might easily result in the biological contamination. After removing all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene drifting into Yuetai A, the seed purity of Yuetai A and Yuetai B had been raised to 100%.

Development of New InDel Marker to Detect Genotypes of Rf-1a Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility in Rice

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id^ntifio.~tinn ~nd m^rkp.r-~.~.~i^fp.d .~.tAr.tinn （MA.~ in hr~=dinn i^nnnir~ r^fnr~=r lin==~

Analysis of restoring gene in Heizhenmi

The purple pigment is controlled by three domi-nant genes.According to the theory ofdominance-super dominance in Fgeneration, breeding purple black hybrid rice can be an effec-tive way to overcome lower yield and weak resist-ance to disease of purple black rice.

抗稻瘟病水稻恢复系的分子标记辅助选育及抗性鉴定

【目的】开展抗稻瘟病水稻恢复系的分子标记辅助选育及抗性鉴定,为育成持久、广谱抗病优质杂交水稻新品种提供优异的遗传资源和技术参考。【方法】利用分子标记辅助选择和回交育种技术,将持久、广谱的抗稻瘟病基因Pi1、Pi2和Pigm导入恢复系桂R1703,获得了单基因、双基因和三基因导入系,并对其进行稻瘟病鉴定和农艺性状调查,分析不同抗稻瘟病基因间的抗性效应及抗性基因导入对受体亲本农艺性状的影响。【结果】通过对杂交后代和回交群体的Pi1、Pi2和Pigm基因相关分子标记检测,发现抗稻瘟病基因Pi1、Pi2和Pigm已成功导入到桂R1703中,筛选获得7个携带有抗稻瘟病基因且抗性和农艺性状综合表现优良的导入株系,表现出较强的稻瘟病抗性。单基因导入系中,抗稻瘟病基因的抗性效应排序为为Pigm>Pi2>Pi1;双基因和三基因导入系的稻瘟病抗性均强于单基因导入系,尤其以三基因导入系抗性最强,不同基因聚合的抗性效应排序为Pi1+Pi2+Pigm>Pi2+Pigm>Pi1+Pigm>Pi1+Pi2。7个瘟病抗性基因导入株系与桂R1703在穗长和千粒重方面无显著差异(P>0.05,下同),有2个瘟病抗性基因导入系的株高显著高于桂R1703(P<0.05,下同);有1个瘟病抗性基因导入系的单株有效穗显著高于桂R1703;有3个瘟病抗性基因导入系的单穗总粒数显著高于桂R1703;有5个瘟病抗性基因导入系结实率显著低于桂R1703;有2个瘟病抗性基因导入系的单株产量显著低于桂R1703;有4个瘟病抗性基因导入系的单株产量显著高于桂R1703。【结论】通过分子标记辅助选育可有效聚合多基因(Pi1、Pi2和Pigm基因),使恢复系桂R1703的稻瘟病抗性得到明显提升,获得一系列抗稻瘟病且与亲本其他性状相近的遗传材料,可作为亲本材料用于选育抗病优质的杂交稻。

辣椒雄性不育分子研究进展

为对辣椒雄性不育分子研究取得的进展有更直观、系统、深入的了解,梳理已鉴定的辣椒核雄性不育(NMS)基因及部分NMS基因分子标记开发,重点阐述近年来核雄性不育基因精细定位及候选基因探究工作;同时回顾辣椒胞质雄性不育(CMS)相关的线粒体不育基因及其关联分子标记和CMS恢复基因分子标记开发,重点概述近年来恢复基因精细定位研究取得的进展。研究指出,精细定位NMS基因,明确候选基因并开发功能标记是辣椒NMS研究的重点,既能为探明辣椒NMS机理奠定基础又可为育种提供高效利用的分子标记。而CMS相较于NMS更为复杂,表现在遗传机制多样且更易受环境影响等方面,因此,从对CMS育性恢复基因的精细定位到基因克隆以及功能研究并探明CMS分子机制还有很长的一段路要走。

不同更新方式下亚热带森林土壤病毒群落结构与功能特征

探讨不同更新方式下森林土壤病毒群落特征及其在生物地球化学循环中作用,以福建上杭白砂国有林场的杉木人工林、天然更新次生林和原生林土壤为研究对象,结合宏病毒组学和生物信息学方法进行病毒群落结构与功能分析。结果表明:森林更新方式对土壤病毒群落结构产生了明显的影响。原生林和天然更新次生林主要病毒类群为长尾噬菌体科(Siphoviridae)(62.60%、31.49%),而杉木人工林主要土壤病毒类群为微小噬菌体科(Microviridae)(27.89%)。在3种土壤中均发现了核质巨大DNA病毒(nucleo-cytoplasmic large DNA viruses,NCLDV),其在天然更新次生林中占比最高(20.83%);杉木人工林和天然更新次生林中最主要的病毒宿主属于变形菌门(Proteobacteria),而原生林则为放线菌门(Actinobacteria)。但3种森林类型土壤病毒宿主均包括戈登氏菌(Gordonia)、红球菌(Rhodococcus)、分枝杆菌(Mycolicibacterium)等感染人和动物的致病菌;病毒功能组中均检测到丰富的辅助碳水化合物活性酶(CAZyme)基因,其中杉木人工林土壤病毒中编码CAZymes的基因丰度(4个)显著低于原生林(210个)和天然更新次生林(69个)。研究结果揭示了森林更新方式对土壤病毒结构、宿主和碳循环功能的潜在影响。

采煤沉陷区复垦对土壤细菌群落组装及固碳功能的影响——以东滩矿区为例

复垦能有效提升矿区生态服务功能,但复垦土壤功能重建的微生物学机制尚不清晰。厘清复垦如何影响土壤细菌群落特征、组装机制及固碳功能,对重塑矿区生态自维持能力至关重要。为此,结合零模型分析,采用MiSeq高通量测序及q PCR芯片技术,探索东滩矿区9 a、12 a、15 a和18 a等4个复垦年限土壤细菌群落组装过程及固碳功能变化。结果表明:(1)复垦和复垦时间对土壤理化和酶活性影响显著,复垦土壤pH、铵态氮(AN)、过氧化氢酶(CAT)及磷酸酶(PO)随复垦时间增长呈显著增加(P<0.05),有机碳(SOC)、有效磷(AP)、硝态氮(NN)及脲酶(UE)、β-葡萄糖苷酶(BG)、蛋白酶(PRO)则相反(P<0.05);(2)随机性过程主导了复垦土壤细菌群落的组装过程,且扩散限制的贡献最大;(3)有机碳、硝态氮、铵态氮、有效磷、β-葡萄糖苷酶及过氧化氢酶与碳循环功能基因丰度有显著性相关,复垦改善土壤理化从而增强固碳功能;(4)结构方程模型显示,复垦年限增加直接影响土壤理化性质,进而间接影响土壤微生物群落的组装过程,这可能是导致碳循环功能基因丰度变化的主要原因。研究结果为矿区复垦土壤生产力改善和固碳功能提升提供理论依据。