Genetic Analysis and Primary Mapping of pms4, a Photoperiod-Sensitive Genic Male Sterility Gene in Rice (Oryza sativa)

To understand the genetic characteristics of a new photoperiod-sensitive genic male sterile line Mian 9S, some reciprocal crosses were made between Mian 9S and six indica rice materials, Yangdao 6, Luhui 602, Shuihui 527, Mianhui 725, Fuhui 838 and Yixiang 1B. Genetic analysis results suggested that the photoperiod-sensitive genic male sterility （PGMS） of Mian 9S was controlled by a single recessive nuclear gene. Thus, the F2 population derived from the cross of Yangdao 6/Mian 9S was used to map the PGMS gene in Mian 9S. By using SSR markers, the PGMS gene of Mian 9S was mapped on one side of the markers, RM6659 and RM1305, on rice chromosome 4, with the genetic distances of 3.0 cM and 3.5 cM, respectively. The gene was a novel PGMS gene and designated tentatively as pms4. In addition, the application of the pms4 gene was discussed.

Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice

Pingxiang-dominant genic male sterile rice （PDGMSR） was the first dominant genic male sterile mutant identified in rice （Oryza sativa L.）, and the corresponding dominant genic male sterile gene was designated as Ms-p. The fertility of PDGMSR can be restored by introduction of a dominant epistatic fertility restoring gene in some rice varieties. In the present study, E823, an indica inbred rice variety, restored the fertility of PDGMSR, and the genetic pattern was found to be consistent with a dominant epistatic model, therefore, the dominant epistatic fertility restorer gene was designated as Rfe. The F2 population from the cross of PDGMSR/E823 was developed to map gene Rfe. The F2 plants with the genotypes Ms-pMs-pRferfe or Ms-pms-pRferfe were used to construct a fertile pool, and the corresponding sterile plants with genotypes Ms-pMs-prferfe or Ms-pms-prferfe were used to con- struct a sterile pool. The fertility restoring gene Rfe was mapped to one side of the microsatellite markers RM311 and RM3152 on rice chromosome 10, with genetic distances of 7.9 cM and 3.6 cM, respectively. The microsatellite markers around the location of the Ms-p gene were used to finely map the Ms-p gene. The findings of this study indicated that the microsatellite markers RM171 and RM6745 flanked the Ms-p gene, and the distances were 0.3 cM and 3.0 cM, respectively. On the basis of the sequence of rice chromosome 10, the physical distance between the two markers is approximately 730 kb. These findings facilitates molecular marker-assisted selection （MAS） of genes Ms-p and Rfe in rice breeding programs, and cloning them in the future.

Characterizations and identification of the candidate gene of rice thermo-sensitive genic male sterile gene tms5 by mapping

Previous study indicated that the thermo-sensitive genic malesterile(TGMS) gene in rice was regulated by temperature.TGMS rice plays an important role in hybrid rice production,because the application of the TGMS system in two-line breeding is laborsaving,timesaving,simple,inexpensive,efficient,and eliminating the limitations of the cytoplasmic male sterility(CMS) system.'AnnongS' is the first discovered and deeply studied TGMS rice lines in China.'AnnongS-1' and 'Y58S',two derivatives of TGMS line AnnongS,were both controlled by a single recessive gene named tms5,which was genetically mapped on chromosome 2.In this study,three populations('AnnongS-1' × 'Nanjing11','Y58S' × 'Q611',and 'Y58S' × 'Guanghui122') were developed and used for the molecular fine mapping of the tms5 gene.By analyzing recombination events in the sterile individuals using a total of 125 probes covering the tms5 region,the tms5 gene was physically mapped to a 19-kb DNA fragment between two markers 4039-1 and 4039-2,which were located on the BAC clone AP004039.After the construction of the physical map between two markers 4039-1 and 4039-2,a member(ONAC023) of the NAC(NAM-ATAF-CUC-related) gene family was identified as the candidate gene of the tms5 gene.

Characterization and fine mapping of RTMS10, a semi-dominant reverse thermo-sensitive genic male sterile locus in rice

The discovery and application of environment-sensitive genic male sterile(EGMS) rice germplasm provide an easy method for hybrid rice breeding and have made great contributions to hybrid rice production. Typically, the photoperiod-and thermosensitive GMS(P/TGMS) lines utilized in two-line hybrid systems are male sterile under long day or/and high temperature but fertile under short day or/and low temperature conditions. However, Yannong S(Yn S), a reverse TGMS(rTGMS) line, is sterile under low temperature(<29℃) and fertile under high temperature(>29.5℃). Here, we report a genetic study on the rTGMS trait in Yn S. Interestingly, the F1 plants of the cross between Yn S and a cultivar, L422, were male sterile at 22℃ and completely fertile at 27℃. Moreover, the segregation ratio of fertile and sterile individuals in Yn S/L422 F2 populations changed from 1:3.05 to 2.95:1 when the ambient temperature increased, showing that the rTGMS trait exhibits semidominance in Yn S. We further found a locus on chromosome 10, termed RTMS10, which controls the rTGMS trait in Yn S. We then finely mapped RTMS10 to a ~68 kb interval between markers ID13116 and ID1318 by Yn S/L422 BC6 F2 populations. A near iso-genic line(NIL) NL1 from the BC6 F3 generation was developed and the pollen of NL1 became abnormal from the meiosis stage under low temperature. In summary, we identified an rTGMS locus, RTMS10, and provided co-segregated markers, which could help to accelerate molecular breeding of rTGMS lines and better understand the rTGMS trait in rice.

Breeding double low super hybrids with dominant genic male sterility three lines in Brassica napus

A double low homozygous two-type sterile line ZY-2AB, its near-isogenic temporary maintainer line ZY-4, and the full sterile line ZY-3 derived from the cross of ZY-2A with ZY-4, were developed to breed three-line dominant GMS hybrid in Brassica napus. Three years data showed that the ratios of sterile and fertile plants fit the expected 1 ： 1 ratio in ZY-2AB, and the 1：0 ratio in ZY-3, respectively. Some double low super hybrid combinations were screened out when the full sterile line ZY-3 was used as female parent, among which the combination 01Z24 increased 36.88 percent more yield than control and the conbination 98Z131 has been passed through the Sichuan Provincial Regional Test and registered in Sichuan Province. Two years data showed that the agronomic performance of the two-line hybrids derived from crosses of ZY-2A with restorer lines versus that of the three-line hybrids produced by ZY-3 was not different for any of the nine most important agronomic characteristics, revealing that near-isogenic line of homozygous two-type line was used as temporary maintainer could effectively avoid the disadvantage of threeway cross hybrid in breeding three-line dominant GMS hybrid.

Differentially Expressed Genes Related to Fertility Conversion in Thermo-sensitive Genic Male Sterile(TGMS) Lines of Brassica juncea

[Objective] This study was performed to screen functional genes related to the fertility conversion of thermo-sensitive genic male sterile （TGMS） lines of Brassica juncea L. [Method] A B. juncea TGMS line K121S was selected as the experimental material. The total RNAs were isolated from fertile and sterile pollens at different development stages, including mother cell stage, tetrad stage, tricellular pollen stage and maturity stage. DDRT-PCR was carried out to identify differentially expressed genes. [Result] A total of 44 differentially expressed cDNA fragments were identified with Dot blot. And seven candidate genes related to fertility conversion of K121S were screened out by BLASTN, including callose synthase gene, aldehyde dehydrogenase gene and RNA polymerase I transcription factor RRN3 gene which were differentially expressed at the transcriptional level, H＇-ATPase gene, fructose diphosphate aldolase -class I gene, teucine-rich repeat receptor-Jike serine/threonine- protein kinase gene and alkaline/neutral invertase gene, which were differentially expressed at the post-transcriptional level. [Conclusion] The results of this study will help to explain the molecular mechanism of thermo-sensitive genic male sterility of B. juncea.

Studies on Pingxiang dominant genic male sterile (PDGMS) rice

So far,14 examples of dominant genic malesterility on 10 crop species have been discov-ered in the world.In crossing sterile plants ofnon-pollen-type Fof cultivar-Wild combina-tlon(Ping＇ai 58/Huaye)as female parent withfertile plants Fof the wild-cultivar combination(Huaye/Ping＇ai 58)as male parent,a typ-ical abortive plant was found in the progeny.By excluding the Probability of balancedlethal,gametophyte sterility,and chromo-some aberration,studies showed that thesterility was controlled by a dominant malesterile gene in the nucleus.The material was

Mapping of a gene for photoperiod-sensitive genic male sterility in Nongken 58s on chromosome 5 of rice

A photoperiod-sensitive genic male sterile (PGMS) rice was found in 1973 as a spontaneous mutant of Nongken 58, a japonica variety. Pollen fertility of Nongken 58s (N58s) is completely sterile when grown under long-day conditions, whereas fertile under short-day conditions. This PGMS was found to be controlled by one or two recessive gene(s), of which one gene(pms)was linked to a marker gene(d-1) on chromosome 5. In order to identify a more precise location of the pms, we analyzed the populations of BCFand BCFof N58s//N58s/KL211(v-10, virescent) and N58s//N58s/KL520 (gh-1, gold hull). The marker genes v-10 and gh-1 are located on the flanking region of d-1. The F, plants of two crosses were fertile. The number of fertile and sterile individuals in BCFfit

Development of Japonica Male Sterile Lines Integrating Cytoplasmic Male Sterility and Photosensitive Genic Male Sterility

Acknowledgement It has been previously established that the BT type of cytoplasmic male sterility （CMS） is induced by high temperatures, while photosensitive genic male sterility （PGMS） seed sets by low temperatures induce. In the current study, we have bred photosensitive cytoplasmic male sterility （PCMS） lines （2308SA and 2310SA） by crossing the CMS line with the PGMS japonica line with maintainer genes. The sterility of PCMS japonica was consequently controlled by two groups of male sterile genes resulting from the integration of PGMS and CMS genes. The results on plant fertility, at different sowing times, were as follows： （a） Under conditions of natural long-day photoperiod and at temperatures above 35~C, the PGMS gene regulated PCMS japonica sterility - the higher the temperature, the lower the pollen fertility. However, bagged seed sets of PCMS japonica, not exposed to high temperatures, induced the CMS seed set. （b） Exposure to long-day photoperiod and temperature conditions between 35℃ and the critical sterility inducing temperature of PGMS resulted in both PGMS and CMS gene controlled sterility of PCMS japonica, which exhibited stable characteristics. （c） When exposed to critical sterility inducing temperatures or short-day photoperiod and daily high temperatures below 32℃, the BT type of the CMS gene regulated PCMS sterility. Under these conditions, the PGMS gene rendered male sterility insusceptible to occasional cool summer days when this PCMS line, adopted for hybrid seed production, develops into panicle differentiation stage. The present study also investigated the fertility restoration, seed production and combining ability of PCMS japonica so as to optimize its use.

Progresses and Strategies of Gene Mapping and Isolating of Photo(thermo) sensitive Genic Male Sterile Rice

Considering the research on classical genetics of photoperiod(therm) sensitive genic male sterile rice, it is important to select the sterile lines and their segregating population controlled by one pair of gene in mapping and isolating sterile genes. It is discussed the advantages, disadvantages and the reasons leading to various mapping results of chromosome location of sterile genes through gene marker, isozyme marker and DNA marker techniques. In comparison to isolation of photo(thermo) sensitive sterile genes via various plant gene cloning techniques, it was concluded that map based cloning was acceptable, but it is still difficult to locate the loci of sterile genes within 1cM. On the other hand, “sensitivity to environment”, an important characteristic of sterile lines can be fully utilized by DD PCR and (or) RDA techniques. Therefore, these two techniques were considered as the effective ways to isolate sterile genes.

Rapid Constructing a Genetic Linkage Map by AFLP Technique and Mapping a New Gene tms5

In this study, we reported the repaid construction of a molecular marker linkage map of rice (Oryza sativa L.). An F-2 population from the cross between Annong S-1 and Nanjing 11 was used to construct a genetic linkage map of rice. Total of 142 newly screened AFLP markers and 30 anchor markers (25 SSR markers and 5 RFLP markers) were mapped on the 12 chromosomes covering 1537.4 cM of rice genome. The average interval between these markers was 9.0 cM. The total work which usually was finished in more than one year was finished within only 3 months by one person. This is the first plant AFLP map developed in China. A new thermosensitive genic male sterile gene in rice, tms5, was Egged and mapped onto chromosome 2 during the development of the linkage map.

Isolation and Analysis of the Promoter of OsRacD from PhotoperiodSensitive Genic Male Sterile Rice

By using OsRacD cDNA as probe to screen the genomic library of photoperiod sensitive genic male sterile rice line Nongken 58S, a positive clone containing 2 kb promoter and 396 bp coding region of OsRacD was obtained. Compared with the promoter of OsRacD cloned by reverse PCR from normal rice variety Nongken 58 （Nongken 58N）, the homology was 99.8%, and the different nucleotides were outside the predicted response elements in promoter, suggesting that the fertility between rice varieties Nongken 58S and Nongken 58N under the long-day conditions was not attributed to the difference in the structure of OsRacD upstream regulation sequences, but to the developmental regulation of gene differential expression.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Fertility Expression of TGMS-Genes in the Backgrounds of indica CMS-lines,B-lines and R-lines of Hybrid Rice

The generation fertility of 51 F1, 19 F2 and 6 BC1 between 3 thermo-sensitive genic male sterile lines （TGMS-lines） Pei＇ai 64S, 6311S and 360S, and the three lines of hybrid rice including 7 indica cytoplasmic male sterile lines （CMS-lines） and their corresponding maintainer lines （B-lines） and 3 indica restorer lines （R-lines） were investigated to study the expression of TGMS-genes in the backgrounds of the three lines of hybrid rice. Pei＇ai 64S has stronger fertility restoring （Rf） genes for CMS-lines and its TGMS trait is governed by 2 pairs of independent recessive genes; The TGMS trait of 6311S is governed by a single recessive gene with weaker Rf-gene in 6311S and the TGMS trait of 360S is governed by a single recessive gene with no Rf-gene in 360S. The investigation on the fertility of F1 plants between 5 CMS-lines and 4 TGMS generations selected from F2 plants of 4 CMS-lines x 6311S confirmed that the expression of TGMS-gene was controlled by Rf-gene in the genetic background of cytoplasm of CMS-lines, but not affected by Rf-gene in the genetic background of normal fertile cytoplasm. The potential breeding strategies of TGMS-lines with cytoplasm of CMS-lines and CMS-lines with the nucleus of TGMS-genes were discussed.

Elongation of the Uppermost Internode for Changxuan 3S,a Thermo-Sensitive Genic Male Sterile Rice Line

Changxuan 3S, a thermo-sensitive genic male sterile （TGMS） rice line with eui gene, is derived from the TGMS rice line Pei＇ai 64S by irradiation with 350 Gy of ^60Co γ-ray. To elucidate the uppermost internode elongation of the TGMS line with eui gene, Changxuan 3S and its parent Pei＇ai 64S were used to study the effects of temperature on panicle exsertion. At 24℃, the uppermost internode of Changxuan 3S elongated the fastest from the 4^th day before flowering to 0 day （flowering）, being 2.1-fold as that of Pei＇ai 64S, whereas it elongated slowly during the 12^th day to the 4^th day before flowering and the 1^st to the 3^rd day after flowering. The uppermost internode of Changxuan 3S exserted from the flag leaf sheath at 22℃, 24℃ and 26℃, and the length of elongated uppermost internode increased with the decreasing temperatures. At 28℃, though the panicles of Changxuan 3S were still enclosed in the leaf sheath, the degree of panicle enclosure was significantly lower compared with Pei＇ai 64S. Cytological studies on Changxuan 3S showed that the uppermost internode elongation was attributed to the increase of cell number and cell elongation, and the latter was more significant. Moreover, the numbers of outermost and innermost parenchyma cells and the cell length of the uppermost internode reduced with the increasing temperatures.

辣椒GMS育性相关候选基因的克隆及表达分析

【目的】对辣椒差减文库中挑选出的4个育性相关候选基因进行克隆与表达分析,探讨其与辣椒细胞核雄性不育的关系。【方法】通过半定量RT-PCR技术分析候选EST在辣椒可育株和不育株中的表达情况。利用RACE技术获得4个育性相关基因的cDNA全长,结合生物信息学方法进行序列比对和蛋白理化性质分析。采用半定量RT-PCR检测基因在不同器官(花药、子房、花瓣、萼片和叶片)和花药小孢子不同发育时期(四分体时期、单核早中期、单核靠边期和双核期)的表达量。【结果】根据比对注释结果,将4个基因分别命名为CaSEP1、CaPROF、CaOle e 6和CaPCP。CaSEP1全长1 108 bp,编码221个氨基酸,含有一个MADS结构域和一个K结构域;CaPROF全长767 bp,编码131个氨基酸,含有一个PROF结构域;CaOle e 6全长523 bp,编码85个氨基酸,含有一个Ole e 6结构域;CaPCP全长563 bp,编码66个氨基酸。氨基酸序列比对及系统发育树分析表明,CaSEP1与番茄SEP1的氨基酸序列相似性最高,并且亲缘关系最近;CaPROF与茄科作物烟草和番茄的前纤维蛋白相似性较高,且与番茄的前纤维蛋白亲缘关系最近;CaOle e 6与番茄中的对应蛋白存在一定的相似性,亲缘关系也最近;CaPCP则与茶树中的对应蛋白相似性最高,亲缘关系最近。表达分析显示,CaSEP1在可育株花药、子房、花瓣等花器官中表达量一致,且高于萼片和叶片;CaPROF在可育株花药中的表达明显高于叶片,在其他器官中不表达;CaOle e6在可育株花药中的表达明显高于其他器官;CaPCP只在可育株的花药中表达。CaSEP1在可育株小孢子不同发育时期表现为先逐渐升高,至单核靠边期达到最高,而后在双核期表达量明显下降,在不育株中则表现为伴随着小孢子发育表达量逐渐升高;该基因在四分体时期两材料的表达量相当,在单核早中期和单核靠边期可育株中的表达量明显高于不育株,而双核期可育株中的表达量比不育株低;CaPROF、CaOle e 6和CaPCP均在可育株小孢子发育后期(主要为单核靠边期和双核期)表达,在不育株花药发育的各个时期均不表达。【结论】4个候选基因的序列比对和表达分析结果表明它们与辣椒雄蕊育性关系密切,为揭示雄性不育机理和调控辣椒育性提供了重要信息。

甘蓝型油菜核不育材料Shaan-GMS不育基因的RAPD标记

以825条10碱基随机引物对甘蓝型油菜Shaan-GMS转育的杂合近等基因系220AB的可育群体和不育群体进行PCR扩增,其中632条引物共扩增出2043条带,引物BA1102在220AB的可育群体及不育群体间扩增出2条多态性条带,BA1102.500只在220AB的不育群体中出现,BA1102.1000在220AB的不育群体中缺失。进一步对单株PCR分析表明,15株不育株都扩增出了BA1102,500条带;未扩增出BA1102.1000条带,而15株可育株的扩增结果相反,说明BA1102.500是与Shaan-GMS的显性核不育基因Ms相连锁的阳性带。在恢保关系相同的双显性核不育材料6CA的F_1可育和不育群体中,BA1102未获得多态性。

甘蓝型油菜隐性上位互作核不育基因(Ms1)精细定位

甘蓝型油菜细胞核雄性不育是杂种优势利用的重要途径。隐性上位互作核不育系9012A已经广泛用于杂交种子生产,其不育性受两对隐性重叠不育基因(ms1和ms2)与一对隐性上位抑制基因(rf)互作控制。ms1和ms2同时纯合(ms1ms1ms2ms2)表现不育,但隐性纯合rf(rfrf)对ms1ms1ms2ms2的表达起抑制作用,又可使其表现可育(TAM系,ms1ms1ms2ms2rfrf)。本研究利用AFLP和SRAP分子标记技术,分别筛选了884对AFLP引物和506对SRAP引物,筛选到了14个与不育基因Ms1基因紧密连锁的标记,其中4个标记与育性共分离。Ke等发表的AFLP标记E-ACA/P-CTG在本研究群体中与Ms1基因相距0.1cM。紧密连锁标记测序序列BLASTN结果表明,其和拟南芥第五染色体高度同源,并且通过和已测序的甘蓝C基因组序列进行比对,将其定位在C9染色体的末端。本研究结果有助于该基因的分子标记辅助选择和克隆。本文还对前人发表的与Ms1基因连锁的标记在本研究群体中的分离情况作了探讨。

水稻光敏核不育基因pms3的精细定位

为了进一步研究水稻晚粳品种农垦 5 8转变为光敏核不育水稻农垦 5 8S的突变位点 ,并精细定位光敏不育基因pms3,我们利用农垦 5 8S/15 14杂交组合的 F1 进行花药培养构建了一个 DH群体 ,验证了在该群体中光敏不育受 pms1、pms3两对基因的控制 ,并根据分子标记分析选择第 12染色体是 15 14基因型、第 7染色体农垦 5 8S基因型的 DH系 DH80与农垦 5 8S杂交构建一个 pms3光敏不育单基因分离的群体。根据 F3家系的育性分离情况推测出 F2 的基因型 ,结合分子标记分析进一步验证了 pms3在第 12染色体上的位置 ,并将其定位在 RFL P标记 M36和 RZ2 6 1之间 ,与两标记的遗传距离分别为 1.5 c M和 3.0 5 c M,为启动 pms3区域的染色体步查打下了基础。同时还比较分析了 RFL P标记在 DH群体及F2 群体中的分离情况 ,发现第 12染色体上 pms3区域在 F2 群体中正常分离的多个标记在 DH群体中发生了严重的偏分离 ,另外还发现 DH群体第 3、 7、

甘蓝型油菜核不育材料Shaan-GMS恢复基因的筛选及其遗传分析

从国内外77份甘蓝型油菜材料中筛选出1份Shaan-GMS的恢复材料"96-803",对其的遗传研究结果表明,Shaan-GMS育性恢复遗传机制符合2对核基因互作控制假说,假设不育基因为Ms,其对应的隐性可育等位基因为ms,Rf为Ms的显性抑制基因,rf为Rf的隐性等位基因,基因型Ms-rfrf表现为雄性不育,其他7种基因型都表现为雄性可育,则Shaan-GMS的基因型为Msmsrfrf,"96-803"的基因型为msmsRfRf,隐性测交系"84004"的基因型为msmsrfrf。"96-803"和其他8份材料的测交结果表明,Shaan-GMS同6CA恢保关系相同。