Effect of High Temperature on Outcrossing Characteristics at the Fertility Sensitive Period of Photo-thermo Sensitive Genic Male Sterile (PTGMS) Rice Lines

Four sterile lines （Peiai64S, Y58S, Guangzhan 63-2S and H638S） and the restoring line R527 were used as materials. Five temperature gradients （24, 27, 30, 33 and 36 ℃ in artificial climate chamber） and the natural temperature （as control） were treated to the four sterile lines for 6 d in the fertility sensitive period of heading to flowering stage, respectively, to study the effects of temperature on physiological biochemical characteristics of young panicles and outcrossing characteristics. The results showed that the percentages of exerted stigma of Peiai 64S and Y58S were the highest at 27 ℃, which were 86.81% and 86.06%, respectively, while the percentages of exerted stigma of Guangzhan 63-2S and H638S were the highest at 24 ℃, which were 76.24% and 81.76%, respectively; the stigma viability of Peiai 64S, Y58S, Guangzhan 63-2S and H638S were the best at 24 ℃, which were 1.96, 2.12, 1.74 and 1.94, respectively; the outcrossing rates of Peiai 64S, Guangzhan 63-2S and H638S were the highest at 24 ℃, which were 58.87%, 54.22% and 50.50%, respectively, while the outcrossing rate of Y58S was the highest at 27 ℃, which was 58.96%; and the contents of peroxidase （POD） of the four sterile lines increased significantly at 33 ℃ compared with the control, while the contents of malondialdehyde （MDA） and proline increased significantly at 36 ℃ compared with the control. There were differences in temperature sensitivity between the male sterile lines, and the 24 ℃ treatment during the sensitive period was the best for the fertility sensitive period of Peiai 64S, while 27 ℃ was the best temperature for Y58S, Gangzhan 63-2S and H638S.

Preliminary Study on Inheritance of Stigma Exertion in Wheat Thermo-photo Sensitive Genic Male Sterile Line

Stigma exertion is one of the key factors for improving the outcrossing ability of wheat thermo-photo sensitive genic male sterile（TPSGMS） line. A DH population derived from K239S/K92 S was constructed to investigate the inheritance of stigma exertion. K239 S and K92 S are TPSGMS lines with higher and lower stigma exertion rates（SER）, respectively. The SERs of parents, reciprocal crosses and the DH population were evaluated for two consecutive years. The results showed that no significant difference was observed in SER between F1 s of K239S/K92 S and K92S/K239 S,implying that stigma exertion was a trait controlled by nuclear gene（s）. In the DH population, the segregation of low and high SERs fitted to a ratio of 3 ∶1 by Chisquare test, suggesting that the stigma exertion of K239 S was controlled by one pair of recessive genes. In addition, the effects of temperature and humidity on the expression of stigma exertion were also discussed.

Comparative Studies on the Changes of Microtubule Distribution and Reorganization During the Meiotic Stages of Development in Normal (IR36) and a Temperature/photoperiod Sensitive Male Sterile Line (Peiai 64S) of Rice ( Oryza sativa )

Changes in the pattern of organization of microtubules in the meiotic stages of development of pollen (i.e. from pre-meiotic interphase to more or less metaphase I) of a normal (IR36) and a temperature/photoperiod sensitive male sterile line (Peiai 64S) of rice were studied using immunofluorescence confocal microscopy. In IR36, from pre-meiotic interphase to metaphase I, the pattern of microtubule distribution in the meiocytes underwent a series of changes. Some new organizational patterns of microtubules (that have not been described before) were observed during microsporogenesis, including the existence of a broad band of perinuclear microtubules at the diakinesis stage of development. The pattern of microtubule distribution in the meiocytes of the male sterile line, Peiai 64S, was quite different front that seen in IR36. In Peiai 64S, the microtubules showed abnormal patterns of distribution from pre-meiotic interphase to metaphase I. For example the broad band of perinuclear microtubules seen at diakinesis in IR36 was much disorganized and loosened in Peiai 64S. The spindles formed were also very abnormal and different from the normal spindle. The appearance of abnormal microtubule distribution in the early stages of microsporogenesis may contribute to the malformation and ultimate abortion of pollen in Peiai 64S.

Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S （9.09%）. On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

Inheritance of the Male Sterility in a New Photo/Thermo-Sensitive Genie Male Sterile Line B06S of Rice

The major male sterile genes in a new photo/thermo-sensitive genie male sterile (PTGMS) line B06S of rice were analyzed by the manipulation of mixture distribution theory. The results indicated that a pair of major male sterile nuclear genes with large effects were responsible for controlling the male sterility of B06S.

Study on the Pollen Thermo-Sensitive and Stigma Exsertion Male Sterile Line Tomato (Lycopersicon esculentum Mill.) cv. Da107

To accelerate the breeding process, male sterile line is used to leave out the troublesome procedures of the artificial emasculation in tomato breeding. However, the fertility of the pollen thermo-sensitivity male sterile line (PTMSL) and the stigma exsertion male sterile line (SEMSL) are affected easily by the environments when used alone. The trial materials were Da107 and the control was First. This study was conducted to create a new male sterile line of tomato characterized by pollen thermo-sensitivity and long style by genic recombination through the hybridizing of the PTMSL and SEMSL. Research on the statistics of the pollen germination rate, the contamination rate in F1 and the flower organics indicated that Da107 was an ideal TS and SE male sterile line with the sterility of 95%, as well as it also could be used as fertile line at low temperature. Meanwhile, the results showed that hybrid-seed contamination risk with selfed seeds from residual fertility in Da107 was low.

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.

Mechanism of Sterility and Breeding Strategies for Photoperiod/Thermo-Sensitive Genic Male Sterile Rice

To understand the male sterility mechanism of photoperiod/thermo-sensitive genic male sterile [P（T）GMS] lines in rice, the research progress on genetics of photoperiod and/or temperature sensitive genic male sterility in rice was reviewed. A new idea was proposed to explain the sterility mechanism of P（T）GMS rice. The fertility transition from sterile to fertile is the result of cooperative regulation of major-effect sterile genes with photoperiod and/or temperature sensitive genes, but not the so-called pgms gene in P（T）GMS rice. The minor-effect genes, which exhibit accumulative effect on sterility, are the important factors for the critical temperature of sterility transition. The more minor-effect genes the sterile line holds, the lower the critical temperature of sterility transition is. The critical temperature of sterility transition will be invariable if all the minor-effect genes are homozygous. The strategies for breeding P（T）GMS rice were also proposed. The selective indices of critical photoperiod and temperature for sterility transition should be set according to varietal type and ecological region. Imposing selection pressure is a key technology for breeding P（T）GMS rice with lower critical temperature for sterility, and improving the comprehensive performance of the whole traits and combining ability is vital for breeding P（T）GMS rice lines.

Plant Temperature and Its Simulation Model of Thermo-Sensitive Genic Male Sterile Rice

Plant temperature （Tp） and its relations to the microclimate of rice colony and irrigation water were studied using a thermo-sensitive genic male sterile （TGMS） rice line, Pei＇ai 64S. Significant differences in the daily change of temperature were detected between Tp and air temperature at the height of 150 cm （TA）. From 8：00 to 20：00, Tp was lower than TA, but they were similar during 21：00 to next 7：00. The maximum Tp occurred one hour earlier than the maximum TA, though they both reached the minimum at 6：00. Tp fluctuated less than TA. At the same height, during 6：00-13：00, Tp was higher than air temperature （Ta）, and Tp reached the maximum one hour earlier than Ta. During the rest time on sunny day, Tp was close to or even a little lower than Ta. On overcast day, Tp was higher than Ta in the whole day, and both maximized at the same time. In addition, Tp was regulated by solar radiation, cloudage and wind speed in daytime, and by irrigation water at night. The present study indicated that a TA of 29.6℃ was the critical point, at which Tp was increased or decreased by irrigation water. Tp and the difference between water and air temperatures showed a conic relation. Tp fluctuation was also regulated by the absorption or reflection of solar radiation by leaves during daytime and release of heat energy during nighttime. By analysis on correlation and regression simulation, two models of Tp were established.

New Temperature Sensitive Genic Male Sterile Lines with Better Outcrossing Ability for Production of Two-Line Hybrid Rice

An investigation was carried out with three newly developed temperature sensitive genic male sterile （TGMS） lines for their floral traits, seed production potential and outcrossing ability in ten cross combinations. In the TGMS lines, fertile pollens had an average diameter of 0.89 mm while the sterile pollens was with 0.02 mm diameter.TS-29-150GY produced the biggest fertile pollens with 0.92 mm and other two lines produced relatively smaller pollens with 0.91 and 0.85 ram. Pollen fertility during the fertility reversion period was an average of 60.7%. TS-29-150GY had the maximum of 66.9% spikelet fertility whereas other two lines （TNAU18S and TNAU60S） had relatively lower spikelet fertility of 27.8% and 26.7%, respectively. Average of 17.00 g of seed yield was obtained in the TGMS lines during the fertility reversion period. TS-29- 150GY had the highest value of 21.20 g of seed yield while TNAU18S and TNAU60S produced 16.6 g and 13.2 g of seed yield, respectively. The low seed production ability of these three TGMS lines was attributed only to the environmental conditions prevailing during the period. All three TGMS lines had considerable outcrossing potential of 41.2%, 24.6% and 25.0%, respectively. The cross combinations viz. TNAU18S/IET21508 （36 g/plant）, TNAU18S/IET21044 （13 g/plant）, TNAU18S/IET21009 （26.5 g/plant）, TNAU60S/CB-09-106 （26.2 g/plant）, TNAU60S/IET21009 （14 g/plant） and TS29-150-GY/DRR 3306 （39.2 g/plant） showed perfect synchronization with acceptable hybrid seed yield, indicating suitability of TGMS system under Indian condition. Based on the outcrossing related traits viz. panicle exertion, angle of glume opening, stigma length and pollen size, TNAU18S was identified as the best, followed by TS-29-150GY.

Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.

Factors Influencing the Outcrossed Seed-Setting Obstacles of Photo-thermo Sensitive Genie Male Sterile Rice

Using photo-thermo sensitive genie male rice (PTGMS) Pei' ai 64S, W7415S, W6154S, N26S, Annong S, Nongken 58S, 7001S and 5088S as female parents and conventional indica lines 8258 and U89 as male parents, the factors affecting outcrossed seed-setting were analyzed. The PTGMS had obstacles in outcrossed seed setting influenced by inheritance and environment at varying degrees. Environmental temperature was regarded as the main factor that resulted in the outcrossed seed-setting obstacles. The sensitive stage was at the early stage of grain filling for outcrossed seed setting. There existed remarkable differences at the sensitivity stage, the duration of sensitive period, the sensitive level and the effective level of outcrossed seed-setting obstacles caused by environmental temperature among different PTGMS lines. Therefore, attention should be paid to outcrossed seed-setting obstacles in selection and utilization of PTGMS lines.

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.

MAPPING OF THE RICE (ORYZA SATIVA L.) THERMO-SENSITIVE GENIC MALE STERILE GENE TMS5 WITH EST AND SSR MARKERS

With the cDNA suppression subtraction hybridization method, a spikelet-specific cDNA library was constructed that expressed at meiosis stage in rice. A total of 121 cDNA fragments were selected from the library and used as EST (expressed se-quence tags) markers to detect the polymorphism between Annong N, a normal fertile Indica rice line and Annong S-1, its spontaneous mutant with thermo-sensitive genic male sterility, using the RFLP (restriction fragment length polymorphism) technique. HN57, one of the EST probes, could detect poly-morphism between them. The results of segregation analysis with the F2 population developed from An-nong S-1 and Annong N indicate that HN57 co-seg- regates with the thermo-sensitive genic male-sterility controlled by tms5, the recessive gene in Annong S-1. This marker is located on the 31.2-cM region of the chromosome 2 of RGP (rice genome research pro-gram) genetic map. To further determine the location of tms5, 80 SSR (simple sequence repeat) markers around this region were developed, and 12 of them were polymorphic. And finally, the tms5 was mapped within region of 181 kb by using these new markers.

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genic male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondria! and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tms 1 gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.

Selection of Submergence Tolerant Homozygous Line by STS Marker and Twice Submergence Stress

One sequence tagged site marker Subl-1 and twice submergence stress method were used in selection of submergence tolerant homozygous line from Sub-lBS, a submergence tolerant, bentazon sensitive and photoperiod-sensitive and/or thermo-sensitive genic male sterile line that developed by our laboratory. The results revealed that the original Sub-lBS was heterozygous in SublA-1 locus even though it was identical in almost all of agronomical traits and the segregation of SublA-1 was in accordance with Mendelian law based on chi-square test. And then the original Sub-IBS was divided into two groups： one was ofSublA-1 introgression and the other was not; and the two groups were tested by twice submergence stress method. After the first submergence stress that lasted for 12 d, the average plant heights were significant difference at the 1% level between the two groups. After recovery for 10 d, the second submergence stress sustained for 18 d was carried on; and the group with SublA-1 gene was found apparently tolerant than the other group in submergence tolerance.

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.

小麦光温敏核雄性不育系BS366和BS1086杂种一代结实率研究

为了解小麦光温敏核雄性不育系BS366和BS1086的育性恢复状况,评估恢复系人工定向改良对其恢复能力的效果,在不育生态区对以55份定向改良的恢复系和33份非改良恢复系(常规小麦)为父本配制的杂种一代的结实率进行了分析。结果表明,BS366和BS1086杂种一代结实率变化范围为15.42%~140.34%和20.28%~119.07%(国际法),平均结实率分别为73.02%和72.21%,二者差异不显著,说明2份母本的育性恢复能力差异不显著。BS366和BS1086杂种一代结实率主要分布于40%~110%和60%~100%间,BS1086杂种一代结实率分布更为集中。BS366和BS1086与改良父本的杂种一代平均结实率分别为84.45%和78.97%,与非改良父本的杂种一代平均结实率分别为53.97%和60.95%,改良父本的平均恢复力高于非改良父本,表明定向改良有利于提高恢复系的恢复力。BS366和BS1086与相同父本的杂种一代结实率差异在-41.66%~61.93%之间,表明相同父本对不同母本的育性恢复力存在差异。BS366和BS1086与相同改良父本的杂种一代中有61.82%的结实率差异分布于-10%~20%之间,与相同非改良父本的杂种一代中有51.52%的结实率差异在-30%~0%之间,表明同一改良父本对不同母本的育性恢复力差异小于非改良父本。BS366与14份父本、BS1086与7份父本的杂种一代结实率高于对照,其中父本14YH261、14YH551、SD036与2份母本杂种一代的结实率均高于对照,说明具有可用恢复力的父本不仅数量极少,而且对不同母本的恢复力存在较大差异。杂种一代主茎穗的平均结实率极显著大于分蘖穗。因此,建议对不同母本分别开展强恢复力的恢复系筛选,并以中选强恢复系为亲本进行定向改良,有效增加杂种一代结实率达对照水平的组合数量,为获得更多强优势杂交组合奠定基础。

茄子反向温敏雄性不育系内源激素含量及相关基因表达分析

雄性不育对杂交育种至关重要,挖掘茄子雄性不育关键候选基因有助于定向改良不育材料。以长茄温敏型雄性不育系05ms及其同源可育系S63为材料,对不育系05ms和可育系S63减数分裂时期(Ⅱ)的花药进行转录组测序,并对05ms春季低温不育(ML)和夏季高温可育(MH)阶段的花粉母细胞时期(Ⅰ)、减数分裂期(Ⅱ)、小孢子时期(Ⅲ)和成熟花粉粒(Ⅳ)4个花药发育时期的内源激素含量进行检测。结果表明,差异表达基因主要富集在戊糖和葡萄糖酸酯的转化、类胡萝卜素的生物合成和植物激素信号转导等代谢通路,Smechr0702283、Smechr0801802、Smechr0600105、Smechr0802213、Smechr0302844、Smechr0601316、Smechr0401804等基因为茄子温敏雄性不育的关键调控基因,实时荧光定量PCR(qRT-PCR)结果表明,转录组数据可靠。激素代谢物检测结果表明,生长素类激素ICAld和乙烯前体ACC含量在4个时期中ML均显著高于MH,而ML的生长素IAA含量低于MH。从代谢水平和转录水平解析了茄子花药的败育机制,研究结果为进一步揭示茄子温敏雄性不育调控机制奠定了理论基础。

光温敏两系不育系‘稆S’的选育与应用

[目的]选育优良的光温敏两系不育系,以提高其杂种后代的产量和品质。[方法]采用光温敏两系不育系‘RGD-7S’为母本与三系保持系‘中浙B’杂交,经5年9代的系谱法选育,育成不育性稳定、稻米品质优、异交特性好、株叶形态优良的光温敏两系不育系‘稆S’,并分析了其主要特征特性及杂种优势。[结果]‘稆S’不育性稳定,育性转换临界温度<23℃,稳定不育期127 d左右。该不育系制种产量高,异交特性好,柱头外露率78.62%,其中双边外露率39.74%。采用‘稆S’配组的高产、优质两系杂交稻组合‘稆两优质美’先后通过了福建省和海南省农作物品种审定。[结论]光温敏两系不育系‘稆S’不育性稳定,育性转换临界温度低,稻米品质优,配合力强,应用前景广泛。