两系杂交水稻应用化学杂交剂保纯的研究 Ⅰ.“保纯灵”诱导光温敏核不育水稻雄性不育的效果

为了探讨化学杂交剂对光温敏核不育水稻雄性不育的影响效果，１９９３～１９９６年对光温敏核不育水稻衡农Ｓ－１和培矮６４Ｓ进行了化学杂交剂筛选和应用技术的研究．结果表明，湖南农业大学生化教研室研制的无毒化学杂交剂“保纯灵”在光温敏核不育水稻衡农Ｓ－１和培矮６４Ｓ可育状态下叶面喷施（适宜时期为花粉充实早期），诱导雄性不育效果较好，辅助制种能明显提高杂种纯度，在衡农Ｓ－１和培矮６４Ｓ育性恢复、自交结实率２０％左右时，每６６６．７ｍ２分别用“保纯灵”１６０和２４０ｇ，对水２００ｋｇ叶面喷施１次，一般空壳率达９８％，杂种纯度９５％左右；Ｆ１杂交种子发芽率正常，秧苗生长旺盛；根芽比提高，细胞色素氧化酶、过氧化物酶和多酚氧化酶活性增强；“保纯灵”对水稻杀雄有效的剂量范围广，活性期长，处理后水稻开花集中，午前花比例提高，雌蕊柱头生活力正常．但处理后的水稻植株对赤霉素有轻度钝感，生育期延长２～３ｄ．

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.

Correlation between Stigma Characteristics and Outcrossing Rate of Five Photo-thermo Sensitive Genic Male Sterile(PTGMS) Rice Lines

[Objective] This study aimed to investigate the correlation between stigma characteristics and outcrossing rate of photo-thermo sensitive genic male sterile（PTGMS） rice lines. [Method] The stigma exsertion rate, stigma size and stigma vitality of Chuang 5S, Yun 9S, Peiai 64 S, P88 S and Y58 S were analyzed. Using R608 as a male parent, outcrossing rate of these five PTGMS lines was determined. [Result]There were differences in stigma exsertion rate, stigma size and stigma vitality among five PTGMS lines. P88 S exhibited the best stigma characteristics and relatively strong stigma vitality, with the stigma exsertion rate of 95.94%, stigma vitality coefficient of 2.71, outcrossing rate of 84.02% in the field. Peiai 64 s had the minimum stigma length（2.41 mm） and stigma width（1.10 mm） and the weakest stigma vitality, with the stigma exsertion rate of 93.04%, outcrossing rate of 65.47%. Yun 9S,Y58 S and Chuang 5S displayed medium stigma characteristics, among which Yun9 S exhibited higher outcrossing rate than other two PTGMS lines. Correlation analysis showed that stigma exsertion rate, stigma length, stigma width, stigma area and stigma volume were significantly correlated to outcrossing rate（P〈0.05）, with correlation coefficients of 0.897, 0.934, 0.934, 0.939 and 0.937, respectively. However,there was no significant correlation between stigma vitality coefficient and outcrossing rate. [Conclusion] Based on stigma characteristics of five PTGMS lines, auxiliary pollination measures should be adopted reasonably to save production costs and improve hybrid rice seed yield.

Studies on GA_3 Spraying Dosage for eui TGMS Rice Changxuan 3S in Its Hybrid Seed Production

[Objective] Changxuan 3S was thermo-sensitive genicmale sterile（TGMS）rice selected from irradiated seeds of Peiai 64S by 350 Gy^（60）Coγ-ray.The aim of the study was to confirm GA3 spraying dosage of Changxuan 3S with eui gene in its hybrid seed production.[Method] Changxuan 3S possessing eui gene and its parent Peiai 64S were chosen as materials.Comparison studies on sensitivity to GA3 in their hybrid seed production were carried out.[Result] The suitable stage for spraying GA3 in the hybrid seed production of Changxuan 3S was at 10% of panicles headed;The optimal dosage was 90 g/hm2 with 2 split sprayings,the first spraying of 45 g/hm2 at heading of 10% panicles and the second one of 45 g/hm2 on the following day.Under the condition of spraying GA3 at the rate of 90 g/hm2,the panicle neck exsertions of Changxuan 3S was ＋1.78 cm,and exserted stigma rate and seed setting rate of Changxuan 3S were 96.87% and 36.44%,being 21.46% and 16.33% more than those of Peiai 64S,respectively.The theoretical yield of ＂Changxuan 3S/9311＂ reached 2 931.90 kg/hm2,which was increased by 1 259.40 kg/hm2 comparing with ＂Peiai 64S/9311＂.[Conclusion] Compared with Peiai 64S,Changxuan 3S is more sensitive to GA3,which results in no or little using GA3 in seed production of Changxuan 3S.Moreover,Changxuan 3S showed higher yield potential than Peiai 64S.

Primary Study of Rice AFLP Analysis Optimization of Reaction Conditions and Analysis of Thermo sensitive Genic Male Sterile Rice Allelic Mutant Lines

AFLP analysis was performed between a pair of thermo_sensitive genic male sterile (TGMS) rice allelic mutant lines (5460S and 5460F). The reaction conditions for rice AFLP assay were optimized. The relative efficiencies for polymorphism detection of RFLP, RAPD and AFLP were compared. The results indicated that the efficiency for polymorphism detection in rice was in the order of AFLP>RAPD>RFLP, and also indicated that AFLP was a powerful DNA molecular marker technique for polymorphism detection, especially in the case of extremely low polymorphism, such as isogenic lines and allelic mutant lines. Some of the AFLP products between the TGMS rice allelic mutant lines were cloned. Three of them were used as mixed probes to screen BAC library of rice line 5460S. 12 positive clones were screened out. In addition, the advantages and disadvantages of these three molecular marker systems were discussed.

Study on Seed Vigor of Rice Photo-ThermoSensitive Genic Male Sterile(PTGMS) Line with Low Temperature Storage

To explore the change rule of seed vigor and seed application technology of rice PTGMS lines with low temperature storage,Zhun S,Y58S,Feng S and Guangzhan 63-4S stored in the seed bank with the temperature of 8-10 ℃ and with the relative humidity of 55% for different years were used as the materials to study the properties of seed vigor,growth and development in the field, etc. The re- sults showed that the germination potential, germination rate, germination index and vigor index of rice PTGMS lines seeds showed downward trends as the time of low temperature storage prolonged,and all of the germination rates of the seeds within five years were over 60%. The conductivity in the soaked solution of seeds was significantly increased with the prolonged low temperature storage time. Seedling rate,root length,white root number,base stem width and seedling dry weight of seeds showed downward trends as the time of low temperature storage prolonged. There was no significant difference between two integral seedling qualities of seeds within 4-6 years in low temperature storage. Compared with the germination rate of low temperature storage seeds soaking for 6,12 and 24 h,the appropriate soak time of long time low temperature storage or low vigor seeds was 12 h. A serious natural aging phenomenon occurred in low temperature storage seeds after out of the storage. The germination rate of seeds placed under natural condition for 50 d decreased obviously. The duration from seeding to heading of Zhun S,Y58S and Feng S shortened as the time of low temperature storage prolonged, while the vari- ation of panicle duration of all PTGMS lines was not obvious. In conclusion, rice PTGMS line seeds with low temperature storage in 5 years could still be used nor- mally. However.seeds after low temperature storage should be sowed as soon as possible, and the soaking time of low vigor seeds should be shortened,and the possibility that the decreased duration from seeding to heading should also be con- sidered.

Cloning of the APRT Gene from Rice and Analysis of Its Association with TGMS

Adenine phosphoribosyltransferase (APRT) is the major enzyme that converts adenine into adenosine-3'-phosphate (AMP). APRT-deficient mutant caused by APRT gene mutation results in the male sterility in Arabidopsis thaliana L. In order to confirm the existence of rice APRT gene and to investigate its association with thermo-sensitive genic male sterile (TGMS) phenotype of rice, a APRT gene was identified from BLAST search of the rice genome database using APRT gene sequences from other plant species as probes. Further, the gene was cloned from rice and named APRT(GenBank accession number AY238894) using the combination of bioinformatic and experimental approaches. The rice APRT was located in the 56 000 bp to 63 000 bp region of a rice bacterial artificial chromosome (BAC) clone (AL606604) on chromosome 4 and was deduced by software from the positive DNA clone. Its cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed according to the sequence of the putative gene. The full-length cDNA was obtained by rapid amplification of cDNA ends (RACE) procedure and was sequenced. Open reading frame (ORF) analysis indicated that the rice APRT gene encodes a peptide of 212 amino acid residues, including seven exons and six introns. Using reverse position specific BLAST (RPS-BLAST), the APRT domain was identified in the polypeptide. The homology comparison demonstrated that the polypeptide exhibits 54.9%, 54.9%, 49.6% and 59.5% identity with that from Hordeum vulgare, Ttriticum aestivum, and A. thaliana (APRT types 1 and 2), respectively. Comparing the sequence of APRT gene from TGMS mutant lines 'Annong S-1' (Oryza sativa subsp. indica) with that from its corresponding wild type 'Annong F' (Oryza sativa. subsp. indica), we found that there are five single nucleotid polymorphism (SNP) sites in the gene of 'Annong S-1', which locate mainly in the second intron. However, the result of cDNA sequencing showed that these SNP sites do not damage the successful splicing of intron 2. Qualitative RT-PCR and Northern blot indicated that the gene tran-scription in the 'Annong S-1' young panicles that were verified to be the thermo-sensitive organ at the early stage of pollen fertility alternation is down-regulated by high temperature stress (28 V), which is the critical temperature causing 'Annong S-1' fertility conversion. These results revealed that the change of expression pattern of APRT in young particles of 'Annong S-1' in high temperature conditions is perhaps related to the TGMS of 'Annong S-1'.

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.

Breeding and Application of Indica PTGMS Line Yan 161S and Its Hybrid Yanliangyou 1618 in Rice

In 2006, the indica photoperiod- and thermo-sensitive genic male sterile （PTGMS） line Yan 161S was successful y bred by crossing the Guangzhan 63S （♀） and the medium indica breeding material Yan 229 （♂）, which was bred by the Institute of Agricultural Sciences in Coastal Region of Jiangsu Province, after years＇ and generations’ selection in different areas. It passed the identification by Jiangsu Provincial Crop Variety Appraisal Committee in 2013. Crossing the Yan 161S with Yanhui 888, a new high-yielding and high-quality two-line hybrid rice combination Yanliangyou 1618 was developed, and it passed the variety identification in 2014. This paper introduced the breeding processes and characteristics of Yan 161S and Yanliangyou 1618 and the cultivation and seed production techniques of Yanliangyou 1618.

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.

Breeding and Application of Indica PTGMS Line Yan 220S in Rice

Yan 220S, which is derived from a natural variant plant discovered among the population of Guangzhan 63S and developed by Jiangsu Agricultural Research Institute for Coastal Regions through pedigree selection over years, is an indica photo- and thermo-sensitive genic male sterile （PTGMS） line in rice. In 2013, it was technically identified by Jiangsu Crop Variety Appraisal Committee. Its hybrid Yan-liangyou 2208 （Yan 220S/Yanhui 888）, a new two-line hybrid rice combination with high yield and good grain quality, was subsequently developed and released for commercial production by the National Crop Variety Appraisal Committee in 2013.

Characterization of TGMS Line 33S in Rice

The research expIored morphoIogical characteristics, fertiIity, and tralts re-Iating to outcrossing of 33S which was of good shapes in pIant and Ieaf, with Iow infertiIity temperature, earIy fIowering period, high fIowering rate before noon, and good tralts reIating to outcrossing.