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.

A Preliminary Study on the Relationship Between the Indica-Japonica RAPD Differentiation of Parents and Heterosisin Dian Type Hybrid Rice

The relationship between the indica-japonica differentiation of parents and heterosis of hybrid in rice was studied with 10 sterile lines, 10 maintainer lines of the sterile lines, 38 restorer lines of Dian type hybrid rice and 40 hybrids derived from the sterile lines and restorer lines. The results indicated that there was a parabolic correlation between the indica-japonica differentiation of parents detected by RAPD markers and heterosis of hybrid rice. When certain indica-japonica differentiation of parents exists, the percentage of combinations with positve heterosis and the mean heterosis of hybrids were high. But that didn't mean too much difference of parents could lead to stronger hetesosis, that meant overdiffernt parents seldom bring positve het-erotic hybrids. Suitable difference of percent of indica alleles of parents of stronger heterotic hybrid was 12 -16%.

Analysis of Indica-Japonica Differentiation in Rice Parents and Derived Lines Using ILP Markers

Revealing the indica-japonica differentiation in parents of hybridization between indica and japonica rice and their derived lines can provide theoretical and practical bases for the breeding of practical inter-subspecific hybrid rice. Using subspeciesspecific molecular markers ILP （intron length polymorphism） and Cheng＇s index, the indica-japonica differentiation was analyzed with special materials including 18 indica-japonica hybrid parents and 39 derived lines, which accumulated different wide compatibility and restoring genes by convergent cross method in 21 years spanning four breeding phases. The indica-japonica differentiation was detected on all tested loci in 57 materials. Among the 18 parental lines, 4 were japonica type, 5 japonicaclinous type, 8 indicaclinous type and one indica type. The japonica proportion indexes in indica restorer lines Minghui 63 and 9308 were 12.50 and 33.33 %, respectively, while that in japonica restorer line C418 was only 31.25%. Among the 39 derived lines from indica-japonica hybridization, one was japonica type, 11 japonicaclinous type, 20 indicaclinous type and 7 indica type. The japonica proportion index in Minghui 502 was only 10.42%. The results of indica and japonica classification by ILP molecular markers and Cheng＇s index were relatively consistent. The correlation coefficient between the japonica proportion index and morphology index was 0.794＊＊, while that between the indica proportion index and morphology index was -0.7662＊＊. ILP markers could be used to accurately detect the proportion of indica/japonica content in the genome of a rice variety. The results of indica-japonica differentiation analysis could make reasonable explanation for that the hybrids obtained from indica-japonica type restorer lines had obvious heterosis. This conclusion would provide important guidance in efficient use of beneficial genes of inter-subspecific hybrid rice.

Molecular identification of the cytoplasmic male sterile source from Dongxiang wild rice(Oryza rufipogon Griff.)

The use of cytoplasm male sterility （CMS） is crucial for three-line hybrid seed production. Two types of CMS have been discovered from Dongxiang wild rice, namely the wild abortion type CMS （CMS-WA） and the Dongxiang wild type CMS （CMS-DW）. In this study, we show the molecular identification of the two types of CMS in Dongxiang wild rice. WA352, which conferred CMS-WA, was not detected in Dongxiang wild iice, implying Dongxiang wild rice does not carry the CMS- WA source. Further analysis of WA352 in DYIA, a CMS-DW line, by PCR amplification and sequencing, revealed two insertion-deletion polymorphisms occurred in CMS-DW compared to WA352 of CMS-WA. It was reported that WA352 was comprised of an unknown origin sequence and partial sequences of three open reading frames （ORFs）, orf284, orf224 and orf288. The 42-bp insertion was located between the two segments of orf224 and orf288, which created a new chimeric ORF, orf216. This new ORF was also detected in CMS-HL. Based on the 9-bp deletion in orf284, a specific mitochon- drial marker of DW-M1 was developed, which could be used to specifically distinguish the DW-type source. Moreover, semi-quantitative RT-PCR analysis preliminarily suggested that both orf216 and orf284 could be considered as candidates for CMS-DW. These findings present a preliminary understanding of CMS-DW at the molecular level.

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.

Relationship of Parental Indica-Japonica Indexes with Yield and Grain Quality Traits of Japonica Hybrid Rice in Northern China

Taking the main parents （10 male sterile lines and 10 restorer lines） and their 100 combinations of japonica hybrid rice in northern China as materials, the relationships of parental indica-japonica indexes determined by the methods of the Cheng’s index as well as simple sequence repeat （SSR） markers with yield and grain quality traits of hybrid rice were studied. For the parents, the Cheng’s index （Chi） ranged from 13.5 to 19.3 and the indica index in SSR markers （ADi） were from 0.12 to 0.38. The classification of parents by Chi was not completely consistent with that by ADi. The Chi of male parent was more closely related to hybrid traits than that of female parent, as contrasted to ADi. At the same time, the difference between parents （PD） in Chi was more closely related to hybrid traits than that in ADi. The indica-japonica indexes of parents and their difference between parents appeared quadratic relationship to hybrid traits with the critical extremum. The directions of the correlation of indica-japonica indexes of parents and their differences with hybrid yield traits were on the opposition to those with hybrid grain quality traits. Therefore, the female parent should match the male parent moderately in indica-japonica index to obtain the optimum of hybrid traits, high yield as well as good quality.

Effects of chilling tolerance induced by spermidine pretreatment on antioxidative activity,endogenous hormones and ultrastructure of indica-japonica hybrid rice seedlings

Spermidine（Spd） is known to be involved in the regulation of plant responses to chilling stress and counteract the adverse effect of stress conditions.Antioxidant activities,endogenous hormones and ultrastructure change under chilling stress were investigated in indica-japonica hybrid rice seedlings.12-d-old seedlings were subjected to exogenous Spd（1 mmol L^（-1）） and then a chilling stress（6℃,4 d） was induced,followed by a subsequent recovery（25℃,4 d）.Results showed that malondialdehyde（MDA） and proline content were enhanced significantly,whereas shoot fresh and dry weights decreased during chilling stress and after recovery;chlorophyll content of chilling-stressed seedlings increased slightly but declined after recovery;additionally,total soluble sugar,sucrose,fructose and starch contents increased significantly during chilling stress,and only soluble sugar and fructose contents were observed in increase after recovery;chilling stress-induced increases in superoxide dismutase（SOD）,peroxidase（POD） and catalase（CAT） activities,but declined after recovery,and the level of ascorbate peroxidase was lower during chilling stress and after recovery;however,endogenous indole-3-acetic acid（IAA）,zeatin riboside（ZR）,gibberellic acid（GA_3）,and abscisic acid（ABA） levels were induced decreased compared with Spd pretreatment.The microscopic analysis revealed that chilling stress-induced destruction of the chloroplast envelope during chilling stress and increased the number of plastoglobuli along with aberrations in thylakoid membranes after recovery.In contrast,exogenous Spd protected rice seedlings from chilling-induced injuries in terms of lower malondialdehyde,proline and carbohydrates accumulation coupled with increased endogenous hormones metabolism.After recovery,Spd pretreatment chilling-exposed seedlings showed higher activities of antioxidant enzymes and normal physiological function of chloroplasts.These results suggest that Spd could promote effectively chilling tolerance which might be largely attributable to the integrity of cell structure and normal metabolism of endogenous hormones in indica-japonica hybrid rice seedlings.

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%.

Genetic Mechanism of Dominant Earliness in Kefeng A,a New Rice Cytoplasmic Male Sterile Line

Kefeng A is an early maturing indica cytoplasmic male sterile （CMS） line of rice. Combinations derived from Kefeng A and late maturing indica restorer lines showed dominant earliness to various extents. To understand the genetic basis of dominant earliness, the genotype of photoperiod-sensitive genes in Kefeng A was analyzed using a complete set of heading time near isogenic lines （NILs） EGO to EG7, ER, LR, T65, T65E^b, T65E^bm, T65m, NIL（Hd1） and NIL（Hd4）. Results indicated that Kefeng A contained two dominant photoperiod-sensitive alleles E1 and Se-1^U on E1 and Se-1 loci, respectively, and the genotype of photoperiod-sensitivity genes for heading time in Kefeng A was E1E1e2e2E3E3Se-1^USe-1UEf-1Ef-1. Based on the detected heading time genotype, in combination with the heading time of Kefeng A and the early maturing phenomenon in its derived F1 hybrids, it is speculated that Kefeng A might carry a dominant inhibitor gene Su-E7 for the dominant photoperiod- sensitive gene E1, and a recessive inhibitor gene i-Se-1 for another dominant photoperiod-sensitive gene Se-1. The reason why F1 hybrids from Kefeng A exhibited early maturing was hereby analyzed and the breeding value of dominant earliness related genes in Kefeng A was discussed.

Comparison of Tissue Transglutaminase Activity During Young Panicle Development in Honglian-type Cytoplasmic Male Sterile Rice

To investigate the changes of tissue transglutaminase activity, the leaves and young panicles of rice at different developmental stages were excised from the Honglian-type cytoplasmic male sterile line, Yuetai A and its maintainer line, Yuetai B, respectively. An ELISA measurement protocol for tissue transglutaminase activity detection in rice was well established. The results indicated that the tissue transglutaminase activity was regulated positively by calcium cation, and the tissue transglutaminase activity in senescent leaves was remarkably higher than that in young leaves. No distinct difference was noted between Yuetai A and Yuetai B. Moreover, from the tetrad to binucleate stages the tissue transglutaminase activity increased gradually with the progression of the young panicle development and up to maximum at binucleate stage in Yuetai A. However, no similar changes were observed in Yuetai B. This indicates that the tissue transglutaminase is involved in cell programmed death in abortive pollen.

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==~

Studies of the Energy Release of Mitochondria from Sporophyte Cytoplasmic Male Sterile Rice by Differential Scanning Calorimetry

The differential scanning calorimetric (DSC)curves of the mitochondria isolated from two varieties of sporophyte cytoplasmic male sterile and their fertile lines of Yie Bai and Ma Xie type rice have been determined. The curves show that the energy is released continuously as temperature rise to 70℃. Some thermodynamic and kinetic parameters of the energy release of the mitochondria have been obtained. The presented results showed that the mitochondria from cytoplasmic male sterile rice released more heat and they had higher energy barrier, less rate, and more complicated mechanism than that of their fertile lines in the energy release process.

Developmental Analysis of Genetic Behavior of Brown Rice Width in indica-japonica Hybrids

The developmental genetic behaviors of brown rice width （BRW） have been studied in indica-japonica hybrid rice （Oryza sativa L.）, in which seven indica male sterile lines and five japonica restorer lines were applied, by using the developmental genetic models and corresponding statistical approaches for quantitative traits of triploid in cereal crops. The BRW of indica-japonica hybrid rice was co-determined by gene expression of tdploid endosperm, cytoplasm, diploid maternal plant and their genotype × environmental interaction effects. Unconditional analysis showed that the endosperm additive and matemal additive effects were predominant for the development of BRW from early- to late-stage of the grain development, but the endosperm dominant effect together with matemal effect and cytoplasmic effect became the major factor determing the BRW at the ripening stage. Moreover, conditional analysis found that there were new onset and offset of gene expression at different developmental stages of BRW in indica-japonica hybrid rice. Maternal and cytoplasm general heritabilities and their interaction heritabilities were more important compared to other components of heritability for BRW at all the five developmental stages.

Development of Maweizhan cytoplasmic male-sterile line in rice(Oryza sativa L.)

A male sterile mutant plant was found from the rice field of the local rice variety which was cultivated for a long time in the west of Hubei Province. The plant has more vigorous tillering ability. Its plant height of 95 cm was a bit shorter than that of the normal ones. The plant type, the leaf shape and the color of leaf sheath and glume tip of this mutant were identieal to that of the normal plant whereas the color of arrowhead-shape anther was milky white. This mutant set no seed during self-pollination. When it was crossed with IR24. IR26, Minhui 63 or Xieqing-zaoxuian. the Ffertilities were normal. The fertility of this mutant could be restored by IR24, IR26 and Minhui 63 and maintained by Xieqing-zaoxian. Maxie A was developed in 1987 by the successive backcrossing of the mutant with Xieqing-zaoxian. The percentage of abortive pollen in Maxie A in uninucleate microspore to early binucleate microspore stage is

Genetic Analysis of Leucine Content in Indica-Japonica Hybrid Rice (Oryza sativa L.)

Genetic control of leucine content in indica-japonica hybrid rice (Oryza sativa L.) was studied in 35 crosses of F1 and F2 generations, which were derived from crossing 7 male sterile indica rice lines with 5 restorer japonica rice lines along with their parents. Two genetic models and their corresponding statistical methods for quantitative traits of triploid seeds in cereal crops were used for the analysis. The first was the unconditional genetic model, which refers to the analysis of cumulative measurements (from flowering to a specific time) along the developmental stages, while the second was the conditional genetic model, which relates to analysis from one developmental stage to another stage (t - 1→t). The results showed that leucine content of indica-japonica hybrid rice was controlled by the expression of triploid endosperm effect (endosperm additive effect and endosperm dominant effect), cytoplasm effect, diploid maternal plant effect (maternal additive effect and maternal dominant effect) and their environmental interaction effects. Of these effects, endosperm dominant effect and maternal dominant effect were more important at the earlier stages, while endosperm additive effect and maternal additive effect were more important at the later stages of rice grain development under both unconditional and conditional genetic analyses. Due to the high heritabilities, which came from endosperm, maternal and cytoplasm effects for leucine content at different developmental stages, selection for leucine content of indica-japonica hybrid rice would be more efficient at early generations in breeding programs.

Distinguishing male sterile and fertile cytoplasms in indica rice(Oryza sativa L.) by OPA 12

The majority of off-type plants in in-dica hybrid rice were CMS and itsmaintainer lines.Discrimination ofmaintainer and CMS could only beconducted at heading stage.If main-tainer and CMS line could be distin-guished earlier,it would be useful toavoid yield losses caused by usingcontaminated seeds.In this study,three pairs of A and B,Minghui 63,and Fand Fplants of Zhenshan97A/Minghui 63 were used to find

Genetic analysis of fertility restoration of Maxie cytoplasmic male sterility in rice(Oryza sativa L.)

The genetic behavior of fertility restorationgenes of the cytoplasmic male sterile line Max-ie A was studied to facilitate the use ofMaweizhan male sterile cytoplasm.The F,F,and Fof Maxie A/Minhui 63 were grownin paddy field,1993-1995.Meanwhile,someof the Fplants were testcrossed with themaintainer of Maxie A.The fertilities were

Analysis of Heterosis, Combining Ability and Heritability of Cadmium Content in Brown Rice of Three-line Indica Hybrid Rice

Five cytoplasmic male sterile （CMS） lines were used as parents in an incomplete diallet cross and six restorer lines of rice design （Nc II design）. Thirty hybrid combinations with the same growth period were selected as experimental ma- terials to investigate the heterosis, combined ability and heredity of Cd content in brown rice of indica hybrid rice. According to the results, Cd content in brown rice showed a significantly negative heterosis; the general combining ability and specific combination ability of Cd content in CMS and restorer lines both reached extremely significant level （P〈0.01）, indicating that both genetic improvement of parents and e- valuation of combinations are important to the breeding of hybrid combinations with low accumulation of Cd; the broad-sense heritabitity and narrow-sense heritability of Cd content were both relatively high with slight differences, which respectively reached 97.73% and 80.10%, indicating that Cd content in brown rice mainly de- pends on the additive action of genes; in addition, parent improvement showed bet- ter effect on the selection of early generation.

Discussion on strategy of grain quality improvement for super high yielding japonica rice in Northeast China

japonica rice is mainly distributed in Northeast China and accounts for 44.6% of the total cultivated area of japonica rice in China. The comprehensive using of inter-subspecies heterosis is the main breeding mode of super japonica rice varieties in this region. Improving rice quality at relative high yielding level is the current research focus. Performing crosses between indica and japonica lines allows for the recombination of regulatory genes and genetic backgrounds, leading to complicated genetic rice quality characteristics, which can be used to explore patterns of quality improvement. In the present study, we utilize recombinant inbred lines （RILs） derived from indica-japonica hybridization to analyze the effect factors of rice quality derived from genetic factors, which contain both regulatory genes concerning rice quality and genetic backgrounds＇ random introduction frequency coming from indica （Di value）, and the improvement strategy was further discussed. The regulatory genes involved in amylase content （Wx） and nitrogen utilization efficiency （NRT1.1B） were the major factors affecting the amylose content （AC） and protein content （PC） in RILs, respectively. Both the Di value and the major grain width gene （GS5） had regulatory effects on milled rice width （MRW） in RILs, and their interaction explained the major variance of MRW in the RILs. With the mediation of MRW and chalkiness degree （C）, Di value had a further impact on head rice rate （HR）, which was relatively poor when the Di value was over 40%. In Northeast China, the Di value should be lowered by backcrossing or multiple crosses during the breeding of indica-japonica hybridization to maintain the whole better HR and further to emphasize the use of favorable genes in individual selection.