Disruption of Energy Metabolism and Reactive Oxygen Species Homeostasis in Honglian Type-Cytoplasmic Male Sterility(HL-CMS)Rice Pollen

Honglian type-cytoplasmic male sterility(HL-CMS)is caused by the inter-communication between the nucleus and mitochondria.However,the mechanisms by which sterility genes regulate metabolic alterations and changes in mitochondrial morphology in the pollen of HL-CMS remain unclear.In this study,we compared the morphological differences between the pollen of the male sterile line YA and the near-isogenic line NIL-Rf6 using hematoxylin-eosin staining and 4ʹ,6-diamidino-2-phenylindole(DAPI)staining.HL-CMS is characterized by gametophytic sterility,where the aborted pollen grains are empty,and the tapetal layer remains intact.Transmission electron microscopy was employed to observe mitochondrial morphological changes at the microspore stage,revealing significant mitochondrial alterations,characterized by the formation of'large spherical mitochondria',occurred at the binucleate stage in the YA line.Additionally,metabolomics analysis revealed decreased levels of metabolites associated with the carbohydrate and flavonoid pathways.Notably,the decrease in flavonoids was found to contribute to an elevation in reactive oxygen species(ROS)levels.Therefore,we propose a model in which rice fertility is modulated by the levels of pollen carbohydrates and flavonoid metabolites,with impaired mitochondrial energy production and reduced flavonoid biosynthesis as the main causes of ROS accumulation and pollen abortion in rice.

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

Characterization of Fertility Alteration in 8 Environment-sensitive Genic Male-sterile Lines of indica Rice

Eight indica ( Oryza sativa L.) environment-sensitive genic male-sterile (EGMS) lines, 2-2S, K1405S, F131S, 2136S, Pei-Ai 64S, 1290S, GD-IS and N17S, were sequentially seeded with 10-15 d interval at three sites, Wuhan in 1997, Guiyang in 1997 and Sanya in 1997 and 1998, China. The results of investigations on self-sterilities showed that all of eight EGMS lines had stable sterile periods of longer than 30 d at Wuhan. They can be used for seed production of two-line hybrid rice, but can not reproduce themselves. Their stable sterile periods were shorter than 30 d at Guiyang, they can reproduce themselves and can not be used for hybrid seed production. In Sanya, their stable sterile periods were longer than 150 d, all of eight lines can be used for seed production in summer and autumn and reproduce themselves in winter. The fertility of all eight lines were sensitive to temperature. The sensitive stages, sensitive duration and critical point of temperatures (CPT) of fertility alteration in various lines were different. The sensitive stages of 2-2S and K1405S were from 18 d to 9 d before heading, the sensitive durations were 7-10 d and the CPTs were 23.7-24.5 degreesC. The sensitive stage, sensitive duration and CPT of F131S were from 17 to 5 d before heading, 13 d and 24.3-24.7 degreesC, respectively. The sensitive stage, sensitive duration and CPT of 2136S were from 18 to 12 d before heading, 7 d and 24.6-25.1 degreesC:, respectively. The sensitive stages, sensitive durations of Pei-Ai 64S, 1290S, N17S and GD-1S were from 24 to 13 d before heading and 10-13 d. And their CPTs were 24.6-25.1 degreesC, 25.5-26.2 degreesC, 25.4-26.1 degreesC,, and 24.1-24.7 degreesC, respectively.

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.

Brassinosteroids mediate the effect of high temperature during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile rice lines

Brassinosteroids(BRs)play critical roles in a wide range of plant developmental processes.However,it is unknown whether and how BRs mediate the effect of high temperature(HT)stress during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile(PTSGMS)rice(Oryza sativa L.)lines.This study investigated the question.Three pot-grown PTSGMS rice lines were subjected to HT stress during anthesis.The contents of 24-epibrassinolide(24-EBL)and 28-homobrassinolide(28-HBL),the major forms of BR in rice plants,and levels of reactive oxygen species(ROS)or antioxidants(AOS),hydrogen peroxide(H2O2),1-aminocylopropane-1-carboxylic acid(ACC),ascorbic acid(AsA),and catalase activity in pistils,were determined.HT stress significantly reduced the contents of both 24-EBL and 28-EBL relative to those under normal temperatures,but the reduction varied by PTSGMS line.A line with higher BR contents under HT stress showed lower contents of ACC and H2O2,higher catalase activity and AsA content in pistils,and higher fertilization rate,seed-setting rate,and seed yield when the line was crossed with a restorer line,indicating that higher levels of BRs increase HT stress resistance.Applying 24-EBL,28-HBL or an inhibitor of BR biosynthesis confirmed the roles of BRs in response to HT stress.The results suggest that BRs mediate the effect of HT stress on pistil activity during anthesis and alleviate the harm of HT stress by increasing AOS and suppressing ROS generation.

Application of hybrids with cytoplasmic male-sterility in Zea mays L.in China

The abortive behaviour and the cytoplasm quality of cytoplasmic male-sterility(CMS)lines of homocaryon were investigated and the existence of C race of Bipolaris maydiswere discussed from the point of view of genetic breeding,physiology and pathology in this paper.Then the countermeasures to prevent the danger from disease of B. maydis in CMS hybrid pro-duction were proposed.

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.

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.

Relationship between rice heading sensitivity and male-sterile sensitivity to photoperiod in view of developmental genetics

We studied the relationship between the head-ing sensitivity and the male-sterile sensitivityin photoperiod-sensitive genie males-sterile rice(PGMSR)and conventional rice.Materialsused in this study were 7001S,a late maturingPGMS japonica strain derived from Nongken58S; Akihikari and Youmang-zaojing(YMZJ),two early maturing conventional Japonica rice;and CPSLO-17,a Wide compatible convention-al indica rice.The heading date and seed-set-ting rate of Fand Fof three cross combina-tions between PGMSR and conventional riceswere examined in phytotron which was adjust-ed at 25±1℃ with day-length of 14.5 h(LD)and 12 h(SD).The segregation ratio of fertile:sterile in Funder LD was in accordance

Effect of day length and temperature on the pollen fertility in photo(thermo)-sensitive genic male-sterile rice

The effect of day length and temperature on the pollen fertility of five photoperiod-sensitive genic male-sterile japonica rice lines (PGMSR) and three temperature-sensitive genic malesterile indica rice lines (TGMSR) were investigated in phytotron. The light source used for illumination was xenon lamp, and the light intensity which plant accepted on the leaf surface was 300—350μmol photons ms. The results indicated that pollens of PGMSR 7001S and E47S aborted completely whereas a little part of 31116S pollens appeared normal under long day photoperiod (LD,25℃,15h) (Table 1). High temperature (HT, 30℃, 12h) and lower temperature (LT,

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

Differences in Nuclear DNA Between Male-Sterile and Male-Fertile Lines of Sorghum bicolor

Cytoplasmic male sterility(cms)is determined by nuclear-cytoplasmic interactions. Up to now, most studies are focused on the comparison of cytoplasmic DNAs of male-sterile lines and male-fertile lines, and analysis of nuclear DNA has not been documented yet. In order to find out the possible difference in nuclear genome of male-sterile line A1 Tx623 and corresponding male-fertile line Tx623 of sorghum, random amplified polymorphic DNA(RAPD)approach was used to analyze their cytoplasmic and nuclear genomes. Total DNAs of them were amplified at first to screen primers, which were able to generate reproducible bands specific to male-sterile line or male-fertile line. Then the selected primers were used to amplify their mitochon-drial DNA(mtDNA)and chloroplast DNA(cpDNA). The origins of all the polymorphic fragments were analyzed. After ruling out those amplified from cytoplasmic DNA, seventeen polymorphic fragments were determined to be amplified from nuclear DNA. These fragments originated from nuclear DNA indicate that differences in sequence exist between the nuclear DNA of male-sterile line and male-fertile line of sorghum, which do not agree with the traditional standpoint that they have identical nucleus.

Differential RNA Editing of Mitochondrial Genes in WA-Cytoplasmic Based Male Sterile Line Pusa 6A, and Its Maintainer and Restorer Lines

RNA editing changes the nucleotides at the transcript level of mitochondrial genes which results in synthesis of functional proteins.This study was designed to find the editing sites which could be implicated in male fertility restoration and to develop editing based markers for differentiation of cytoplasmic male sterility and maintainer lines from each other.DNA and RNA from young panicles were isolated from three-line system of hybrid rice PRH10,wild abortive(WA)cytoplasm based male sterile(A line Pusa 6A),maintainer(B line Pusa 6B)and restorer(R line PRR78)lines.Pusa 6A and PRR78 having the same WA cytoplasm are allo-nuclear and iso-cytpolasmic lines.The genomic and cDNA amplicons for eight mitochondrial genes(18SrRNA,atp6,atp9,cobII,coxI,coxIII,nadI and rps3)were sequenced and compared.Differences in genomic and cDNA sequences were considered as editing.Two hundred and thirty editing sites having base substitution or insertion/deletion were identified with the highest in 18SrRNA(5.74%)and the lowest in coxI(0.60%).The highest editing sites were observed in fertile maintainer Pusa 6B followed by PRR78 and Pusa 6A,of which random five editing sites in five different rice mitochondrial transcripts namely atp9,cobII,coxIII,rps3 and 18SrRNA were chosen and validated through cleaved amplified polymorphism sequence(CAPS)analysis and found to be partially edited in four genes.The identical editing sites of different mitochondrial genes from maintainer and restorer lines might reflect their possible contribution to fertility restoration of sterile WA cytoplasm.

Studies on the Recross Heterosis Effect of Male-sterile Single-hybrids in Corn

Re-cross was conducted between five ES cytoplasm male-sterile hybrids and four high-oil hybrids, and five normal hybrids, respectively. The oil content, yield, 1000-kernel weight and kernel size were estimated. Results were as follows: The average kernel oil content in the male-sterile hybrid ×high-oil hybrid was 1.6% higher than that of the female parent. The xenia effect value of oil genes was 0.30. The yield and 1000-kernel weight of male-sterile hybrids were 10.1% and 5.8% higher than that of corresponding fertile hybrids respectively, which showed significant cytoplasm effect. The yield of the male-sterile hybrid × high-oil hybrid was similar to that of the female parent, but its 1000-kernel weight was 7.3% lower than that of the female parent. Compared with the high-oil male parent, grain yield, 1000-kernel weight and kernel size of the male-sterile hybrid× high-oil hybrid were higher. Seed-filling rate, the days of seed-filling and IAA content in the male-sterile hybrid × high-oil hybrid showed average heterosis tending the female parent, which may be perhaps one of the reasons resulting in the average heterosis tending to the female parent on kernel oil content, yield, 1000-kernel weight and kernel size.