Development of four tobacco cytoplasmic male sterile sources using in vitro techniques

In the present paper are summarized our results on obtaining tobacco male sterile forms through interspecific hybridization. The wild species Nicotiana velutina, N. benthamiana, N. maritima, N. paniculata were used as cytoplasm donors and N. tabacum as the donor of the nucleus. Completely sterile hybrids from these combinations were obtained whose sterility was overcome through the use of tissue culture. Stem parenchyma grown in vitro on MS medium was used for inducing callus, and for organogenesis and rooting. The regenerants obtained were mixoploid. Male sterile plants were obtained in BC₁P₂ progenies from the combinations between N. velutina × N. tabacum, N. benthamiana × N. tabacum and in R₂ progenies from N. maritima × N. tabacum and N. paniculata × N. tabacum. The observed male sterility was preserved in BC₁P₂-BC₇P₂ progenies and was identified as cytoplasmic male sterility (CMS) because it was inherited only throuth the female parent.     

Restoration of fertility in cytoplasmic male sterile (CMS) Nicotiana Sylvestris by fusion with X-irradiated N. tabacum protoplasts

Summary Restoration of male fertility was achieved by fusing protoplasts from male sterile (CMS) Nicotiana sylvestris plants with X-irradiated protoplasts derived from fertile N. tabacum plants. The CMS N. sylvestris plants were derived from a previous somatic hybridization experiment and contained alien (Line 92) cytoplasm. About one quarter of the regenerated plants were found to be cybrids. i.e. they consisted of N. sylvestris nuclei combined with all or some components of N. tabacum cytoplasm. In one half of these cybrids male fertility was restored to different levels. The chloroplasts of the two parental donors differ in respect to tentoxin sensitivity: chloroplasts of CMS N. sylvestris are sensitive while those of N. tabacum are insensitive. It could therefore be demonstrated that there was an independent segregation of chloroplast type and male fertility/sterility: several somatic cybrids were male fertile but tentoxin sensitive and others were tentoxin insensitive yet they were male sterile. Only in about one half of the somatic cybrids was male fertility restored together with restoration to tentoxin insensitivity.     

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F₁ hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F₁ hybrids were completely male sterile. We did not obtain any BC₁ progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F₁ hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC₁ plants varied from 28 to 31, and all were male sterile. BC₂ plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC₂ F₂ progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC₂ progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC₂ F₄ progenies 4 were resistant to BPH and 1 to race 6 of BB. F₃ segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.     

Development of a fertile genetic bridge between Trifolium ambiguum M. Bieb. and T. repens L.

  Trifolium ambiguum M. Bieb and T. repens L. are taxonomically related but very difficult to cross. The rare hybrids so far reported between these two species were obtained only by embryo culture. This difficulty has been overcome in the present research by the creation of a “fertile bridge” between T. ambiguum and T. repens. Characters of interest can now be transferred from T. ambiguum to T. repens by using this “fertile bridge” without the use of sophisticated techniques. An array of backcross progenies was generated from crosses between a T. ambiguum×T. repens F₁ hybrid (8x H-435) and its parental species. The 8x hybrid was cross-fertile only with T. repens and resulted in 145 seeds from 1578 reciprocal crosses. Eleven of nineteen initially grown BC₁F₁ plants were all hexaploid with an average pollen stainability of 41.6%. A high frequency of multivalents at metaphase-I indicated that both autosyndetic and allosyndetic pairing occurred. Backcrosses of 6x BC₁F₁ plants to T. repens resulted in 5x BC₂F₁ plants with an average pollen stainability of 59.3%. On the other hand, 6x BC₁F₁×6x T. ambiguum crosses did not produce any seed and only two pentaploid plants were obtained from 6x BC₁F₁×4x T. ambiguum crosses. The difficulty encountered in generating 6x backcross progeny with 6x T. ambiguum was overcome by intercrossing the 6x BC₁F₁ plants and producing 6x BC₁F₂ plants with an average pollen stainability of 65.8%. One of these 6x BC₁F₂ plants was cross-compatible as a female with 6x T. ambiguum and resulted in CBC₂ plants that were all cross-compatible with 6x T. ambiguum. The 6x BC₁F₂ plants are likely to be superior to 6x BC₁F₁ progeny, as they have exhibited better expression of the combined rhizomatous and stoloniferous growth habit, improved fertility, more frequent nodal rooting and heavier nodulation. Consequently, the 6x BC₁F₂ plants can either be used directly in the selection programme or as a “fertile bridge” between the two parental species. The present work has resulted in the development of a series of fertile hybrids by the manipulation of chromosome numbers, combining the agronomic characteristics of the parent species in varying genome balances and at a range of ploidy levels. It is concluded that the initial sterility of the primary interspecific hybrids need not be a barrier to successful inter-breeding. Received: 2 August 1996 / Accepted: 4 April 1997     

Somatic hybridization in Nicotiana: Segregation of organellar traits among hybrid and cybrid plants

Protoplasts from a nitrate reductase-deficient mutant of Nicotiana tabacum L. were fused with protoplasts from a stamen-less, cytoplasmically malesterile cultivar of tobacco containing the cytoplasm from N. suaveolens Lehm. Plants were regenerated from the fused protoplasts and characterized with respect to stamen development, chromosome number, and chloroplast composition. Of 29 regenerated plants, stamen production was restored in 26 plants and pollen production in 22. One plant was male sterile and two plants have never flowered. Analysis of the electrophoretic mobility of ribulose-1,5-bisphosphate carboxylase (RuBPcase) showed that 19 of the plants contained RuBPcase of the N. suaveolens type, six plants contained enzyme of the N. tabacum type, and four plants contained both types. Analysis of resistance to tentoxin in seedlings from 20 of the plants demonstrated that 14 had N. suaveolens-type chloroplasts, three had N. tabacum type, and three contained both types. Many of the plants which produced stamens and pollen still contained chloroplasts of the N. suaveolens type. Thus, the trait of cytoplasmic male sterility in tobacco is not an expression of the type of chloroplast genetic material.     

Expression of nuclear and chloroplastic genes coding for fraction-1 protein in somatic hybrids of Nicotiana tabacum + rustica

In the sexual interspecific cross, Nicotiana rustica L.xN. tabacum L., N. rustica can serve as the female but not as the male parent. By fusion of protoplasts, the barrier to fertilization was overcome and somatic hybrids containing N. tabacum cytoplasm were produced as shown by isoelectric focusing of the Fraction-1 protein (F-1-protein). All somatic hybrids displayed polypeptides of the large subunit of F-1 protein (which is coded by the chloroplast genome) characteristic of only one or the other parental species. Two hybrids had large subunits of the N. tabacum type and two hybrids had those of the N. rustica type. Three hybrids contained three smallsubunit polypeptides (coded by the nuclear genome), one being characteristic of N. rustica, one characteristic of N. tabacum, and one with an isoelectric point common to both species. A fourth hybrid contained only two small-subunit polypeptides of the N. tabacum type but in a F-1 protein macromolecule whose large subunits were of the N. rustica type. One somatic hybrid was self-fertile and its F₂ progeny contained large- and small-subunit polypeptides indistinguishable in their isoelectric points from those in the parent F₁ hybrid. All somatic hybrids showed an aneuploid chromosome number and morphological characteristics intermediate between those of N. rustica and N. tabacum.     

Cytoplasmic male sterility and nuclear restorer genes in a natural population of Beta maritima: genetical and molecular aspects

Summary One natural population (F₀ generation) of Beta maritima situated on the French Atlantic coast has been analysed. It was composed of 62% female, 30% hermaphrodite and 8% intermediate plants. The analysis of half-sib progeny (F₁ generation) obtained from in situ open pollination demonstrates the cytoplasmic determination of male sterility in Beta maritima and the restoration of fertility by nuclear genes. The mitochondrial DNA (mtDNA) and the chloroplast DNA (ctDNA) of sixteen F₁ plants, extracted from offspring of the three sexual phenotypes, were analysed using the restriction enzymes Sal I and Bam HI, respectively. Two cytoplasmic lines with their own peculiar genetic characteristics were distinguished using the restriction enzyme patterns of mtDNA: (i) the S cytoplasmic line was found in segregating progeny of two F₀ plants; all three phenotypes were produced (that is, progeny including hermaphrodite, female and intermediate plants); (ii) the N cytoplasmic line was found in the progeny of one F₀ hermaphrodite plant; this produced only hermaphrodites. Thus, segregating and non-segregating hermaphrodite F₀ plants can be distinguished. The nuclear genes maintaining sterility or restoring fertility are expressed in line S. At the same time the analysis of Beta vulgaris material has been carried out at the molecular level: N cytoplasmic lines of B. vulgaris and B. maritima differed only by 3 fragments of mtDNA; but the S cytoplasmic line of B. maritima was very different from Owen's cytoplasmic male sterile line of B. vulgaris. No variation in the ctDNA pattern was detected within and between the two taxa.     

Chromosomal location of a pollen fertility-restoring gene, Rf, for CMS in Japanese bunching onion (Allium fistulosum L.) possessing the cytoplasm of A. galanthum Kar. et Kir. revealed by genomic in situ hybridization

In a previous study, we developed cytoplasmic male sterile lines of Allium fistulosum possessing the cytoplasm of A. galanthum, a wild species, by continuous backcrossing. Furthermore, we reported the presence of a pollen fertility-restoring gene (Rf) for cytoplasmic male sterility (CMS) in A. fistulosum from segregation of pollen fertility of backcross progenies. In the present study, genomic in situ hybridization (GISH), using genomic DNA of A. galanthum as the probe DNA and that of A. fistulosum as the blocking DNA, was applied to F₁ hybrids between both species and backcross progenies to determine the chromosomal location of the Rf locus. By means of GISH, eight chromosomes from A. galanthum were clearly discriminated from those of A. fistulosum in the F₁ hybrids, and chromosome substitution process through continuous backcrossing was visualized. Interestingly, the chromosome region from A. galanthum, specific to male fertile plants, was detected in one chromosome of BC₄ to BC₇ generations. Based on the karyotype analysis of the male fertile plants, the chromosome was identified as the 5F chromosome. Our results confirm that the Rf locus is located on the 5F chromosome of the male fertile plants. This is the first report that identified the chromosomal location of the pollen fertility-restoring gene in A. fistulosum.     

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Summary Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by -irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) — all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F₁ progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.Abbreviations Km ^r kanamycin resistant - Km ^s kamacysin sensitive - Nop ⁺ nopaline positive - Nop ^– nopaline negative     

Male sterility caused by cytoplasm of Brassica oxyrrhina in B. campestris and B. juncea

Summary Synthetic alloploid Brassica oxyrrhina (2n = 18, OO) x B. campestris (2n = 20, AA) was repeatedly backcrossed with B. campestris to place B. campestris nucleus in the cytoplasm of B. oxyrrhina. Alloplasmic plants, obtained in BC₅ generation, were stably male sterile but mildly chlorotic during initial development. Synthetic alloploid B. oxyrrhina-campestris was also hybridized with B. juncea to transfer B. oxyrrhina cytoplasm. Segregation for green and chlorotic plants was observed in BC₁ and BC₂ generations. By selection, however, normal green male sterile B. juncea was obtained in BC₃. Pollen abortion in both B. campestris and B. juncea is post-meiotic.     

Fertile allotetraploid from the cross betweenPhaseolus coccineus L. andPhaseolus acutifolius A. Gray

Summary As somatic hybridization and genetic transformation are not yet applicable to beans, a programme of hybridization between a male sterile line ofP. coccineus and a wild genotype ofP. acutifolius var.tenuifolius was carried out in order to introduce useful characters from the wild parent into the genome of the cultivated species. This interspecific cross is of particular interest sinceP. acutifolius is a source of resistance to many diseases, drought and high temperature. The difficulties in producing these hybrids were overcome by repeated pollinations and with the help of embryo culture. The F₁ hybrid shows a high sterility which may be explained by the poor pollen quality and the presence of a chromosomic asynapsis at meiosis. Fertile allotetraploids (Co) were successfully produced in progeny from colchicine treated cuttings of F₁ hybrids. Several (C₂) mature seeds were harvested from selfed allotetraploid plants.Abbreviations Co initial allotetraploid plants - C₁, C₂ first and second allotetraploid generation - PMC pollen mother cell     

A study of morphology,cytology and sterility in interspecific hybrids and amphidiploids of Nicotiana knightiana X N. umbratica

Summary Interspecific hybrids and amphidiploids of Nicotiana knightiana Goodspeed (n= 12)x N. umbratica Burbidge (n = 23) resembled either parent in some characters and were intermediate in other characters. The F₁ hybrids (2n = 35) showed mostly univalents during meiosis, while the amphidiploids (2n = 70) formed bivalents almost regularly. The former were completely sterile and the latter fully male fertile but predominantly female sterile. This female sterility was due to disintegration of the embryo sacs leading to collapsed ovules. The few fertile ovules, however, showed normal development of embryo sac and embryo. The occurrence of fertile and sterile ovules was believed to be due to segregation of the genes governing sterility.     

Dwarfism and male sterility in interspecific hybrids of Epilobium

Summary Reciprocal differences for male sterility, dwarfism and morphological traits have been studied in intra- and interspecific crosses of five Epilobium species. Male sterility occurred in two interspecific hybrids with E. montanum as the male parent while dwarfism has been found to varying degrees in three interspecific crosses with E. watsonni. In contrast to transient differences in plant height and leaf morphology in reciprocal hybrids of the cross between E. hirsutum and E. parviflorum, male sterility and dwarfism persistently occur as reciprocally different traits which may be influenced by determinants of the cytoplasm. The molecular characterization of the plastid DNA of the parental lines and the F₁ hybrids indicate that the plastome of male sterile and dwarf plants is identical to that of the female parents. Furthermore, in spite of these developmental disturbances, the expression of plastid genes coding for polypeptides of thylakoid-membrane complexes is unchanged. Thus, it seems unlikely that the genetic compartement of the plastids is responsible for the expression of the male sterile or the dwarfed phenotype.     

普通小麦与鹅观草属间杂种F1及BC1的分子细胞遗传学、育性和赤霉病抗性研究

通过胚拯救, 成功获得鹅观草Roegneria kamoji (2n=6x=42, SSHHYY)和普通小麦中国春Triticum aesti-vum (2n=6x=42, AABBDD)的正反交属间杂种F1, 并对这些杂种F1及其BC1的形态学、减数分裂配对行为、育性和赤霉病抗性进行研究。结果表明, (鹅观草×中国春)F1和(中国春×鹅观草)F1的形态介于双亲之间。杂种F1花粉母细胞减数分裂中期I染色体构型分别为40.33I + 0.78II + 0.03III和40.40I + 0.79II 。杂种F1高度雄性不育, 用中国春花粉与其回交可获得BC1代种子。(鹅观草×中国春) F1×中国春BC1植株的染色体数目主要分布在55~63之间, 单价体较多, 植株高度不育; (中国春×鹅观草)F1×中国春BC₁植株染色体数目也主要分布在55~63之间, 但其中部分植株拥有整套小麦染色体且能正常配对、分离, 可形成部分可育花粉粒, 能收到少量自交结实种子。在 (鹅观草×中国春)F1中有1株穗型趋向中国春, 其染色体数目为2n=63, 经染色体分子原位杂交(GISH)检测, 含有42条小麦染色体和21条鹅观草染色体。该杂种F1在减数分裂中期I平均每个花粉母细胞有26.40I+18.30II, 但植株高度雄性不育, 用中国春花粉回交能收到BC₁种子。(鹅观草×中国春) F₁ (2n=63)×中国春BC₁的染色体数目主要分布在40~59之间, 其中的外源染色体已经逐渐减少, 虽然该BC₁的穗型已接近中国春, 但仍然高度不育。赤霉病抗性鉴定结果显示, 所有杂种F1及大部分BC₁对赤霉病均表现出较好的抗性。     

A new source of cytoplasmic male sterility in maize induced by the nuclear gene,iojap

Summary Cytoplasmic male sterility (cms) was found in plants derived from the F₂ progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.     

Characterization of nuclear and cytoplasmic information in the progeny of a somatic hybrid between male sterile Nicotiana tabacum and N. glutinosa

Summary Several nuclear and cytoplasmic characters of the back-crossed progeny of a somatic hybrid between male sterile Nicotiana tabacum (N. debneyi cytoplasm) and N. glutinosa have been analysed. Progeny were obtained by repeated back-crossing of a somatic hybrid with pollen from either N. tabacum or N. glutinosa. Nuclear ribosomal RNA genes (rDNA) were found to be a reliable marker to determine the constitution of nuclear genomes in the progeny. The progeny obtained by back-crossing with N. tabacum pollen maintained uniformity in leaf morphology. On the other hand, variation in leaf morphology was observed in the second back-cross population obtained with N. glutinosa pollen. This may be due to a variable contribution of N. tabacum chromosomes. Segregation of rDNA was also found in individuals of the same back-crossed progeny, but was not related to the chromosome number. The stable inheritance of chloroplast DNA in the back-crossed generation was confirmed regardless of the type of pollen donor. Male sterility was consistently maintained throughout several generations, suggesting that the nuclear genome of either N. tabacum or N. glutinosa does not influence the expression of cytoplasmic male sterility.     

Production and cytogenetic analysis of BC1, BC2, and BC3 progenies of an intergeneric hybrid between Triticum aestivum (L.) Thell. and tetraploid Agropyron cristatum (L.) Gaertn.

Summary Intergeneric hybrids between Triticum aestivum cv Chinese Spring and Agropyron cristatum 4x (2n= 5x=35, ABDPP genomes) with a high level of homoeologous meiotic pairing between the wheat chromosomes were backcrossed 3 times to wheat. Pollination of the F₁ hybrid with Chinese Spring resulted in 22 BC₁ seeds with an average seed set of 1.52%. Five BC₁ plants with 39–41 chromosomes were raised using embryo rescue techniques. Chromosome pairing in the BC₁ was characterized by a high frequency of multivalent associations, but in spite of this there was no evidence of homoeologous pairing between chromosomes of wheat and those of Agropyron. All of the plants were self sterile. The embryo rescue technique was again essential to produce 39 BC₂ plants with chromosome numbers ranging from 37 to 67. The phenomenon of meiotic non-reduction was also observed in the BC₃ progenies. In this generation male and female fertility greatly increased, and meiotic pairing was fairly regular. Some monosomic (2n=43) and double monosomic (2n=44) lines were produced. Analysis of these progenies should permit the extraction of the seven possible wheat-Agropyron disomic addition lines including those with the added chromosomes carrying the genes involved in meiotic non-reduction and in suppression of Ph activity.     

Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes

An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

Fine mapping and candidate gene analysis of <Emphasis Type="Italic">ptgms2-1</Emphasis>, the photoperiod-thermo-sensitive genic male sterile gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Photoperiod-thermo-sensitive genic male sterile (PTGMS) rice exhibits a number of desirable traits for hybrid rice production. The cloning genes responsible for PTGMS and those elucidating male sterility mechanisms and reversibility to fertility would be of great significance to provide a foundation to develop new male sterile lines. Guangzhan63S, a PTGMS line, is one of the most widely used indica two-line hybrid rice breeding systems in China. In this study, genetic analysis based on F₂ and BC₁F₂ populations derived from a cross between Guangzhan63S and 1587, determined a single recessive gene controls male sterility in Guangzhan63S. Molecular marker techniques combined with bulked-segregant analysis (BSA) were used and located the target gene (named ptgms2-1) between two SSR markers RM12521 and RM12823. Fine mapping of the ptgms2-1 locus was conducted with 45 new Insertion–Deletion (InDel) markers developed between the RM12521 and RM12823 region, using 634 sterile individuals from F₂ and BC₁F₂ populations. Ptgms2-1 was further mapped to a 50.4 kb DNA fragment between two InDel markers, S2-40 and S2-44, with genetic distances of 0.08 and 0.16 cM, respectively, which cosegregated with S2-43 located on the AP004039 BAC clone. Ten genes were identified in this region based on annotation results from the RiceGAAS system. A nuclear ribonuclease Z gene was identified as the candidate for the ptgms2-1 gene. This result will facilitate cloning the ptgms2-1 gene. The tightly linked markers for the ptgms2-1 gene locus will further provide a useful tool for marker-assisted selection of this gene in rice breeding programs.