CMS Lines Research Articles

Cytoplasmic male sterility of tuber mustard is associated with the alternative spliced mitochondrial T gene transcripts

Two transcripts of T gene, T1170 and T1243, were obtained from the mitochondrial cDNA of tuber mustard CMS line. T1243 was a transcript with an intron unspliced, which has the basic characteristics of type II intron. The expressions of the two transcripts were analyzed by reverse transcription PCR (RT-PCR). The results showed that, at seedling stage, the expression of T gene was mainly in the form of T1170 but decreased with the development gradually, while the expression abundance of another transcript, T1243, increased gradually. The T1243 was prevalent at the profuse flowering stage. The expression pattern was confirmed by Northern blot analysis. These results suggested that the alternative spliced mitochondrial T gene transcripts were related to CMS of tuber mustard.

A comparison of cytoplasmic and chemically-induced male sterility systems for hybrid seed production in wheat (Triticum aestivum L.)

A comparison of two male sterility systems was carried out in wheat for hybrid seed production and hybrid performance. Seventeen hybrid combinations based on Triticum timopheevi cytoplasm were compared with their genetically equivalent CHA-facilitated combinations. The same set of restorer lines was used as parents in CMS- and CHA-based hybrids to maintain genetic equivalence. In the first experiment aimed at study of female line behavior and crossed seed production, the CHA treated lines showed significantly shorter heights whereas CMS lines were similar to the control. The two systems were equally effective in sterilizing rate. The outcrossing percentage of the CMS lines was almost twice that of the CHA treated lines. Thousand-grain weight of the crossed seeds on CMS lines was greater than on the CHA treated lines and control. On average, the germination percentage of seeds on CMS lines was double that of the CHA treated lines and the percentage of effective outcrossed seeds in CMS lines was 3 times more than that from CHA treated lines. The second experiment was conducted to examine the yield performance of the hybrids derived from the two systems of male sterility. A total of 40 entries including 20 hybrids and 20 parents were evaluated in the experiment. The mean grain yield of the CMS-based hybrids was greater than that of the CHA-based hybrids,the B and R lines. All the CMS-based hybrids showed significantly higher grain yields than their better parents whereas all the CHA-based hybrids, except two,showed no significant yield increases over their better parents. Possible reasons for differences in CMS- and CHA-based hybrid performance are discussed.

Correlation and Path Coefficients Analysis in Pearl Millet (Pennisetum americanum L.)

An experiment (comprised of 4 CMS lines and 4 open pollinated promising strains of pearl millet and their 16 hybrids) was conducted at MMRI, Sahiwal, during Kharif 1998 to ascertain the information of genetic variability, correlation, path and regression coefficients. Mean squares for plant height, leaf area, head length, head girth, biological yield per plot and grain yield per plot were highly significant and significant for number of leaves per main tiller. Genotypic coefficient of variability (GCV%) and phenotypic coefficient of variability (PCV%) were higher for plant height, biological yield/plot, harvest index and grain yield/ plot. The PCV% was higher than GCV% for all the traits studied. High genetic advance as percentage of mean and high heritability estimates 93.88, 86.83, 74.81 and 68.62% were observed for grain yield/plot, biological yield/plot, harvest index and plant height, respectively. Genotypic correlation coefficients were higher than phenotypic correlation coefficients for almost all the traits. Grain yield/plot was positively and significantly correlated with all traits. Positive direct effect of biological yield/plot (0.7238), harvest index (0.5976) and plant height (0.1419) on grain yield/plot was noted. Regression coefficients of biological yield and harvest index were significant and positive. This study indicated the presence of very high genetic variability for grain yield, biological yield, harvest index and plant height. Similarly analysis of correlation path and regression coefficients revealed the importance of biological yield, harvest index, head girth and plant height.

Characterization of atpA and orf220 genes distinctively present in a cytoplasmic male-sterile line of tuber mustard

SummaryIn the present investigation, the atpA (Accession Number: AY211265, AY211266) gene, encoding the a subunit of F1-ATP synthase, as well as the open reading frame orf220 (Accession Number: AY208898), presumably encoding a hydrophobic membrane-associated polypeptide with deduced 220 amino acids, were found to be distinctively present in a cytoplasmic male-sterile (cms) line of tuber mustard. They were preferentially cloned by using mitochondria-specific primers and were partially characterized. The atpA gene cloned from cms and its fertile maintainer (m) lines were sequenced to be 1,454 and 1,073 bp-sized in fragments, respectively. They had a high similarity of approximately 70% in terms of nucleotide constituents. However, alignment of these two genes suggested that the atpA gene from the cms line had an insertion of 383 bp-sized fragment in 5’-flank spanning from 141th to 523th base site, compared with that of its m line. Besides, the open reading frame, orf220, was found to be exclusively present in the cms line of tuber mustard. It was deduced to possibly encode a hydrophobic N-terminus membrane-associated polypeptide with a deduced molecular weight of 26 kD. By Southern hybridization, it was further shown that orf220 existed only in the cms line and was absent from the fertile m line with no hybridization signal detected. Although the atpA gene was blotted in either cms or m line, they were of different fragment sizes when blotted with DNA digested with Hind III and Xba I, respectively. Both atpA and orf220 genes were supposed to be low coped in mitochondria genomes. In addition, by phylogenetic analysis, it was manifest that the orf220 gene had a closer genetic relationship with orf222 in Brassica nap cms than both of orf224 in Brassica pol cms and orf240 in Arabidopsis thaliana cms. The direct evidence of the involvement of these two genes in the expression of cms of tuber mustard needs further investigation.

A common sequence difference between cytoplasmic male sterile lines and their maintainer lines existing in rice (Oryza sativa L.) chloroplast tRNA-Leu gene region

DNA polymorphism between a wild-abortivecytoplasmic male sterile line Zhenshan97Aand its maintainer line Zhenshan97B wasanalyzed using AFLP technique. Among the1953 amplified bands, a stable polymorphicfragment was identified, and designated asA1 in Zhenshan97A and B1 inZhenshan97B. Sequence and homology analysisshowed that A1 contained a more tandemrepeat of 6 base pairs (AGAAAA) thanB1 and the repeat was located 7-baseupstream to the transcription start site ofthe rice chloroplast tRNA-Leu gene.According to this difference, a pair ofspecific primers was designed toinvestigate the distribution of thedifference in 5 pairs of CMS and theircorresponding maintainer lines by PCR. Itwas demonstrated that a single band couldbe amplified from all the 5 CMS lines whilenone from the maintainer lines. Sequencesanalysis of the differential regions of the5 pairs of CMS and maintainer lines showedthat all the 5 CMS lines contained this 6bprepeat, whereas the correspondingmaintainer lines did not. It was proposed thatthis differential fragment was likelyassociated with cytoplasmic malesterility.

Characterization of CMS and maintainer lines in indica rice (Oryza sativa L.) based on RAPD marker analysis

Total DNA from WA type CMS lines: Zhenshan 97 A, Longtepu A and theirmaintainers Zhenshan 97 B, Longtepu B wasextracted by CTAB method. One hundredprimers were used for screening RAPDmarkers to distinguish CMS line (A) andmaintainer (B) plants at seedling stage.Results showed that under the conditions of37 °C annealing temperature and1.5 mM MgCl2 concentration, in Zhenshan97 A, Longtepu A there was a 1600 bp DNAfragment in product amplified by primerOPA12, while in Zhenshan 97 B, and LongtepuB no 1600 bp fragment was found. The 1600 bpfragment was also found in DA type CMS lineXieqingzao A, but was absent in XieqingzaoB. Also in the restorer line, Minghui 63the 1600 bp fragment was absent. In F1and F2 generation of Zhenshan97A/Minghui 63, all plants investigated hadthe 1600 bp fragment. When mitochondrial DNA(mtDNA) was isolated from the three CMS (A)and their B lines and amplified by OPA12,results showed that the 1600 fragment wasfound in all the three A lines and wasabsent in the three B lines. In DA typeXieqingzao A, two other fragments (700 bp,1000 bp) were also found except the 1600 bp.These results indicate that the 1600 bpfragment derived from CMS mitochondrial DNAcan be used as a RAPD marker to distinguishA and B plants at seedling stage, and thefragments (700 bp, 1000 bp) can be used todistinguish WA and DA cytoplasms.

Silencing of fertility restoration genes in sunflower

Single dominant genes for fertility restoration of cytoplasmic male-sterile Cms ANN2 have been identified in PI 413178, P21, and RCMG3, while PI 413180 P21, RHA801, RCMG1, and RHA280 were identified as restoration sources for cmsANN3. However, some cms plants were observed in progenies of homozygous restoration lines crossed with HA89, RHA266, and RHA274. It is believed that HA89, RHA266, and RHA274 possess gene(s) which suppress, or silence the expression of some Rf genes. HA821 does not have silencing gene(s), thus producing all male-fertile progenies after crossing with the restoration lines. F2 progenies of the half-diallel crosses among the three restoration lines for cmsANN2 and among the five restoration lines for cmsANN3 were nearly all male-fertile, suggesting a single Rf gene for each of the cms sources. However, testcross progeny families from crosses of half-diallel F1 with respective cmsANN2 and cmsANN3 were not all male-fertile, with most families having all male-sterile plants or segregating for both male-sterile and male-fertile plants. This could happen if cmsANN2(HA89) and cmsANN3(HA89) contain silencing gene(s) obtained from backcrossing with HA89. Restoration lines containing silencing genes can be identified and the silencing genes eliminated. Hidden silencing gene(s) in cms lines is a problem, and lines without these genes, such as HA821, should be used to develop maintainer lines. Gene silencing appears to be simply inherited. Molecular marker development would assist in identifying lines without silencing genes.

Induction of microspore embryos in a CMS line of Brassica juncea and formation of the androgenic plantlets

Anther culture of two wide hybrids (Diplotaxis erucoides × Brassica campestris) × B. juncea and (D. berthautii × B. campestris) × B. juncea, their CMS lines and the parent species elicited a range of responses highlighting the importance of the genotype. Androgenesis was expressed in cultured anthers of CMS (D. erucoides) B. juncea (22.8%), in restored pollen fertile plants of this CMS line (1.66%), and in the parent, B. juncea cv Pusa Bold (13.02%). AgNO3 was essential for androgenic response in the CMS lines, and it markedly increased the frequency of androgenesis in the cultivated species. Multiple crops of microspore embryos were obtained from responsive anthers of CMS plants in anther recultures. As high as92% microspore embryos of the CMS line germinated on basal B5medium and formed normal plantlets.

Sequence and transcript analysis of the orf140-nad3-rps12-atp1 gene cluster in the mitochondrial DNA of mung bean (Vigna radiata L. (Wilzek) cv. Tainan No. 5).

An orfl4O-nad3-rpsl2-atpl gene cluster, which spans a 7.1 kb region of the mung bean mitochondrial genome, was identified and completely sequenced. The coding regions of nad3, rpsl2. and atpl genes are highly conserved between mung bean and other plant species. The nucleotide sequences of orfl4O and its 5'-flanking region are homologous with ORFBs in sunflower and Oenothera, and are also found to be conserved in several chimeric ORFs of various plant CMS lines. The region 3'-downstream from the mung bean atp1 is similar to its counterparts in soybean and common bean, which has been shown to be specific to a subset of legumes. Genes of this cluster are present as a single copy in mung bean. Northern-blot hybridization and RT-PCR suggested that the four genes are co-transcribed and then processed into transcripts of different sizes. Variation of accumulation levels of different transcripts was observed.

Identification of Hybrid Sterility Gene Loci in Two Cytoplasmic Male Sterile Lines in Rice

In hybrid rice (Oryza sativa L.) breeding, spikelet sterility is a barrier to attaining enhanced heterosis in crosses between different groups or subspecies of rice. Our objective was to identify the hybrid sterility gene loci (HSGLi) in two indica cytoplasmic male sterile A lines (‘IR58025A’ and ‘IR62829A’) and their corresponding maintainer B lines (‘IR58025B’ and ‘IR62829B’). To detect the HSGL, three testers, ‘IR36’ (indica cultivar), ‘Dular’ (indica wide compatibility variety [WCV]) and ‘Nekken 2’ (japonica WCV) were crossed to the two groups in the form of IR36/WCV//B and A//IR36/WCV. The level of spikelet fertility in the F1 progeny of three-way crosses was differentiated by morphological and isozyme marker genes at the known HSGLi, S5, S7, S8, and S15 on chromosomes 6, 7, 6, and 12, respectively, and by two new HSGLi, on chromosomes 3 and 11. The levels of pollen fertility were differentiated by specific marker alleles among segregating genotypes in the cytoplasmic male sterile (cms) background. The presence of marker alleles from IR36 was associated with high pollen fertility, while marker alleles from Dular and Nekken 2 were associated with low pollen fertility due to the effect of HSGLi in the cms background. The effect of dominant and recessive restorers from IR36 was not estimated due to lack of appropriate markers. To alleviate problems of hybrid sterility, the level of sterility should be tested in crosses between maintainers and restorers before the former are used for backcrossing to create new cms lines.

Development of new cytoplasmic source of male sterile lines in rice for shallow lowland

Two shallow lowland male sterile lines, MotiA and PadminiAwith Miz.4 cytoplasmic source were developed throughintervarietal hybridization and recurrent backcrossing withtheir respective parents. In all probability, MotiA and PadminiA of Miz.4 source may be genetically different from the Wild abortive CMS source (V.20A). These two CMS lines are semitall in stature, photosensitive, of late duration in flowering with easy fertility restoration ability, and thus could be useful for developing hybrid rice suitable for shallow lowland areas.

High regeneration potential in vitro of sunflower (Helianthus annuus L.) lines derived from interspecific hybridization.

Successful selection of interspecific hybrid progenies with superior ability to regenerate shoots from apical meristems was performed in sunflower which now allows for the development of lines for improved biotechnological applications. Early generations of interspecific hybrids originating from crosses between the two H. annuus CMS lines ‘HA89’ and ‘Baso’, and 9 wild species were screened for their ability to regenerate in vitro. Evaluation of 36 progenies allowed to identify seven progenies from crosses involving H. mollis, H. giganteus, H. strumosus, and H. decapetalus which showed a significantly higher regeneration potential than the commercial hybrid ‘Albena’ regarding the number of shoots per explant. Among these progenies, 47.2 to 62.4% of explants produced shoots with an average of 2.3 to 3.5 shoots per cultured explant. Regeneration in vitro was significantly determined by the genotype. More than half of the investigated interspecific hybrids performed better than the inbred ‘HA89’ demonstrating that the high regeneration potential available in the wild species can be efficiently transferred to cultivated sunflower. The seven progenies with high regeneration potential in vitro were characterised by agronomic performance in the field. Two of the interspecific hybrids derived from H. strumosus and H. decapetalus not only showed a superior regeneration potential but also proved to be competitive to commercial hybrids with regard to important agronomic traits, e.g. fat content and TGW.

New procedure for the differential display of mitochondria RNA

A novel approach for the differential display of mitochondria RNA has been described. The total mitochondria RNAs are ligated to a single-strand linker with T4 RNA ligase and then reversely transcripted into the first strands of cDNA. The first strands of cDNA that are ligated to another single-strand linker also with T4 RNA ligase can be directly used as the templates of AFLP. After preamplification, selective amplification and then separating the selectively amplified products on 5% PAGE gel, the display patterns of mtRNA can be generated and the RNAs that represent the differentially expressed mitochondria genes may be identified. This new procedure has been used to analyze the total RNAs that were prepared from the mitochondria of CMS line, maintainer line and F1 hybrid in wild abortive type rice; and one differential band has been detected and then verified.

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.

Agro-morphological characterization of ovary culture-derived plants of rice (Oryza sativa L.)

Plants derived from unpollinated ovary culture of ten rice genotypes showed significant variability in agro-morphological characteristics. The ovary-derived plant (H1) populations were completely haploid, doubled haploid or haploid-doubled haploid mixture. Haploids had very drastic reduction in plant height, panicle length, grain length, breadth and number and spikelet fertility (0.0%–2.1%). Doubled haploids from the hybrid of UPRI 95–121 × UPRI 95–165 were normal with fertility ranging between 69.6% and 97.7%. A genetic segregation in ratio of 1:1 was observed for five pigmentation characters in the H1 population derived from hybrid UPRI 95–122 × UPRI 95–165. Plant height showed the largest coefficient of variability (28.5%) followed by the number of spikelets per panicle (24.2%), number of grains per panicle (22.0%), percent seed set (9.2%) and panicle length (9.0%). The range of variation in the H1 population from fully fertile hybrid PMS 2A (CMS) × IR 31802 (restorer) was similar to its corresponding F2 population for plant height, spikelet fertility and number of grains/panicle. A single clone of plants from the cultivar BG 1321 exhibited complete male sterility but normal female fertility when pollinated with other varieties. Ovary-derived plants from the CMS lines PMS 2A and IR 58025A showed stable male sterility and those from thermosensitive genetic male sterile line UPRI 95–140 showed thermosensitive genetic male sterility. These lines have potential in the hybrid breeding program and are being currently exploited.

Chloroplast substitution overcomes leaf chlorosis in a Moricandia arvensis-based cytoplasmic male sterile Brassica juncea

A male sterile Brassica juncea line based on Moricandia arvensis cytoplasm was developed previously by backcrossing the somatic hybrid M. arvensis+B. juncea, and the gene for restoring fertility was introgressed. The CMS line is very severely chlorotic because of the presence of alien chloroplasts and flowering is delayed by 30–40 days, making it unsuitable for the exploitation of heterosis. We have resorted to another cycle of protoplast fusion between green fertile B. juncea and chlorotic male sterile B. juncea, and developed green male-sterile plants. Molecular analysis revealed that in green male-sterile plants chloroplasts of M. arvensis origin were substituted by those from B. juncea, giving rise to intergeneric cytoplasmic hybrids with mitochondria of M. arvensis origin. With the development of dark-green male-sterile plants, the CMS fertility restoration system is suitable for the production of hybrid mustard.

Sterility and variation resulting from the transfer of polima cytoplasmic male sterility from Brassica napus into Brassica chinensis

Male sterile combinations made from interspecific crosses between the polima CMS line of Brassica napus and varieties of B. chinensis were backcrossed to BC3. Twenty-six selfing lines from B. chinensis were tested for their ability to either maintain complete sterility or to restore fertility in crosses with the polima male sterile line. Results show that four of these hybrids were completely male sterile and two were fertile. The sterility of the B. chinensis with polima cytoplasm was much more stable than male sterile lines with B. campestris and B. chinensis cytoplasm, which were sterile before full flowering but progressively became fertile as flowering proceeded. The results suggest that polima cytoplasm could be a suitable male sterile-inducing allocytoplasm for B. chinensis, as both maintainers and restorers are available, and could supply a reliable pollination control system for hybrid seed production in this species.

Heterosis and combining ability for plant height and its components in hybrid wheat with Triticum timopheevi cytoplasm

The heterosis and combining ability for plant height and its components of hybrid wheat were investigated in an incomplete diallel experiment including 5 CMS lines and 4 restorer lines. The results showed that heterosis (HS) and heterobeltiosis (HBS) occurred in plant height (PH) and length of the first internode (LFI), second internode (LSI), third internode (LTI), basal internode (LBI) and the spike (LS) of hybrids, but their values varied among crosses and characters; the HS and HBS of LBI were larger than those of other characters, the HS and HBS of LSI and LTI contributed a lot to those of PH. There were significant relationships between internode lengths and PH for specific combining ability (SCA) and general combining ability (GCA), and among lengths of the adjacent internodes for SCA and/or GCA effects. However, the relationships of LS with the lengths of internodes and PH were insignificant for GCA, SCA, HS and HBS. The SCA effects were more important than GCA effects for LFI, the reverse was true for LSI, LTI, LS and PH, and the SCA effects was nearly equal to the GCA effects for LBI. So, LFI was mainly influenced by non-additive effect of genes, while LSI, LTI, LS and PH were mainly controlled by additive gene effects, LBI was controlled equally by additive and non-additive effects of genes. The genes that control the length of specific internode not only affect PH, but also the length of the adjacent internode. The genetic system in charge of lengths of internodes and plant height is independent of that for length of spike. Thus, it is possible to develop new wheat cultivars or hybrid combinations having long spike but dwarf plant height.

Characterization and expression of rpoC2 in CMS and fertile lines of sorghum

A 165 bp deletion in the middle of rpoC2, the plastid gene which encodes the RNA polymerase beta" subunit, was identified in the small-anthered types of CMS sorghum, Sorghum bicolor (L.). Moench, containing A1, A2, A5, and A6 cytoplasms. It was previously shown that the amino acid sequence deleted in these CMS lines is in a monocot-specific region that contains several protein motifs that are characteristic of several transcription factors. Using primers flanking the deletion in PCR analyses, various types of CMS lines, some of which are used in hybrid sorghum production, were classified into two groups. CMS lines containing A1, A2, A5, A6 cytoplasms display the deletion in rpoC2. These lines have small anthers in which pollen development is arrested at an early stage and in which usually only empty exines are found. CMS lines containing A3, A4, and 9E cytoplasms do not possess the deletion. These lines have large anthers in which pollen degenerates at a later stage. Run-on transcription assays using 15 chloroplast genes showed that chloroplast gene transcription rates are similar in CMS and fertile (maintainer and restorer) lines and F1 in seedling leaves. Analyses of RNA blots indicated that rbcL, rpoB and rpoC2 transcripts are accumulated mainly in the leaves and low in the inflorescence tissues and pollen. These data document plastid gene expression in leaves and non-photosynthetic tissues from CMS and fertile lines of sorghum.

A new dominant earliness character of CMS line Zaoxian A and its application value in rice (Oryza sativa L.)

A distant cross between a maintainer Zhenshan 97B and an early maturing F2 palnt of O. longistaminata x O. rufipogon was made in 1985, and a new CMS line Zaoxian A was bred in 1989 through continuous backcross of new maintainer plants derived from the distant cross to the leading CMS line Zhenshan 97A. Several experiments were carried out in Sichuan during 1990–1992, to analyze the inheritance of earliness of Zaoxian A and its application value to hybrid rice production. The results showed that Zaoxian A was as early-maturing as Zhenshan 97A, the earliness of Zaoxian A was incompletely dominant to the lateness of restorer lines, and was different from the earliness of Zhenshan 97A which was suppressed by the lateness of restorer lines. The F1 hybrids between Zaoxian A and late maturing restorers combined the early maturity and high yielding capacity, and significantly outyielded the provincial and national checks with same or longer growth duration, because the larger genetic distance between early and late maturing ecotypes remained unchanged and the new earliness could express in F1 generation. Therefore, the new dominant earliness character of Zaoxian A could enhance the regional and seasonal adaptability of rice. It is also valuable to hybrid rice production and the formation of new cropping systems with high yielding capacity and high economical profit.