RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton

Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited trait and CMS-associated genes are known to be located in the mitochondrial genome. However, CMS-inducing genes in CMS-D2 and CMS-D8 of Upland cotton (Gossypium hirsutum L., AD1) are currently unknown. The objective of this study was to identify potential candidate DNA or gene sequences for CMS-D2 and CMS-D8 through restriction fragment length polymorphism (RFLP) analysis. Seven mtDNA gene probes and five restriction enzymes were first used to compare D2 (from G. harknessii Brandegee) and AD1 cytoplasms. With cox1, cox2, and atp1 as probes, RFLP polymorphisms were detected with one or more restriction enzyme digestions. The most notable difference was an additional fragment in the normal AD1 cytoplasm detected by cox2 in digests of three enzymes, and by cox1 and atp1 in digests with PstI. The RFLP analysis was then conducted among CMS-D2, CMS-D8 (from G. trilobum (DC.) Skovst.), and AD1 cytoplasms. Two probes from maize, atp1 and atp6, detected polymorphism among the different cytoplasmic lines. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D2 or D8 cytoplasm, indicating that the presence of the D2 or D8 restorer gene does not affect mtDNA organization in Upland cotton. The results demonstrate that RFLP using atp1 and atp6 as probes can distinguish the three cytoplasms. The atp1 and atp6 in CMS-D8 and these two genes together with cox1 and cox2 in CMS-D2 could be the candidates of CMS-associated genes in the mitochondrial genome, providing information for further molecular studies and developing PCR-based markers for the CMS cytoplasms in breeding. This research represents the first work using RFLP to analyze the genetic basis of CMS in cotton.     

Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.)

The main objective of the present study was to identify mitochondrial DNA based marker, which can distinguish male sterile and fertile counterparts of the cytoplasmic male sterile (CMS) lines used in production of rice hybrids. Amplified fragment length polymorphism (AFLP) analysis in CMS lines: IR58025A & IR62829A and their respective maintainers: IR58025B & IR62829B identified a polymorphic DNA fragment of about 510 bp size that was present in both CMS (A) and absent in their maintainer (B) lines. Sequencing followed by database analysis of the polymorphic fragment indicated about 97% similarity with mitochondrial NADH gene subunits of rice, maize and wheat. Based on the variable sequence regions, a site specific primer pair (BF-STS-401) was designed. PCR analysis showed that BF-STS-401 could amplify a strong band of 464 bp size in CMS and a faint band of the same size in maintainer line. To act as a positive control and avoid possible errors in PCR, BF-STS-401 was multiplexed with a new primer pair (BF-STS-402), derived from mitochondrial atp9 subunit of rice, producing monomorphic amplification indiscriminately in both CMS and maintainer lines. Both the primer pairs in combination clearly differentiated CMS lines from their corresponding maintainer lines. This primer combination was validated in a set of diverse genotypes consisting of different sources of CMS lines, restorer lines, hybrids, varieties and mixed samples from private seed companies. Our results suggested that the multiplex primer pairs developed in this study can be effectively utilized to assess the genetic purity in commercial seed lots of CMS lines and hybrids of rice.     

瓣化型胡萝卜胞质雄性不育相关片段的分离及序列分析

以128对引物组合对瓣化型胡萝卜胞质雄性不育系和保持系进行cDNA-AFLP分析，筛选与不育性相关的特异片段，对其中两个表达丰度较高的cDNA差异片段进行克隆测序。序列分析表明BY1949与拟南芥质子依赖性的类肽转运蛋白在核苷酸水平有87%的同源性，蛋白质水平有58%的同源性。BY2562与瓣化型胞质雄性不育（CMS）胡萝卜线粒体ATP8、ATP9、NAD6和cob-sp基因在核苷酸水平有99%的同源性，期望值为0.0；在蛋白质水平与CMS向日葵线粒体ORFB基因，COXIII 5'端未知蛋白YMF19以及CMS胡萝卜ATP-8，orfB-F1有97%的同源性；另外与胡萝卜orfB-CMS、orfB-F2、烟草orfB、油菜orf224、拟南芥线粒体orfB、萝卜orf158以及芥菜线粒体膜蛋白、甘蓝型油菜orf474也有90%以上的同源性。     

Development of markers for the use of the PEF1 cytoplasm in sunflower hybrid breeding

The use of the new cytoplasmic male sterility (CMS) source PEF1 in sunflower hybrid breeding requires markers closely linked to the restorer gene Rf_PEF1 necessary for fertility restoration of hybrids based on the PEF1 cytoplasm as well as diagnostic markers to distinguish the PEF1 cytoplasm from other cytoplasms. Bulked segregant analyses of 256 AFLP primer combinations identified 35 polymorphic primer combinations with 1–3 polymorphisms, resulting in 40 polymorphisms. Eighteen AFLP markers mapped together with the Rf_PEF1 gene covering 119.9 cM. The closest markers, E39M51_300R and E44M56_112A, mapped 3.9 and 6.0 cM to the Rf_PEF1 gene, respectively. Six SSR markers, which belong to the linkage group 13, were screened for polymorphisms between the parental lines. Only ORS630 was polymorphic, but did not map to the same linkage group as Rf_PEF1, indicating that Rf_PEF1 is not located on linkage group 13 where the restorer gene Rf1 for the PET1 cytoplasm is located. Diagnostic markers to distinguish the PEF1 cytoplasm from the PET1 and the fertile cytoplasm in sunflower were obtained using primer combinations for the atp9 gene and orfH522.     

大白菜细胞质雄性不育的分子鉴定及序列分析

为了获得3种大白菜细胞质雄性不育系Ogu CMS,Pol CMS,CMS96和保持系间的多态性以及定位大白菜CMS96不育系所属的不育类型,利用设计的atp6,orf222单一和混合引物PCR扩增3组11份同核异质大白菜细胞质雄性不育系和保持系mtDNA。结果表明,atp6引物在大白菜Ogu CMS不育系扩增的200 bp片段为其特异带;orf222引物仅在大白菜Pol CMS和CMS96不育系有扩增产物,但二者有3点完全不同:大白菜Pol CMS不育系扩增产物为675bp,CMS96不育系扩增产物为669 bp,二者相差6个核苷酸,后者定名为大白菜CMS96-orf222。大白菜CMS96-orf222与甘蓝型油菜Nap CMS的nad5c基因和Nap-orf222基因同源性均为99%,E值为0.0;大白菜Pol CMS的675 bp序列具有ORF224开放阅读框,没有保守结构域,而大白菜CMS96的669 bp序列具有ORF222开放阅读框和保守结构域YMF19。另外,atp6和orf222混合引物多重PCR扩增产物存在明显多态性:800 bp为大白菜保持系的差异带型;2 300 bp和1 500 bp为大白菜Pol CMS不育系特异带型;200 bp为大白菜Ogu CMS不育系特异带型;690 bp为大白菜CMS96不育系特异带型。该方法仅用一次PCR反应快速地将3种大白菜细胞质雄性不育系和保持系一次性全部区分开,为大白菜分子育种和常规育种更好地相结合提供了简单、快速、准确和重复性好的方法和手段。     

Molecular characterization of fertile and sterile cytoplasms in Beta spp.

Mitochondrial DNA (mtDNA) from sugar beet carrying fertile (F) and male sterile (CMS) cytoplasms, and from male sterile accession of Beta maritima collected in Brittany (France) were characterized and compared by restriction fragment length polymorphism (RFLP) and Southern hybridization with coxII. The F and CMS cytoplasms could be clearly distinguished from each other by RFLP when XhoI, EcoRI and BamHI endonucleases were used. Southern hybridization with the coxII gene provided further evidence that mitochondrial genome organization differs between fertile and sterile plants. All cytoplasmic male sterile lines from different breeding stations showed the same restriction and hybridization patterns, which confirms the uniformity of mitochondrial genomes within the materials used for hybrid seed production in several European countries. No visible differences were found between the maintainer lines studied. However, comparisons of XhoI restriction profiles of mtDNA from maintainer lines and from fertile monogerm populations revealed slight differences, which were reflected by the appearance of a unique 0.9 kb fragment in the latter. Analysis of mtDNA from male sterile plants of the wild beet B. maritima showed different restriction and hybridization patterns in comparison with normal and sterile sugar beet cytoplasms. This shows the unique nature of cytoplasmic male sterility in this species.     

A short review of research on male sterility and prospects for F1 hybrid varieties in field beans (Vicia faba L.)

Summary It has not yet been possible to utilise cytoplasmically inherited male sterility (CMS) in Vicia faba, in the commerical production of F₁ hybrids. The main problem has been a rapid increase in the proportion of fertile revertants during multiplication. Investigations, mainly in France, have clarified environmental causes of phenotypic instability, CMS and maintainer lines with improved stability have been selected, and two new cytoplasms have been produced by mutation of the first two. CMS is known to be associated with cytoplasmic spherical particles, and recognisable types of double stranded RNA, and there may also be an association with mitochondrial DNA, and polypeptides of mitochondrial origin.The better understanding of CMS may eventually lead to more easily managed forms, either through genetic manipulation or biochemical assays that breeders can use for screening for stability.     

Molecular characterization of cytoplasmic male sterility conditioned by <Emphasis Type="Italic">Gossypium harknessii</Emphasis> cytoplasm (CMS-D2) in upland cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F₁ seed lots.     

The use of wheat aneuploids for the chromosomal assignment of microsatellite loci

Summary The chromosomal assignment of 64 PCR-amplified microsatellite loci and 29 additional fragments amplified by the same primer pairs is described for bread wheat (Triticum aestivum). The distribution over the different chromosomes and chromosome arms appears to be random. The highest proportion of microsatellite loci is found on the B genome, followed by the A and D genome. About half of the primer pairs amplified unique fragments, while the other half amplified additional fragments. 25% of the primer pairs, mostly designed to clones of a PstI-library, amplify fragments on homoeologous chromosomes. In some cases, more than one fragment on a single chromosome or fragments on non-homoeologous chromosomes occurred. The use of an automated DNA sequencer accounts for the accurate resolution of multiple fragments and enables to differentiate between fragments, amplified by a single primer pair, with size differences as small as two base pairs.     

Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats

Sequence databases were screened to identify simple sequence repeats (SSRs) in Brassica oleracea sequences. A total of 512 B. oleracea DNA sequences were screened and 43 potential SSRs were identified. Thirty-six primer pairs were designed to amplify target sequences. Of the 36 primer pairs, six failed to amplify fragments of expected sizes, and 17 primer pairs failed to generate polymorphisms. Thirteen SSRs were used to assess genetic similarity between 54 B. oleracea cultivars, belonging to 3 variteal groups (cabbage, cauliflower, and broccoli). Pairwise genetic similarities were calculated for cultivars, and a dendrogram of relationships was produced. All cabbage cultivars were distinguished from each other and clustered in two separate groups. Five cauliflower cultivars could not be distinguished with SSR markers used in the study. Three broccoli cultivars clustered with cauliflower cultivars, and two cauliflower cultivars grouped with broccoli cultivars. The varietal group with the narrowest genetic variation in the study was cauliflower (B. oleracea var. botrytis) followed by broccoli (B. oleracea var. italica) and cabbage (B. oleracea var. capitata) groups. Polymorphism information content (PIC) values and number of alleles produced per marker ranged between 0.25 to 0.86 and 1 to 8, respectively, for database derived SSR markers.     

Relative susceptibility of different male-sterile cytoplasms in sorghum to shoot fly, <Emphasis Type="Italic">Atherigona soccata</Emphasis>

Summary The shoot fly, Atherigona soccata is an important pest of sorghum, and host plant resistance is one of the most effective components for managing this pest. Most of the hybrids grown in India based on milo cytoplasm (A₁ cytoplasm) are highly susceptible to shoot fly. Therefore, the present studies were undertaken to evaluate different male-sterile cytoplasms (CMS) for their relative susceptibility to sorghum shoot fly. Oviposition and deadheart formation were significantly lower on the maintainer lines as compared to the corresponding male-sterile lines. Among the cytoplasms tested, A₄M cytoplasm showed antixenosis for oviposition and suffered lower deadheart formation than the other cytoplasms tested. The A₄G₁ and A₄M cytoplasms suffered lower deadhearts in tillers than the other cytoplasms. Recovery following shoot fly damage in A₄M, A₃, and A₂ cytoplasms was better than in the other cytoplasms tested. The larval and pupal periods were longer and male and female pupal weights lower in A₄M and A₄VzM CMS backgrounds compared to the other CMS systems. Fecundity and antibiosis indices on CMS lines were lower than on the B-lines. The A₄M cytoplasm was found to be relatively resistant to sorghum shoot fly, and can be exploited for developing shoot fly-resistant hybrids for sustainable crop production in future.     

Efficiency of PCR-RF-SSCP marker production in Brassica oleracea using Brassica EST sequences

Efficiencies of SCAR, CAPS and PCR-RF-SSCP marker production were investigated using two combinations of breeding lines in Brassica oleracea. Published EST sequences of B. oleracea, Brassica rapa, Brassica napus, and Arabidopsis thaliana and newly determined nucleotide sequences of anther cDNA clones from B. oleracea were used for designing primer pairs to amplify genes. The percentage of primer pairs yielding DNA amplification of a single gene was higher in primer pairs of B. oleracea (91%) than those of B. rapa (56%) and A. thaliana (17%). Single DNA fragments amplified by 9% of the primer pairs showed polymorphism as SCAR markers between a broccoli line and a Chinese kale line by agarose-gel electrophoresis. CAPS analysis showed different band patterns in 32% of the same-sized DNA fragments, and PCR-RF-SSCP analysis revealed DNA polymorphism in 52% of those showing no DNA polymorphism by CAPS. In total, 71% of the single DNA fragments were converted to DNA markers. The frequency of DNA polymorphism between parental lines of a cabbage F₁ hybrid was lower, 5% by SCAR and 12% by CAPS. However PCR-RF-SSCP analysis revealed DNA polymorphism in 21% of the DNA fragments showing no polymorphism by CAPS. These results suggest that PCR-RF-SSCP analysis enables highly efficient DNA marker production for mapping of genes in Brassica using progeny, even progeny of closely related parents. Analysis of selfed seeds of broccoli F₁ cultivars using PCR-RF-SSCP markers indicated that PCR-RF-SSCP analysis is also applicable to seed purity tests.     

Complete mitochondrial genome sequence of Brassica oxyrrhina and comparative analysis of the region containing orf108, a male sterility gene for mustard (Brassica juncea), among B. oxyrrhina,Diplotaxis erucoides and Sinapis species

To date, several cytoplasms of wild species have been introduced to Brassica juncea, for inducing cytoplasmic male sterility (CMS). One of the causal genes of CMS is orf108, which is widely distributed in Brassicaceae including Brassica oxyrrhina. To further understand the origin of orf108, we assembled the complete mitochondrial genome sequence of B. oxyrrhina. We also determined the DNA sequences upstream of atp1 including orf108 for D. erucoides and five Sinapis species. The orf108 was definitively placed in the mitochondrial genome of B. oxyrrhina consisting of 247,936 bp. In S. alba, previously reported to possess orf108, the gene was not present, whereas Sinapis arvensis and Sinapis turgida contained orf108. The orf108 sequence in the two Sinapis species showed a novel nucleotide substitution compared to D. erucoides. Northern hybridization suggested, furthermore, that orf108 mRNA was processed in the two species similarly to that in B. oxyrrhina. The results clarified the interspecific differentiation of orf108‐apt1 region in Sinapis.     

A PCR marker system for monitoring the frequencies of normal and sterility-inducing cytoplasm types in German chive varieties

Random amplified polymorphic DNAs and atp9‐related sequences were amplified in cytoplasmic male sterile (CMS) and maintainer lines drawn from a backcross programme to represent all five known cytoplasm types in chives. From these PCR amplifications, markers associated with CMS‐inducing cytoplasm types, (S1) and (S₂), and for two of the three known normal cytoplasm types, (N₂) and (N₃), were developed. These newly developed PCR markers were used to determine the cytoplasm types in 126 plants representing 12 German chive varieties. The dependability of these PCR markers was confirmed by analysis with previously described and marker‐trait linked restriction fragment length polymorphisms. Two to five cytoplasm types were found in each of the 12 German chive varieties investigated. While the (S1) cytoplasm occurred, on average, at a frequency of 5% and the (S₂) cytoplasm at 12%, the three normal cytoplasms (N1), (N₂) and (N₃) were present at 30, 29 and 24%, respectively. Thus, the prospects of finding maintainers for both CMS systems are relatively high in this population, if the frequency of non‐restoring alleles for the nuclear genes involved is also high enough.     

Induction of cytoplasmic male sterility in the seed progeny derived from artificially-synthesized interspecific chimera in Brassica

We produced artificial interspecific chimeras by in vitro grafting, and obtained cytoplasmic male sterile (CMS) variants in the seed progenies derived from backcrossing the chimera with one of the mother plants, B. campestris cv. Komatsuna. The induced CMS has been stably inherited by crossing it with `Komatsuna', not with `Ruby Ball' cabbage. The nuclear component of CMS is complete `Komatsuna' type in morphology, chromosome number (2n = 20) and Southern blot using ribosomal 17S RNA gene as a probe. PCR analysis by using mitochondrial atpA primer showed the complete `Ruby Ball' type, suggesting nuclear-cytoplasmic exchange. However, Southern blot patterns were different among those of the CMS and both parents by using atpA. Recombination or some unknown change is supposed in the mitochondrial genome via the processes of synthesis and propagation of the chimeras.     

烟草ATP合酶F_0部分4个亚基基因转录本编辑位点分析

RNA编辑是高等植物线粒体基因转录后表达调控的一种重要方式。为探究ATP合酶F_0部分的4个亚基基因与植物雄性不育性的关系,本研究以3个烟草雄性不育系(MS中烟90、MS云烟85和MS K326)及其同型保持系为供试材料,比较分析atp6、atp9、orf25和orf B线粒体基因转录本的编辑位点。结果表明,orf25和orf B基因转录本在不育系和保持系中发生的编辑位点是一致的。atp6基因在不育系中未发生编辑,在保持系中共有6处发生了RNA编辑。与保持系相比,atp9基因在不育系中除8处共同的C→T编辑外,还缺少2个C→T的特异编辑位点,其中1个导致氨基酸类型的改变。推测不育胞质因缺少特异的线粒体基因转录本编辑而导致烟草的细胞质雄性不育。     

Haplotype analysis of CMS-associated DNA markers in sweet peppers

Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) is an economical and efficient system to produce F₁ hybrid seeds. Although the CMS/Rf system has been used to produce hybrid seeds of hot peppers, this system has never been used for sweet pepper seed production, presumably due to the inability to select stable restorer lines during the breeding process. To test the feasibility of the CMS/Rf system in sweet pepper breeding, we investigated the distribution of haplotypes of previously developed, CMS-associated markers (orf456, ψ atp6-2, CRF-SCAR, OPP13-CAPS, PR-CAPS, and PR-SNP) in 27 commercial sweet pepper F₁ hybrids and 12 breeding lines. When CMS-associated cytoplasmic markers orf456 and ψ atp6-2 were applied, male sterile cytoplasm was not detected in commercial sweet pepper cultivars. When nuclear haplotype markers linked to Rf were applied, all sweet pepper cultivars showed haplotype 3, haplotype 1, and the rf genotype for OPP13-CAPS, PR-CAPS, and CRF-SCAR, respectively. In contrast, we were able to detect male sterile cytoplasm in some breeding lines, and we were also able to detect polymorphisms for PR-CAPS between stable and unstable maintainer lines. The 17T7-SNP also showed polymorphisms between unstable and stable maintainer (or restorer) lines. In conclusion, we expect that it will be possible to develop stable A, B, and C sweet pepper lines using CMS-associated markers and that this will eventually lead to successful implementation of the CMS/Rf system to produce F₁ hybrid sweet pepper seeds.     

Identification of ‘Sib’ plants in hybrid cauliflowers using microsatellite markers

Hybrid cauliflowers have been developed to exploit heterosis and to improve uniformity of production. Two breeding systems are commonly employed, self-incompatibility (SI) and cytoplasmic male sterility (CMS). Sibs, assumed to be self-inbred, often contaminate hybrid seed lots in the SI system and whilst self-inbreeding is not possible in the CMS system, plants that look like sibs occur. The objective of this study was to develop microsatellite markers for male and female cauliflower parent lines of both SI and CMS systems and to use them to screen sibs and aberrant plants in F₁ hybrids. Fifty six pairs of microsatellite primers were screened and 8 primer pairs produced co-dominant markers in parent plants and two pairs of markers were chosen for purity testing of F₁ hybrid seeds. Controlled pollinations were conducted in the glasshouse to produce hybrid and selfed-seeds. These seeds were grown in a field trial to identify morphologically normal and sib plants and to assess the reliability of microsatellite markers in detecting sib plants. Microsatellite analysis of morphological sib plants from the SI system revealed that these were not always self-inbred, in contrast, most self-inbred plants showed normal growth. Similarly, all morphological sibs from the CMS system showed hybrid bands. This suggests that morphological sibs were not always due to selfing but possibly to an interaction between genetic and environmental factors and this requires further investigation.