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.     

甘蓝型油菜NCa CMS育性相关候选线粒体基因及其受恢复基因的转录调控

用10个线粒体基因为探针, 对NCa不育系、保持系和可育F₁幼蕾的线粒体RNA进行了Northern检测。结果表明，atp6、atp1、cox1、cox2、cob、rrn5S和rrn26S等7个线粒体基因探针在不育系、保持系、可育F₁中的转录没有差异；只有orf222、orf139和atp9等3个探针检测到转录本的差异。orf222和orf139分别在不育系和可育F1中产生相同大小和丰度的转录本，但是在保持系中没有检测到转录本；orf222检测到的3条转录本分别为1.1、0.9和0.6 kb，orf139检测到0.8和0.6 kb 2条带。atp9探针在不育系和保持系中都检测到1条0.6 kb转录本，而在可育F₁中检测到0.6和1.2 kb的转录本。NCa CMS不育的形成可能与orf222、orf139和atp9基因的表达有关；讨论了恢复基因在F₁育性恢复过程中对育性相关候选基因的可能调控方式。     

PCR-based markers to differentiate the mitochondrial genomes of petaloid and male fertile carrot (Daucus carota L.)

Cytoplasmic male sterility (CMS) is essential for the development of highly adapted and uniform hybrid varieties of carrot and other crops. The most widely used type of CMS in carrot is petaloidy, in which the stamens are replaced by petals or bract-like structures. We have developed a series of mitochondria-specific PCR markers to distinguish cytoplasms inducing petaloidy (Sp) and male-fertility (N). The markers target the atp1,atp6, atp9, orfB (atp8), nad6 and cob loci from the mitochondrial genomes of a diverse collection of male fertile and petaloid carrots. We report 14 primer pairs that amplify marker fragments from either Sp or N cytoplasms and three primer pairs that amplify fragments with length polymorphism. The amplification products span sites of insertions, deletions or recombinations adjacent to or within the coding regions of the targeted genes. The markers reported here are useful tools to identify the type of cytoplasm in cultivated carrot and to evaluate variation in the mitochondrial genomes within the genus Daucus. This revised version was published online in August 2006 with corrections to the Cover Date.     

甘蓝型油菜NCa CMS育性相关候选线粒体基因及其恢复基因的转录调控

用10个线粒体基因为探针，对NCa不育系、保持系和可育F1幼蕾的线粒体RNA进行了Northern检测。结果表明，atp6、atp1、cox1、cox2、cob、rrn5S和rnn26S等7个线粒体基因探针在不育系、保持系、可育F1中的转录没有差异，只有orf222、orf139和atp9等3个探针检测到转录本的差异。orf222和orf139分别在不育系和可育F1中产生相同大小和丰度的转录本，但是在保持系中没有检测到转录本；orf222检测到的3条转录本分别为1．1、0．9和0．6kb，orf139检测到0．8和0．6kb2条带。atp9探针在不育系和保持系中都检测到1条0．6kb转录本，而在可育F1中检测到0．6和1．2kb的转录本。NCaCMS不育的形成可能与orf222、orf39和atp9基因的表达有关。讨论了恢复基因在F1育性恢复过程中对育性相关候选基因的可能调控方式。     

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.     

Effect of alien cytoplasm and fertility restorer genes on agronomic and physiological traits of Brassica juncea (L.) Czern

To assess the effect of alien cytoplasm and fertility restorer genes on agronomic and physiological traits in Brassica juncea, cytoplasmic male sterile (CMS) and fertility restorer lines involving five alloplasms in three nuclear backgrounds were constituted through repeated backcrossing. These lines were evaluated along with euplasmic lines for agronomic traits in field experiments. Respiration efficiency of in vitro cultured hypocotyls, and chlorophyll content in leaves were also estimated. Significant adverse effects of trachystoma and catholica cytoplasms on yield‐contributing traits and yield were noticed. The restorer gene corrected the floral defects in CMS trachystoma and restored yield to the level of euplasmic line, while it did so only partly in CMS catholica. Restorer genes had no adverse effect on traits in any of the systems. On the contrary, the Rf gene of moricandia CMS system exerted a positive effect on leaf chlorophyll content. Likewise, in vitro assay indicated the moricandia CMS system as vigorous. The moricandia CMS plants gave higher seed yield perhaps due to overall better vigour and higher leaf chlorophyll content.     

Molecular advancements in male sterility systems of Capsicum: A review

In recent years, plant molecular research on genetic mapping, gene tagging and cloning, and marker-assisted selection (MAS) have gained importance in crop improvement programmes. In Capsicum, several inter- and intra-specific genetic maps with wide distribution of markers covering the whole genome have been developed. Recently, whole genome of the hot pepper C. annuum, its wild progenitor C. annuum var. glabriusculum and C. baccatum has been sequenced. The Capsicum genome size has been estimated to be approx. 4× (3.48 Gb) the genome size of cultivated tomato (Solanum lycopersicum L.) (900 Mb). Breeders’ access to the pepper genomic information would facilitate the choice of markers from different linkage groups, thus paving the way for gene cloning and its introgression into the elite breeding lines through MAS. Till date, approx. 20 independently inherited nuclear male sterility (NMS) genes have been reported. Linked markers have been identified for ms1, ms3, ms8, ms10, ms_k, msc-1 and an undesignated gene. However, markers tightly linked to ms8 and ms10 are still lacking. Except ms1, ms3, ms8 and ms10, the map position of other NMS genes is not known. In cytoplasmic male sterility (CMS), markers for the mitochondrial gene atp6 have been developed and the gene cloned. Number of markers some very tightly linked to the restorer-of-fertility (Rf) gene have been identified. However, the actual map position of the Rf locus is still not determined. Another CMS-associated nuclear gene “pr” responsible for restoring partial fertility has been identified and tagged. In this review, we have compiled up-to-date information about the marker technology relating to the NMS and the CMS-associated genes in Capsicum. This information can be useful when screening Capsicum germplasm, developing NMS lines through MAS, improving efficiency of the NMS system, transferring rf gene for maintainer line breeding and Rf genes for restorer line breeding in CMS and assessing genetic purity of the hybrid seed.     

Mitochondrial DNA variation in finger millet (Eleusine coracana L. Gaertn)

Summary This study was conducted to classify 26 lines of finger millet from Africa and India into cytotype groups based on the Southern blot hybridization patterns obtained with maize and sorghum mitochondrial cloned gene probes. Five restriction endonuclease enzymes were used for single digestions on total cellular DNA, giving a total of 20 enzyme/probe combinations. There was a low level of polymorphism, with identical RFLP banding patterns in 23 of the 26 lines. However, mtDNA clone atp9 hybridized to a 3.6 Kb Xba1 fragment in ecotype SDFM-1143 from Malawi; but did not hybridize to a 3.0 Kb fragment present in all other ecotypes. Two Zimbabwean lines, SDFM 63 and SDFM 77, had an extra, low intensity 6.5 Kb Xba1 fragment hybridized by mtDNA clone cox1. This data enabled classification of the lines into 3 cytopype groups.     

小麦T、 K、 V型胞质不育系和杂种mtDNA的RAPD分析及育性相关片段的克隆

用RAPD技术对细胞质分别来源于粘果山羊草（Ae.kotschyi）、偏凸山羊草（Ae.ventricosa）、提莫菲维小麦（T.timopheevii）的三种普通小麦雄性不育系--K型、V型、T型及相应的保持系、恢复系以及杂种F1代的线粒体DNA（mtDNA）进行了比较分析。结果如下：（1）发现在mtDNA组织结构上K型、V型、T型不育系之间以及与保持系之间均     

棉花新型D8胞质雄性不育的细胞质效应研究

 利用3个不育系、3个同质保持系和3个恢复系配制不育系×恢复系、保持系×恢复系、恢复系×保持系三种类型组合,研究新型三裂棉（D8）不育胞质对棉花杂交种产量性状和纤维品质的影响。结果表明：D8不育胞质对子棉和皮棉产量有极显著的负效应,子棉平均减产15.3%～25.6%,皮棉减产25.0%～39%;但D8胞质对不同产量因素的影响不同,平均降低铃重0.89～1.00 g,降低衣分4.59%～7.08%,对单株结铃数无明显影响。D8胞质不育对纤维长度和整齐度没有影响,还可以提高纤维比强度1.0～1.1 cN·tex^-1,降低麦克隆值1.4～1.5,增加纤维伸长率0.4。不同的基因型受D8不育胞质的影响不同,通过选择合适的基因型,可以减小胞质对铃重和衣分的负面影响,选育出优良的胞质不育三系杂交棉\{品种。     

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.     

哈克尼西棉细胞质雄性不育相关线粒体基因多态性分析

【目的】研究棉花细胞质雄性不育的相关线粒体基因。【方法】利用线粒体基因atp A、atp6、atp9、cob、coxⅠ、coxⅡ、coxⅢ、nad3、nad6、nad9探针,对哈克尼西棉细胞质雄性不育系S、保持系F、杂种一代H进行限制性片段长度多态性分析。【结果】atp A、coxⅢ、nad3、nad6在3个材料间具有多态性。atp A、nad6基因探针与所有酶的杂交结果均显示出多态性,且在不育系与杂种一代中表现一致,而在保持系中差异明显。atp A/Eco RⅠ在3个材料中的杂交结果均显示2条带,其中1条2.2 kb的杂交带在3个材料中相同,另一条在不育系和杂种一代中大小为3.2 kb,而在保持系中为4.8 kb;atp A/PstⅠ杂交结果中,在不育系和杂种一代中的条带大小为17.0kb,而在保持系中为10.2 kb。nad6探针与4个酶的杂交结果均为不育系和杂种一代比保持系多1~2条杂交带。coxⅢ/Eco RⅠ在3个材料中均有1条2.5 kb的杂交带,但在保持系与杂种一代中比不育系多1条1.7 kb的弱带。nad3/Bam HⅠ的杂交结果在不育系与保持系中表现一致,而在杂种一代中缺少1条9.5 kb的杂交带。【结论】推测atp A、nad6基因参与调控不育系的形成,而coxⅢ、nad3可能受到恢复系核基因的调控,在不育系育性恢复过程中发挥较为重要的作用。     

Introgression of a gene restoring fertility to CMS (Trachystoma) Brassica juncea and the genetics of restoration

A dominant gene restoring fertility to a cytoplasmic male sterile (CMS) line of Brassica juncea was derived from the somatic hybrid Trachystoma ballii+B. juncea. Its introgression resulted from forced pairing between chromosomes of the cultivar ‘Pusa Bold’ and chromosomes of the fusion hybrid. Segregation ratios of this fertility restorer gene followed a monogenic pattern. The introgression of the fertility restorer gene did not cause any abnormalities, such as reduced fertility; pollen and seed fertilities of the restored plants were over 90%. Restored fertile and CMS plants exhibited similar Southern hybridization patterns when probed with the mitochondrial probe atp6.     

Assessment of cytoplasmic effects of cytoplasmic male-sterile lines in upland cotton

Paired A- and B-lines, cytoplasmic male-sterile (CMS)-lines and main-tainer lines, respectively, were crossed with R-lines (restorer) to produce A × R, R × B and B × R hybrids, which were used in 1993 and 1996 to predict the performance of three lypes of CMS cotton lines available in China. A significant difference in yield and yield components was revealed between paired A × R and B × R hybrids. This difference was greatly influenced by both CMS cytoplasm and the interaction between cytoplasm and nuclear genotypes. It is suggested that there are detrimental effects of CMS cytoplasm on yield and yield components. General combining ability of near-isogenic CMS lines was also affected by this negative effect. The detrimental effect was closely related to an increased number of immature seeds per boll, which might be caused by partial female sterility associated with CMS cytoplasm. The possibility of developing specific combinations of CMS Upland × Upland restorer hybrids that express enough heterosis for yield to overcome the detrimental effect of the CMS cytoplasm is discussed.     

A unique introgression from Moricandia arvensis confers male fertility upon two different cytoplasmic male-sterile lines of Brassica juncea

A Brassica juncea line carrying an introgression from Moricandia arvensis restored male fertility to two cytoplasmic male‐sterile (CMS) B. juncea lines carrying either M. arvensis or Diplotaxis catholica cytoplasm. Genetics of fertility restoration was studied in the F₁, F₂, F₃ and backcross generations of the cross between CMS and fertility‐restorer lines. No male‐sterile plants were found in F1‐F₃ generations of the cross between CMS [M. arvensis] B. juncea and the restorer. However, a 1: 1 segregation for male sterility and fertility was observed when the F1 was pollinated with non‐restorer pollen from a euplasmic line. These results clearly show that restoration is mono‐genic and gametophytic. In CMS lines carrying D. catholica cytoplasm, the restorer conferred male fertility to the F1 and showed 3: 1 and 1: 1 segregations for male fertility and sterility in F₂ and BC1 generations, respectively, indicating a monogenic, sporophytic mode of fertility restoration. The results were also supported by pollen stainability in the F1 which was about 65% in M. arvensis‐based CMS and >90% in D. catholica‐based CMS. The above results are discussed in the light of previous molecular studies which showed association between CMS and atpA in both systems.     

Chromosomal location of fertility restoring genes for ‘wild abortive’ cytoplasmic male sterility using primary trisomics in rice

Summary Identification and location of fertility restoring genes facilitates their deployment in a hybrid breeding program involving cytoplasmic male sterility (CMS) system. The study aimed to locate fertility restorer genes of CMSWA system on specific chromosomes of rice using primary trisomics of IR36 (restorer), CMS (IR58025A) and maintainer (IR58025B) lines. Primary trisomic series (Triplo 1 to 12) was crossed as maternal parent with the maintainer line IR58025B. The selected trisomic and disomic F₁ plants were testcrossed as male parents with the CMS line IR58025A. Plants in testcross families derived from disomic F₁ plants (Group I crosses) were all diploid; however, in the testcross families derived from trisomic F₁ plants (Group II crosses), some trisomic plants were observed. Diploid plants in all testcross families were analyzed for pollen fertility using 1% IKI stain. All testeross families from Group I crosses segregated in the ratio of 2 fertile: 1 partially fertile+partially sterile: 1 sterile plants indicating that fertility restoration was controlled by two independent dominant genes: one of the genes was stronger than the other. Testcross families from Group II crosses segregated in 2 fertile: 1 partially fertile+ partially sterile: 1 sterile plants in crosses involving Triplo 1, 4, 5, 6, 8, 9, 11 and 12, but families involving triplo 7 and triplo 10 showed significantly higher X² values, indicating that the two fertility restorer genes were located on chromosome 7 and 10. Stronger restorer gene (Rf-WA-1) was located on chromosome 7 and weaker restorer gene (Rf-WA-2) was located on chromosome 10. These findings should facilitate tagging of these genes with molecular markers with the ultimate aim to practice marker-aided selection for fertility restoration ability.     

Development of InDel markers for the restorer gene <Emphasis Type="Italic">Rf1</Emphasis> and assessment of their utility for marker-assisted selection in cotton

The cytoplasmic male sterility (CMS) system is ideal for exploiting heterosis in crops such as cotton. However, CMS-D2, which is based on Gossypium harknessii cytoplasm, is still not widely used for cotton production. In this study, we developed an efficient marker-assisted selection method based on insertion/deletion (InDel) markers that can identify restorer lines carrying Rf1. Whole-genome resequencing was first completed for restorer [N(Rf1Rf1)] and maintainer [N(rf1rf1)] lines with normal fertile cytoplasm (N). Comparisons with the TM-1 reference genome sequence resulted in the identification of 292,065 and 183,657 InDels for the restorer and maintainer lines, respectively. Most of the InDels in the restorer line were distributed on Chromosome_D05, which carries Rf1. Of the 12 InDel markers near the Rf1 target region that were further validated, four co-dominant markers (i.e., InDel-1891, InDel-3434, InDel-7525, and InDel-9356) co-segregated with Rf1, as verified by a segregation analysis in an F₂ population. We subsequently used InDel-1891 to determine the allele status at the Rf1 locus in a backcross scheme for transferring Rf1. In this study, we developed new markers to increase the marker density in the Rf1 target region, which will be useful for the fine mapping of Rf1. The development of convenient and inexpensive co-segregating InDel markers will facilitate the marker-assisted selection of restorer lines carrying Rf1.     

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.     

Are ordinal rating scales better than percent ratings? a statistical and “psychological” view

Cytoplasmic male sterility (CMS) is considered an efficient genetic tool in pearl millet hybrid breeding. Of the several CMS sources available in pearl millet, A₁ is the only CMS widely exploited to produce commercial hybrids in India. To explore the possibility of using alternate CMS sources, we studied the cytoplasmic effects of different CMS sources on agronomic characters in pearl millet. Five CMS (A) lines representing A₁, A₂, A₃, A₄ and A₅ cytoplasms, their respective maintainer (B) lines and eight restorer (R) lines were used to generate 40 A × R and B × R experimental crosses. The experimental material was evaluated at two different locations in India. Analysis of combining ability and heterosis revealed that A₄ and A₅ cytoplasms had desirable effects for earliness. The A₅ CMS was found to be particularly promising, as compared to other CMS sources for improving grain yield. The study also indicated that the cytoplasmic effects on general combining ability (GCA) for various agronomic characters were largely non-significant. However, cytoplasmic effects on specific combining ability and heterosis were found to be modulated by cytoplasmic-nuclear interactions and influenced by the environmental conditions. The study also demonstrated the advantage of utilizing diverse male-sterile and restorer combinations in maximizing the productivity as well as for genetic and cytoplasmic diversification of hybrids in pearl millet.     

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.