Male Fertile Lines Research Articles

Mapping and Preliminary Analysis of ABORTED MICROSPORES (AMS) as the Candidate Gene Underlying the Male Sterility (MS-5) Mutant in Melon (Cucumis melo L.).

Melon is an important agricultural and economic vegetable crop worldwide. The genetic male sterility mutant (ms-5) has a recessive nuclear gene that controls the male sterility germplasm. Male sterility could reduce the cost of F1 seed production in melon, but heterozygous fertile plants should be removed before pollination. In this study, bulked segregant analysis combined with specific length amplified fragment sequencing was applied to map the single nuclear male sterility recessive gene. A 30-kb candidate region on chromosome 9 located on scaffold 000048 and spanning 2,522,791 to 2,555,104 bp was identified and further confirmed by cleavage amplified polymorphic sequence markers based on parental line resequencing data and classical mapping of 252 F2 individuals. Gene prediction indicated that six annotated genes are present in the 30-kb candidate region. Quantitative RT-PCR revealed significant differences in the expression level of the LOC103498166 ABORTED MICROSPORES (AMS) gene in male-sterile lines (ms-5) and male-fertile (HM1-1) lines during the 2-mm (tetrad) and 5-mm (the first pollen mitosis) periods, and negative regulation of the AMS candidate gene transcription factor was also detected. Sequencing and cluster analysis of the AMS transcription factor revealed five single-nucleotide polymorphisms between the parental lines. The data presented herein suggest that the AMS transcription factor is a possible candidate gene for single nuclear male sterility in melon. The results of this study will help breeders to identify male-sterile and -fertile plants at seeding as marker-assisted selection methods, which would reduce the cost of seed production and improve the use of male-sterile lines in melon.

Recessive male sterility in cabbage (Brassica oleracea var. capitata) caused by loss of function of BoCYP704B1 due to the insertion of a LTR-retrotransposon.

The LTR-retrotransposon insertion in BoCYP704B1 is proved to be the primary cause of the male sterility in cabbage. Effective allele-specific markers were developed for marker-assisted selection of male sterile gene. 83121A is a spontaneous male sterile mutant identified from cabbage. Genetic analysis indicated that male sterility is controlled by a single recessive gene. Pollen wall formation in the 83121A mutant was severely defective, with a lack of sporopollenin or exine. To understand the mechanisms of male sterility in 83121A, transcription analysis using RNA-Seq was carried out in the buds of the male sterile line 83121A and the male fertile line 83121B, which are near-isogenic lines differing only in the fertility trait. Via expression analysis of differentially expressed genes involved in pollen exine development before the bicellular pollen stage, BoCYP704B1 was identified as a candidate gene, which was approximately downregulated 30-fold in 83121A. BoCYP704B1 is a member of the evolutionarily conserved CYP704B family, which is essential for sporopollenin formation. The BoCYP704B1 transcript is specifically detected in the developing anthers of wild-type cabbage. Further sequence analysis revealed that a 5424-bp long terminal repeat-retrotransposon (LTR-RT) was inserted into the first exon of BoCYP704B1 in 83121A, which is not found in wild-type plants. The insertion of LTR-RT not only reduced the expression of BoCYP704B1 but also altered structure of protein encoded by BoCYP704B1. Moreover, linkage analysis showed that the homozygotic mutational BoCYP704B1 always cosegregated with male sterility. These data suggest that the LTR-RT insertion in BoCYP704B1 hinders sporopollenin formation in 83121A leading to male sterility. The allele-specific markers developed in this study were effective for marker-assisted selection of the male sterile gene.

Different lengths, copies and expression levels of the mitochondrial atp6 gene in male sterile and fertile lines of carrot (Daucus carota L.)

The male-sterile carrot is an effective material for carrot breeding. The atp6 gene is involved in carrot fertility. However, the differences in lengths, copies, and expression profiles of the atp6 gene in fertile and male-sterile lines of carrot are unclear. In this study, one copy atp6 gene was found in the mtDNAs of ‘Kuroda’ (fertility, 954 bp) and ‘Wuye-BY’ (male sterility, 819 bp) carrot lines, while two copies atp6 genes (Wuye-L and Wuye-D, 954 bp and 819 bp, respectively) were found in the mtDNA of ‘Wuye’ (fertility). Two putative conserved domains have been detected in the carrot atp6 protein. Evolutionary analysis showed that the atp6 protein sequences of Wuye-L and Kuroda were clustered in the same branch, while Wuye-D and Wuye-BY were clustered in the same branch. The atp6 gene was higher expressed in the flowers of ‘Kuroda’ and ‘Wuye’ (Wuye-L), while lower expressed in ‘Wuye-BY’ and ‘Wuye’ (Wuye-D).

Cloning and expression analysis of a new anther-specific gene CaMF4 in Capsicum annuum.

Our previous study on the genic male sterile-fertile line 114AB of Capsicum annuum indicated a diversity of differentially expressed cDNA fragments in fertile and sterile lines. In this study, a transcript-derived fragment (TDF), male fertile 4 (CaMF4) was chosen for further investigation to observe that this specific fragment accumulates in the flower buds of the fertile line. The full genomic DNA sequence of CaMF4 was 894 bp in length, containing two exons and one intron, and the complete coding sequence encoded a putative 11.53 kDa protein of 109 amino acids. The derived protein of CaMF4 shared similarity with the members of PGPS/D3 protein family. The expression of CaMF4 was detected in both the flower buds at stage 8 and open flowers of the male fertile line. In contrast to this observation, expression of CaMF4 was not detected in any organs of the male sterile line. Further analysis revealed that CaMF4 was expressed particularly in anthers of the fertile line. Our results suggest that CaMF4 is an anther-specific gene and might be indispensable for anther or pollen development in C. annuum.

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.

Identification of a nuclear-recessive gene locus for male sterility on A2 chromosome using the Brassica 60 K SNP array in non-heading Chinese cabbage

WS24-3 is a newly bred recessive genic male sterility line of the non-heading Chinese cabbage (Brassica rapa ssp. chinensis). Here, an F2 population was produced from the cross between WS24-3 and a male-fertile breeding line (WS135). The Illumina Brassica 60 K single nucleotide polymorphism (SNP) array was used for SNPs detecting between sterile and fertile bulks from the F2 population, and 62 SNPs were identified. BLAST analysis of the 62 SNPs revealed that the A2 chromosome of Brassica rapa genome contained 22 SNPs, whereas the other chromosomes did not contain more than 6 SNPs each. These data indicated that the potential target gene locus, named Bra2Ms, might be located on A2. Based on 10 of the 22 SNPs, allele-specific-polymerase chain reaction (AS-PCR) primers and single sequence repeat (SSR) primers were designed, 5 AS-PCR primers and 9 SSR primers showed difference between the bulks in electrophoretic determination. Analysis of these markers in F2 population revealed that Bra2Ms was genetically delimited to a region of 1.2 cM. We also detected two co-segregated markers SSRa2-951 and SSRa2-960 in this region. The markers identified in our study might facilitate the transfer of recessive genic male sterility alleles to other favorable genetic backgrounds. Furthermore, these markers will support a map-based clone of Bra2Ms.

Reação de genótipos de cebola à mancha púrpura

RESUMO: A mancha púrpura é uma das doenças mais limitantes da cebola, e a resistência genética é forma de controle mais viável da doença. O objetivo deste trabalho foi avaliar a reação de genótipos de cebola a alternaria porri em campo. Foram avaliados 64 genótipos (cultivares, populações e linhagens do programa de melhoramento da embrapa) em sistema convencional de cultivo. Utilizou-se como testemunha resistente a população cnph 6342 e como suscetível a linhagem macho-fértil 420. A cultura foi implantada pelo método de semeadura direta com população de 500 mil plantas por hectare. Foi utilizado o delineamento de blocos ao acaso com cinco repetições. As parcelas foram constituídas de seis fileiras no sentido longitudinal do canteiro, espaçadas de 15 cm (área total de 0,9 m²). A doença ocorreu de forma natural no campo e, a severidade foi avaliada em dez plantas por parcela aos 80, 110 e 140 dias após a semeadura. A produtividade e a massa média de bulbos comerciais (diâmetro ≥35 mm) foram avaliadas em apenas 45 genótipos, os quais exibiram adaptação às condições de clima local e bulbificaram adequadamente. 'crioula mercosul', '446', 'bola precoce', 'juporanga l2', 'caeté', 'rainha', 'dourada', 'juporanga l7', 'cnph 6342', 'crioula alto vale' e 'roxa do barreiro' apresentaram maior resistência ao patógeno, com áreas abaixo da curva de progresso da severidade da doença (aacpsd) inferiores a 437,33, enquanto a linhagem macho-fértil 420 apresentou aacpsd de 1.177,94. Os genótipos 'brs 367' e 'shinju', por sua vez, associaram alta produtividade de bulbos comerciais e moderada resistência à doença, com produtividades superiores a 43,9 t/ha e aacpsd inferiores a 567,28, sendo considerados os mais tolerantes à mancha púrpura.

Mutations in the mitochondrial orf108 render Moricandia arvensis restorer ineffective in restoring male fertility to Brassica oxyrrhina-based cytoplasmic male sterile line of B. juncea

We reported earlier an evolutionarily conserved mitochondrial orf108 causing cytoplasmic male sterility (CMS) in four alloplasmic lines of Brassica juncea. B. oxyrrhina also carries orf108, but male sterility of B. oxyrrhina-based B. juncea CMS lines is not restored by the Moricandia arvensis restorer that rescues other orf108-containing B. juncea CMS lines. To understand this discrepancy, we characterized B. oxyrrhina-based B. juncea CMS line (oxy-cms) to identify the mitochondrial gene associated with male sterility. Examination of expression patterns of 26 mitochondrial genes in CMS and male fertile (oxy-camp, i.e. amphidiploid B. oxyrrhina × B. rapa, and euplasmic B. juncea) lines revealed polymorphic transcript patterns for six genes: atp1, atp4, ccmfn2, cox3, nad4L and orf108. Detailed analysis showed that orf108 is linked and co-transcribed with atp1. Comparison of orf108-atp1 transcripts in male sterile and male fertile flowers revealed that B. oxyrrhina restorer cleaves orf108-atp1 at a different location within the orf108 coding region as compared with the M. arvensis restorer. A total of eleven SNPs were detected within the orf108 coding region. One of these SNPs was located at the site where M. arvensis restorer processes the orf108-atp1 transcript suggesting that it could be critical for cleavage of orf108-atp1 transcript by the M. arvensis restorer.

Capacidade de combinação de genitores de cenoura para caracteres de produtividade de raízes e tolerância à queima-das-folhas

RESUMO A demanda de mercado por uniformidade e produtividade de raízes comerciais de cenoura tem priorizado materiais híbridos em detrimento às variedades de polinização aberta. Neste sentido, objetivou-se com este trabalho estimar as capacidades de combinação de genitores de cenoura para os caracteres produtividade de raiz e resistência à queima-das-folhas. Os experimentos foram realizados no Gama, DF, nos anos agrícolas de 2012/13 e 2013/14. Foram avaliados 33 híbridos de cenoura, originados de cruzamentos entre três populações macho-estéreis, com 11 linhagens S2 macho-férteis, sendo todos os genitores de origem tropical. Aos 90 dias após a semeadura, foi estimada a severidade da doença queima-das-folhas, nas parcelas. Aos 100 dias após a semeadura, foi realizada a colheita e foram avaliados os caracteres de produtividade de raízes. Foram realizadas análises de variância e análise dialélica parcial, para cada ano e, conjunta, para os dois anos. Verificou-se que genes de efeitos aditivos e não aditivos são importantes na manifestação dos caracteres de produtividade de raízes e de resistência à queima-das-folhas, em híbridos de cenoura. Os genitores macho-estéreis com maior capacidade geral de combinação para produtividade de raízes são as linhagens LM-649 e LM-650 e, dentre os macho-férteis, a linhagem LM-555-2-2. Os melhores híbridos para produtividade de raízes e para resistência à queima-das-folhas são LM-649 × LM-555-11-1, LM-650 × LM-555-7-1 e LM-650 × LM-554-8-1.

Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling.

Tagetes erecta is an important commercial plant of Asteraceae family. The male sterile (MS) and male fertile (MF) two-type lines of T. erecta have been utilized in F1 hybrid production for many years, but no report has been made to identify the genes that specify its male sterility that is caused by homeotic conversion of floral organs. In this study, transcriptome assembly and digital gene expression profiling were performed to generate expression profiles of MS and MF plants. A cDNA library was generated from an equal mixture of RNA isolated from MS and MF flower buds (1 mm and 4 mm in diameter). Totally, 87,473,431 clean tags were obtained and assembled into 128,937 transcripts among which 65,857 unigenes were identified with an average length of 1,188 bp. About 52% of unigenes (34,176) were annotated in Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO (KEGG Ortholog database) and/or GO. Taking the above transcriptome as reference, 125 differentially expressed genes were detected in both developmental stages of MS and MF flower buds. MADS-box genes were presumed to be highly related to male sterility in T. erecta based on histological and cytological observations. Twelve MADS-box genes showed significantly different expression levels in flower buds 4 mm in diameter, whereas only one gene expressed significantly different in flower buds 1 mm in diameter between MS and MF plants. This is the first transcriptome analysis in T. erecta and will provide a valuable resource for future genomic studies, especially in flower organ development and/or differentiation.

Comparative analysis of sequences of mitochondrial genomes of wild abortive male sterile (WA-CMS) and male fertile lines of rice, development of functional markers for WA-CMS trait and their use in assessment of genetic purity of seeds of WA-CMS lines

WA-CMS system based rice hybrids are widely adopted in many rice growing countries, including India. Even though it is well known that the trait is controlled by mitochondria, the genes underpinning the trait remain enigmatic. In the present study, a complete genome-wide comparative sequence analysis was performed using draft mitochondrial genomes of WA-CMS and male fertile lines in a step-wise manner, progressively covering 5–10 kb every time through BLASTN tool. The sequence polymorphisms identified in different mitochondrial regions were targeted to develop two different sets of dominant PCR-based markers, one consisting of six markers targeting WA-CMS mitochondria, the other set consisting of five markers targeting male fertile mitochondria in addition to development of a set of eight co-dominant PCR-based markers targeting both the genomes. When a set of candidate genes/ORFs reported earlier to be associated with WA-CMS trait in rice were analyzed through RT-PCR of RNA isolated from immature rice florets, it was observed that the chimeric ORF, WA352 is expressed only in WA-CMS line and hybrid (i.e. genotypes containing sterile mitochondria), indicating it’s candidacy for the WA-CMS trait. Targeting the functional nucleotide polymorphism between WA-CMS and maintainer mitochondria with respect to WA352, two dominant markers, one targeting sterile and another targeting fertile mitochondria were developed. In addition, a robust, co-dominant functional marker targeting the candidate gene was also developed and validated for its utility in identification of genetic impurities in seed lots of WA-CMS lines.

Is the abaxial palisade parenchyma in phyllaries of the sunflower (Helianthus annuus L.) capitulum a missing trait in modern genotypes?

The involucral bracts (IB or phyllaries) of the sunflower(Helianthus annuus L.) capitulum constitute an example of photosynthesizingorgans that contribute to the photosynthesis budget duringthe generation of crop yield. The anatomy of IB was analyzed in twodomesticated primitive sunflower genotypes, Havasupai and Hopi, inthe sunflower line HA89B, in the wild Helianthus annuus ssp. annuus,in the sunflower male-fertile line R013 and in the commercial hybridDKOP3845. Stomata and trichomes were counted on the adaxial andabaxial epidermis. In all cases, the IB showed a one-layered adaxialand abaxial epidermis, secretory ducts and parenchymatic cells withabundant chloroplast. The vascular system was similar to that of thenomophylls; however, their bundles were smaller, with an abaxial surfaceshowing abundance of glandular and non-glandular trichomesand stomata. IB of Havasupai, Hopi and the male-fertile line strainshowed higher number of adaxial hypodermic strata than those ofHA89B, DKOP3845 and wild sunflower (2-3 vs. 1), and one mesophyllwith inverted polarity with respect to a foliage leaf: the presenceof a spongy parenchyma on the adaxial side was observed with arudimentary palisade parenchyma on the abaxial side. Stomatal densityof the IB was significantly higher in Hopi and Havasupai than inHA89B and DKOP3845, with values ranging from 132 to 156 vs 73to 110 stomata/mm2, respectively. Like the modern commercial hybrid,the IB of male-fertile line showed lower stomatal density (83stomata/mm2) and scarce abaxial trichomes. The anatomical studies ofphyllaries in Asteraceae remain relatively poor in the literature. Fromthe functional point of view, sunflower breeding produced undesiredchanges in the IB anatomy. Future studies for comparing the physiological(photosynthesis and respiration) and biochemical activities ofthe RuBisCO among the IB of the primitive (Havasupai and Hopi),male-fertile lines and modern sunflower genotypes will help to definethe magnitude of their importance as a morphological trait to be consideredin future plans of sunflower breeding.

Comparative Transcriptome Analysis of Recessive Male Sterility (RGMS) in Sterile and Fertile Brassica napus Lines.

The recessive genetic male sterility (RGMS) system plays a key role in the production of hybrid varieties in self-pollinating B. napus plants, and prevents negative cytoplasmic effects. However, the complete molecular mechanism of the male sterility during male-gametogenesis in RGMS remains to be determined. To identify transcriptomic changes that occur during the transition to male sterility in RGMS, we examined the male sterile line WSLA and male fertile line WSLB, which are near-isogenic lines (NILs) differing only in the fertility trait. We evaluated the phenotypic features and sterility stage using anatomical analysis. Comparative RNA sequencing analysis revealed that 3,199 genes were differentially expressed between WSLA and WSLB. Many of these genes are mainly involved in biological processes related to flowering, including pollen tube development and growth, pollen wall assembly and modification, and pollen exine formation and pollination. The transcript profiles of 93 genes associated with pollen wall and anther development were determined by quantitative RT-PCR in different flower parts, and classified into the following three major clades: 1) up-regulated in WSLA plants; 2) down-regulated in WSLA plants; and 3) down-regulated in buds, but have a higher expression in stigmas of WSLA than in WSLB. A subset of genes associated with sporopollenin accumulation were all up-regulated in WSLA. An excess of sporopollenin results in defective pollen wall formation, which leads to male sterility in WSLA. Some of the genes identified in this study are candidates for future research, as they could provide important insight into the molecular mechanisms underlying RGMS in WSLA.

Transcriptome profiling of differentially expressed genes in floral buds and flowers of male sterile and fertile lines in watermelon.

BackgroundMale sterility is an important mechanism for the production of hybrid seeds in watermelon. Although fruit development has been studied extensively in watermelon, there are no reports on gene expression in floral organs. In this study, RNA-sequencing (RNA-seq) was performed in two near-isogenic watermelon lines (genic male sterile [GMS] line, DAH3615-MS and male fertile line, DAH3615) to identify the differentially expressed genes (DEGs) related to male sterility.ResultsDEG analysis showed that 1259 genes were significantly associated with male sterility at a FDR P-value of < 0.01. Most of these genes were only expressed in the male fertile line. In addition, 11 functional clusters were identified using DAVID functional classification analysis. Of detected genes in RNA-seq analysis, 19 were successfully validated by qRT-PCR.ConclusionsIn this study, we carried out a comprehensive floral transcriptome sequence comparison of a male fertile line and its near-isogenic male sterile line in watermelon. This analysis revealed essential genes responsible for stamen development, including pollen development and pollen tube elongation, and allowed their functional classification. These results provided new information on global mechanisms related to male sterility in watermelon.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2186-9) contains supplementary material, which is available to authorized users.

Comparative proteomic analysis of a membrane-enriched fraction from flag leaves reveals responses to chemical hybridization agent SQ-1 in wheat.

The induction of wheat male fertile lines by using the chemical hybridizing agent SQ-1 (CHA-SQ-1) is an effective approach in the utilization of heterosis; however, the molecular basis of male fertility remains unknown. Wheat flag leaves are the initial receptors of CHA-SQ-1 and their membrane structure plays a vital role in response to CHA-SQ-1 stress. To investigate the response of wheat flag leaves to CHA-SQ-1 stress, we compared their quantitative proteomic profiles in the absence and presence of CHA-SQ-1. Our results indicated that wheat flag leaves suffered oxidative stress during CHA-SQ-1 treatments. Leaf O2-, H2O2, and malonaldehyde levels were significantly increased within 10 h after CHA-SQ-1 treatment, while the activities of major antioxidant enzymes such as superoxide dismutase, catalase, and guaiacol peroxidase were significantly reduced. Proteome profiles of membrane-enriched fraction showed a change in the abundance of a battery of membrane proteins involved in multiple biological processes. These variable proteins mainly impaired photosynthesis, ATP synthesis protein mechanisms and were involved in the response to stress. These results provide an explanation of the relationships between membrane proteomes and anther abortion and the practical application of CHA for hybrid breeding.

Defective pollen wall contributes to male sterility in the male sterile line 1355A of cotton.

To understand the mechanisms of male sterility in cotton (Gossypium spp.), combined histological, biochemical and transcription analysis using RNA-Seq was carried out in the anther of the single-gene recessive genic male sterility system of male sterile line 1355A and male fertile line 1355B, which are near-isogenic lines (NILs) differing only in the fertility trait. A total of 2,446 differentially expressed genes were identified between the anthers of 1355AB lines, at three different stages of development. Cluster analysis and functional assignment of differentially expressed genes revealed differences in transcription associated with pollen wall and anther development, including the metabolism of fatty acids, glucose, pectin and cellulose. Histological and biochemical analysis revealed that a major cellular defect in the 1355A was a thicker nexine, consistent with the RNA-seq data, and further gene expression studies implicated differences in fatty acids synthesis and metabolism. This study provides insight into the phenotypic characteristics and gene regulatory network of the genic male sterile line 1355A in upland cotton.

Direct analysis of nectar and floral volatile organic compounds in hybrid onions by HS-SPME/GC–MS: Relationship with pollination and seed production

In this study, a simple and solvent-free method using headspace solid-phase microextraction (SPME) and gas chromatography–mass spectrometry (GC–MS) was developed for determination of the volatile compounds from fresh flowers and nectar of male sterile and fertile lines of Allium cepa L. The SPME parameters were studied; the optimum conditions of 85μm carboxen/polydimethylsiloxane (CAR/PDMS), extraction temperature of 30°C and extraction time of 30min were obtained. The analytical method was applied to characterize different onion lines according to the chemical composition of the volatile fraction of nectar and flowers. On the other hand, it was determined which odor components contribute to the pollination of onions lines and relate the content of specific analytes with foraging behavior and seed production. More than 90 compounds were identified. The samples studied showed differences in the volatile profiles of flowers and nectar among the different lines. The results demonstrated that headspace SPME–GCMS is a simple, rapid and solvent-free method suitable for analysis of volatile compounds emitted from onion plants. Furthermore, a great difference was found for the number of bee visits and seed yield among the onion lines analyzed. This study demonstrates that the combination of chemical information and statistical analysis is able to differentiate onion male sterile lines from fertile lines, as well as among male sterile lines. Repellent compounds, such as dioxolanes, piperidines and organosulfurs may contribute negatively to pollination, since the higher content of these analytes, the less bee visits and seed production yield.

Development of SCAR markers to distinguish male-sterile and normal cytoplasm in bunching onion (Allium fistulosum L.)

SummaryCytoplasmic male sterility (CMS) is one of the most important systems for the production of F1 hybrid seed. Here, we obtained two sequence-characterised amplified region (SCAR) markers that could distinguish between male-sterile (S) and normal (N) types of cytoplasm in bunching onion (Allium fistulosum L.). A 327-bp S band (designated S327) was amplified in the male sterile line 980238A and a 1,412-bp N band (designated N1412) was amplified in its maintainer line 980238B. Both SCAR markers were verified in five male-sterile lines and their five maintainer lines, in four paternal lines and their self-crossed progenies, and in four cross combinations (one male-sterile line as the female parent and the four male-fertile paternal lines as the male parents). All the results agreed with their corresponding cytoplasmic type (S or N). Our findings suggest that the cytoplasm of A. fistulosum can be identified as male-sterile or normal using these two SCAR markers.These findings are expected to make a significant contribution to breeding F1 hybrid cultivars of bunching onion.

Sequencing of the chloroplast genomes of cytoplasmic male-sterile and male-fertile lines of soybean and identification of polymorphic markers

The RN-type cytoplasmic male sterility (CMS) system used to develop Hybsoy-1, the first commercial hybrid soybean, has been subsequently applied to generate nearly all released soybean hybrids. Although more than 3 years are needed to classify sterile (S) and normal male-fertile (F) cytoplasms by conventional crossing, such classifications can be performed rapidly using organellar DNA-based molecular markers. Except for fertility, the agronomic traits of CMS hybrid soybean sterile and maintainer lines are identical. Consequently, it is difficult to distinguish them by routine visual inspection in the mixture arising in the course of field planting and harvesting during breeding. In this study, we performed next-generation sequencing of chloroplast DNAs of F- and S-cytoplasmic soybeans, assembled and annotated the genomes, and identified polymorphisms distinguishing them. Chloroplast DNAs of F and S cytoplasms were very similar in size (152,215 and 152,222 base pairs) and GC contents (35.37%). Among 23 shared SNPs in gene coding regions, we identified four that could be used in conjunction with restriction endonucleases to distinguish S and F cytoplasms. Although CMS is likely associated with mitochondrial DNA, maternal transmission of mitochondrial and chloroplast DNAs allows polymorphisms in either genome to be used to classify soybean cytoplasms, aiding hybrid soybean cultivar development.

Fertility restoration in three CMS systems of eggplant by the Rf genes of each other's systems and their SCAR marker

Three pollen non-formation type cytoplasmic male sterility (CMS) systems of eggplant (Solanum melongena L.) were developed in our previous study, through repeated backcrossing, by using wild Solanum species Solanum aethiopicum L. Aculeatum Group, Solanum anguivi Lam. and Solanum grandifolium C. V. Morton as the cytoplasm donor and eggplant ‘Uttara’ as the recurrent pollen parent. Each CMS system has male fertile (MF) line which carries the fertility restorer (Rf) genes for the pollen non-formation type sterility. In the present study, fertility restoration ability in three CMS systems of eggplant by the Rf genes of each other's systems was evaluated. The male sterile (MS) line of each CMS system was pollinated with the pollen of MF lines of other CMS systems as well as its own CMS system. Among the nine cross combinations, six groups were obtained from crossing MS lines with MF lines of other CMS systems and three groups were obtained from crossing MS lines with MF lines of their own CMS system. The segregation of MF and MS plants was fitted well with a 3:1 ratio in almost all cross combinations. The segregation patterns revealed that two independent dominant Rf genes restore fertility in each CMS system. Pollen stainability and in vitro pollen germination rate of the progenies obtained from different cross combinations were varied from 61.0 to 89.8% and 0.7 to 8.0%, respectively. Each Rf gene of three CMS systems restores fertility of other CMS systems as well as its own CMS system. Moreover, fertility recovery action in these three kinds of CMS is similar. DNA markers linked to Rf genes were screened by bulked segregant analysis (BSA) using random amplified polymorphic DNA (RAPD). A total of 420 primers were used, and more than 65% of the primers yielded a considerable number of RAPD fragments in both MF and MS bulks. Only one Operon primer OPAB10 gave a reproducible polymorphic band of 1.9kbp (OPAB101900) between bulks and was identified as linked to the Rf genes. Additionally, the RAPD marker OPAB101900 was converted to sequenced characterized amplified region (SCAR) marker designated as SCAB101900 by nucleotide sequencing. Findings of the present study may give us a potential way to explore CMS and the corresponding Rf genes from wild Solanum resources for the breeding program of eggplant.