Practical Application of PCR-based Technology to Screen Texas Onion Breeding Lines for Cytoplasmic Male Sterility

Identification and production of onion male-sterile and maintainer lines by conventional breeding takes between 4 to 8 years, due in part to the biennial nature of onion. In addition, male sterile plants and maintainer genotypes occur at a very low frequency in onion populations (Pike, 1986). A significantly shorter and more efficient alternative involves the screening of breeding lines for cytoplasmic male sterility using PCR-based technology. Thirty short-day onion breeding lines from the Texas A&M onion program were screened for type of cytoplasm (normal or sterile). Specific amplification of a fragment of chloroplast genome was achieved using the polymerase chain reaction according to Havey (1991). Forty-eight individual onion plants were screened per line. Out of thirty lines evaluated, 13 showed 100% sterile cytoplasm, 6 showed 100% normal cytoplasm, and 11 showed both types of cytoplasm. Lines showing normal cytoplasm or both cytoplasmic types were kept and reanalyzed. Only plants presenting normal cytoplasm were grown to maturity to help in the identification of maintainer lines as part of the Texas A&M onion breeding program.

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Potentially a new subtype of the cytoplasmic male sterility S- type in maize

Genetika ◽

10.2298/gensr1301145v ◽

2013 ◽

Vol 45 (1) ◽

pp. 145-151

Author(s):

Jelena Vancetovic ◽

Dragana Ignjatovic-Micic ◽

Ana Nikolic ◽

Sofija Bozinovic ◽

Ksenija Markovic ◽

...

Keyword(s):

Cytoplasmic Male Sterility ◽

Male Sterility ◽

Multiplex Polymerase Chain Reaction ◽

Laboratory Experiments ◽

Gene Bank ◽

Chain Reaction ◽

Additional Band ◽

Two Samples ◽

Polymerase Chain ◽

Maize Research Institute

In gene-bank maize collection of Maize Research Institute Zemun Polje (MRI) two samples with untypical mtDNA profile for cytoplasmic male sterility (cms) were identified. These two samples showed typical multiplex polymerase chain reaction (PCR) band for cms-S, but also an additional band of unknown nature. It is assumed that the additional band is the result of a rearrangement of the two mitochondrial episomes characteristic for the cms-S in maize or a duplication of the part of cms-S mitochondrial genome. Additional field and laboratory experiments are necessary in the further lightening of this phenomenon.

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Reference Gene Selection for Real-time Quantitative Reverse-transcription Polymerase Chain Reaction in Flower Buds of Marigold

Journal of the American Society for Horticultural Science ◽

10.21273/jashs05074-21 ◽

2021 ◽

pp. 1-13

Author(s):

Nan Tang ◽

Wuhua Zhang ◽

Liwen Chen ◽

Yan Wang ◽

Daocheng Tang

Keyword(s):

Polymerase Chain Reaction ◽

Male Sterility ◽

Reverse Transcription ◽

Reference Genes ◽

Male Sterile ◽

Chain Reaction ◽

Flower Buds ◽

Qrt Pcr ◽

Polymerase Chain ◽

Suitable Reference

Marigold (Tagetes erecta) is an important commercial plant because of its ornamental, industrial, and medicinal values. Male-sterile two-type lines are important for heterosis utilization and breeding of marigold. Mining of fertility-related genes may help to elucidate the mechanisms underlying male sterility. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a popular and useful tool for analyzing the expression level of a specific gene. Notably, identifying a suitable reference gene is important for data normalization because it affects the accuracy of quantitative analysis. However, at present, no reference genes are available for marigold. During the current study, 10 candidate reference genes were selected and their expression levels in different samples were analyzed by qRT-PCR. The expression level of each gene was analyzed across different developmental stages of male-sterile and male-fertile flower buds by four software programs (geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that different reference genes are required for male-sterile and male-fertile samples, even if they belong to the same line. For male-sterile samples, the ribosomal protein S5/18S ribosomal RNA (RPS5/18S) gene pair was the best reference for qRT-PCR normalization, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be used as an alternative. For male-fertile samples, elongation factor 1-alpha (EF1α) and RPS5 were the most suitable reference genes, and Ubiquitin-conjugating enzyme (UBC) could be used as an alternative. Beta-actin (ACTB), tubulin beta (TUB), and phenylalanine ammonia-lyase (PAL) should not be used as reference genes because they were the most unstable genes in flower buds of marigold. The results of the current study may facilitate the selection of reference genes for analyzing the expression patterns of genes involved in flower development related to male sterility in marigold.

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The Dynamics of Gynodioecy in Plantago lanceolatu L. II. Mode of Action and Frequencies of Restorer Alleles

Genetics ◽

10.1093/genetics/147.3.1317 ◽

1997 ◽

Vol 147 (3) ◽

pp. 1317-1328

Author(s):

Anita A de Haan ◽

Hans P Koelewijn ◽

Maria P J Hundscheid ◽

Jos M M Van Damme

Keyword(s):

Cytoplasmic Male Sterility ◽

Male Sterility ◽

Allele Frequency ◽

Mode Of Action ◽

Male Fertility ◽

Fertility Restoration ◽

Plantago Lanceolata ◽

Allele Frequencies ◽

Male Sterile ◽

Male Fertility Restoration

Male fertility in Plantago lanceolata is controlled by the interaction of cytoplasmic and nuclear genes. Different cytoplasmic male sterility (CMS) types can be either male sterile or hermaphrodite, depending on the presence of nuclear restorer alleles. In three CMS types of P. lanceolata (CMSI, CMSIIa, and CMSIIb) the number of loci involved in male fertility restoration was determined. In each CMS type, male fertility was restored by multiple genes with either dominant or recessive action and capable either of restoring male fertility independently or in interaction with each other (epistasis). Restorer allele frequencies for CMSI, CMSIIa and CMSIIb were determined by crossing hermaphrodites with “standard” male steriles. Segregation of male steriles vs. non-male steriles was used to estimate overall restorer allele frequency. The frequency of restorer alleles was different for the CMS types: restorer alleles for CMSI were less frequent than for CMSIIa and CMSIIb. On the basis of the frequencies of male steriles and the CMS types an “expected” restorer allele frequency could be calculated. The correlation between estimated and expected restorer allele frequency was significant.

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PCR BY THERMAL CONVECTION

Modern Physics Letters B ◽

10.1142/s0217984904007049 ◽

2004 ◽

Vol 18 (16) ◽

pp. 775-784 ◽

Cited By ~ 37

Author(s):

DIETER BRAUN

Keyword(s):

Polymerase Chain Reaction ◽

Thermal Convection ◽

Dna Amplification ◽

Reaction Vessel ◽

Cold Region ◽

Chain Reaction ◽

Heating And Cooling ◽

Highly Sensitive ◽

Specific Amplification ◽

Polymerase Chain

The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

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INHERITANCE OF FERTILITY RESTORATION INVOLVING WILD ABORTIVE CYTOPLASMIC MALE STERILITY SYSTEM IN RICE (Oryza sativa L.)

Bangladesh Journal of Plant Breeding and Genetics ◽

10.3329/bjpbg.v24i1.16997 ◽

2011 ◽

Vol 24 (1) ◽

pp. 33-40

Author(s):

M. J. Hasan ◽

M. U. Kulsum ◽

A. Ansari ◽

A. K. Paul ◽

P. L. Biswas

Keyword(s):

Oryza Sativa ◽

Cytoplasmic Male Sterility ◽

Male Sterility ◽

Gene Action ◽

Oryza Sativa L ◽

Fertility Restoration ◽

Dominant Gene ◽

Recessive Gene ◽

Male Sterile ◽

Male Sterile Line

Inheritance of fertility restoration was studied in crosses involving ten elite restorer lines of rice viz. BR6839-41-5-1R, BR7013-62-1-1R, BR7011-37-1-2R, BR10R, BR11R, BR12R, BR13R, BR14R, BR15R and BR16R and one male sterile line Jin23A with WA sources of cytoplasmic male sterility. The segregation pattern for pollen fertility of F2 and BC1 populations of crosses involving Jin23A indicated the presence of two independent dominant fertility restoring genes. The mode of action of the two genes varied in different crosses revealing three types of interaction, i.e. epistasis with dominant gene action, epistasis with recessive gene action, and epistasis with incomplete dominance.DOI: http://dx.doi.org/10.3329/bjpbg.v24i1.16997

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Low frequency of CYP2A6 gene polymorphism as revealed by a one-step polymerase chain reaction method

Pharmacogenetics ◽

10.1097/00008571-199906000-00007 ◽

1999 ◽

Vol 9 (3) ◽

pp. 327-332 ◽

Cited By ~ 37

Author(s):

Gen-Fu Chen ◽

Yong-Ming Tang ◽

Bridgett Green ◽

Dong-Xin Lin ◽

F. Peter Guengerich ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Gene Polymorphism ◽

Low Frequency ◽

Polymerase Chain Reaction Method ◽

Chain Reaction ◽

Reaction Method ◽

One Step ◽

Polymerase Chain ◽

Cyp2a6 Gene ◽

Step Polymerase Chain Reaction

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Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

Marine and Freshwater Research ◽

10.1071/mf05083 ◽

2005 ◽

Vol 56 (8) ◽

pp. 1127 ◽

Cited By ~ 5

Author(s):

D. G. Bourne ◽

R. L. Blakeley ◽

P. Riddles ◽

G. J. Jones

Keyword(s):

Polymerase Chain Reaction ◽

Environmental Samples ◽

In Situ Hybridisation ◽

Cyanobacterial Blooms ◽

Chain Reaction ◽

Fluorescent In Situ Hybridisation ◽

Cyanobacterial Toxin ◽

Specific Amplification ◽

Polymerase Chain

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

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Evaluation of the expression stability of reference genes in Apis mellifera under pyrethroid treatment

Scientific Reports ◽

10.1038/s41598-020-73125-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Przemysław Wieczorek ◽

Patryk Frąckowiak ◽

Aleksandra Obrępalska-Stęplowska

Keyword(s):

Apis Mellifera ◽

Agricultural Production ◽

Reference Genes ◽

Housekeeping Genes ◽

Chain Reaction ◽

Experimental Conditions ◽

Breeding Lines ◽

Lambda Cyhalothrin ◽

Polymerase Chain ◽

External Stimuli

Abstract Honeybees (Apis mellifera L.), which unquestionably play an economically important role in pollination and agricultural production, are at risk of decline. To study changes in gene expression in insects upon exposure to pesticides or other external stimuli, appropriate reference genes are required for data normalization. Since there is no such gene that is absolutely invariable under all experimental conditions, the aim of this study was to identify the most stable targets suitable for subsequent normalization in quantitative experiments based on real-time polymerase chain reaction in honeybee research. Here, we evaluated the expression of fifteen candidate housekeeping genes from three breeding lines of honeybees treated with pyrethroids to identify the most stable genes. The tested insects were exposed to deltamethrin or lambda-cyhalothrin, and then, changes in the accumulation of selected transcripts were assessed, followed by statistical analyses. We concluded that AmRPL32, AmACT and AmRPL13a were the commonly recorded most stable genes in honeybees treated with the selected pyrethroids.

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Detection of minimal residual disease in acute myelomonocytic leukemia with abnormal marrow eosinophils by nested polymerase chain reaction with allele specific amplification

Blood ◽

10.1182/blood.v84.7.2291.2291 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2291-2296 ◽

Cited By ~ 55

Author(s):

J Hebert ◽

JM Cayuela ◽

MT Daniel ◽

R Berger ◽

F Sigaux

Keyword(s):

Residual Disease ◽

Fusion Transcript ◽

Type A ◽

Chain Reaction ◽

Allele Specific ◽

Acute Myelomonocytic Leukemia ◽

Specific Amplification ◽

Polymerase Chain ◽

Myelomonocytic Leukemia ◽

Minimal Residual

Abstract Acute myelomonocytic leukemia with bone marrow eosinophilia (AML-M4Eo in the French-American-British FAB] classification) is frequently associated with pericentric inversion of chromosome 16, inv(16)(p13q22). Recently, the molecular cloning of teh breakpoints has led to the identification of the two fused genes, CBFB on 16q and MYH11 on 16p. We have analyzed 24 patients with AML-M4Eo at diagnosis and 47 patients with AML of other FAB subtypes, by a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the CBFB/MYH11 fusion mRNAs. Three types of fusion mRNAs were detected in 22 samples of AML- M4Eo (type A, n = 20; type C, n = 1; and type D, n = 1). Among these 22 positive samples, inv(16) was found in the 20 cytogenetically studied cases. No fusion transcript was detected in two patients with AML-M4Eo and in patients with other types of AML. These results confirm that CBFB/MYH11 transcripts (with a predominant type A form) are present in most cases of inv(16) AML. Moreover, detection of the hybrid transcript is closely associated with the finding of abnormal bone marrow (BM) eosinophils in AML-M4Eo as it is not found in other, FAB subtypes of AML, including AML-M4. To assess the presence of type A CBFB/MYH11 fusion transcripts in five AML-M4Eo patients in remission, we designed a sensitive assay combining nested PCR and allele-specific amplification (NPASA). Residual leukemia cells were detected in four patients who were in remission from 4 to 22 months, but not in one patient in long-term remission (5 years). The clinical relevance of persistent CBFB/MYH11 fusion transcripts in remission remains to be established by studying a large prospective series of patients. NPASA provides a useful and sensitive tool for the detection of minimal residual disease in inv(16) AML and, potentially, in other leukemias associated with translocations that result in a predominant fusion transcript.

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Cytoplasmic male sterility in Desmodium

Australian Journal of Agricultural Research ◽

10.1071/ar9690227 ◽

1969 ◽

Vol 20 (2) ◽

pp. 227 ◽

Cited By ~ 5

Author(s):

KS McWhirter

Keyword(s):

Cytoplasmic Male Sterility ◽

Male Sterility ◽

Interspecific Hybrid ◽

Pollen Fertility ◽

Pollen Sterility ◽

Hybrid Origin ◽

F1 Hybrids ◽

Male Sterile ◽

Genetic Stocks

A type of male sterility found in two Desmodium plants of probably interspecific hybrid origin was cytoplasmically inherited. The cytoplasmic male-sterile character was incorporated in the tropical legume Desmodium sandwicense by backcrossing. In this genetic background pollen sterility was complete. The male-sterile character was not graft-transmissible, and it produced no detectable pleiotropic effects on growth and development. Desmodium intortum gave restoration of pollen fertility in Fl hybrids with male-sterile lines of D. sandwicense. Restored F1 hybrids produced apparently normal pollen, but tests of functional ability of the pollen disclosed that pollen fertility was less than that of Fl hybrids with normal cytoplasm. Incomplete restoration of fertility was not due to heterozygosity of fertility-restoring genes with gametophytic expression, since fertility-restoring genes were shown to act sporophytically. The results established the occurrence in the legume Desmodium of a system of determination of the male-sterile, fertility-restored phenotypes that is similar to the cytoplasmic male sterility systems described in many other angiosperm plants. A scheme utilizing the genetic stocks produced in this study for commercial production of the interspecific hybrid D. sandwicense x D. intortum as a cultivar is presented.

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