Development and Validation of Kompetitive Allele-Specific PCR Assays for Erucic Acid Content in Indian Mustard [Brassica juncea (L.) Czern and Coss.]

Brassica juncea L. is the most widely cultivated oilseed crop in Indian subcontinent. Its seeds contain oil with very high concentration of erucic acid (≈50%). Of late, there is increasing emphasis on the development of low erucic acid varieties because of reported association of the consumption of high erucic acid oil with cardiac lipidosis. Erucic acid is synthesized from oleic acid by an elongation process involving two cycles of four sequential steps. Of which, the first step is catalyzed by β-ketoacyl-CoA synthase (KCS) encoded by the fatty acid elongase 1 (FAE1) gene in Brassica. Mutations in the coding region of the FAE1 lead to the loss of KCS activity and consequently a drastic reduction of erucic acid in the seeds. Molecular markers have been developed on the basis of variation available in the coding or promoter region(s) of the FAE1. However, majority of these markers are not breeder friendly and are rarely used in the breeding programs. Present studies were planned to develop robust kompetitive allele-specific PCR (KASPar) assays with high throughput and economics of scale. We first cloned and sequenced FAE1.1 and FAE1.2 from high and low erucic acid (<2%) genotypes of B. juncea (AABB) and its progenitor species, B. rapa (AA) and B. nigra (BB). Sequence comparisons of FAE1.1 and FAE1.2 genes for low and high erucic acid genotypes revealed single nucleotide polymorphisms (SNPs) at 8 and 3 positions. Of these, three SNPs for FAE1.1 and one SNPs for FAE1.2 produced missense mutations, leading to amino acid modifications and inactivation of KCS enzyme. We used SNPs at positions 735 and 1,476 for genes FAE1.1 and FAE1.2, respectively, to develop KASPar assays. These markers were validated on a collection of diverse genotypes and a segregating backcross progeny. KASPar assays developed in this study will be useful for marker-assisted breeding, as these can track recessive alleles in their heterozygous state with high reproducibility.

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The inheritance of erucic acid content in Ethiopian mustard

Canadian Journal of Plant Science ◽

10.4141/p96-074 ◽

1997 ◽

Vol 77 (1) ◽

pp. 33-41 ◽

Cited By ~ 19

Author(s):

A. Getinet ◽

G. Rakow ◽

J. P. Raney ◽

R. K. Downey

Keyword(s):

Erucic Acid ◽

Acid Content ◽

Acid Synthesis ◽

Erucic Acid Content ◽

Human Consumption ◽

Oilseed Crop ◽

High Erucic Acid ◽

Ethiopian Mustard ◽

Low Erucic Acid ◽

Key Words:Brassica

Ethiopian mustard (Brassica carinata A. Braun) is a highly productive oilseed crop in the central highlands of Ethiopia. Cultivars currently in production in Ethiopia produce seed which contains 35–40% erucic acid in its oil which is undesirable for human consumption. Zero erucic acid B. carinata has recently been developed. The objective of this study was to investigate the inheritance of erucic acid in progeny of crosses between the high erucic acid cultivars Dodolla and S-67 with the zero erucic acid line C90-14. The erucic acid content of F1 seed born on either the high or low erucic acid parents was intermediate between the parents indicating embryonic control of erucic acid content in B. carinata. Erucic acid contents of backcross seed derived from the backcross to the zero erucic acid parent segregated into three classes with <0.5%, 6–16% and >16% erucic acid at a ratio of 1:2:1 and F2 seed segregated into five classes with a ratio of 1:4:6:4:1. These segregation patterns indicated that erucic acid in B. carinata was controlled by two genes acting in an additive manner with each locus contributing about 10% erucic acid. It was concluded that the B and C genomes of B. carinata each carry one gene for erucic acid synthesis. The knowledge of the inheritance of erucic acid in B. carinata will assist in the development of zero erucic acid B. carinata cultivars. Key words:Brassica carinata, erucic acid, inheritance

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In Vitro Production of Somaclones with Decreased Erucic Acid Content in Indian Mustard [Brassica juncea (Linn.) Czern&Coss]

Plants ◽

10.3390/plants10071297 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1297

Author(s):

Chitralekha Shyam ◽

Manoj Tripathi ◽

Sushma Tiwari ◽

Niraj Tripathi ◽

Ravindra Solanki ◽

...

Keyword(s):

Erucic Acid ◽

Acid Content ◽

Indian Mustard ◽

Mother Plant ◽

Erucic Acid Content ◽

Oilseed Crop ◽

In Vitro Production ◽

Somaclonal Variations ◽

Low Erucic Acid

Brassica junceais a crucial cultivated mustard species and principal oilseed crop of India and Madhya Pradesh, grown for diverse vegetables, condiments, and oilseeds. Somaclonal variation was explored as a probable source of additional variability for the manipulation of fatty acids, especially low erucic acid contents that may be valuable for this commercially important plant species. The plantlets regenerated from tissue cultures (R0), their R1 generation and respective parental lines were compared for morpho-physiological traits and fatty acid profile for the probable existence of somaclonal variations. The first putative somaclone derived from genotype CS54 contained 5.48% and 5.52% erucic acid in R0 and R1 regenerants, respectively, compared to the mother plant (41.36%). In comparison, the second somaclone acquired from PM30 exhibited a complete absence of erucic acid corresponding to its mother plant (1.07%). These putative somaclones present a source of variation for exploitation in the development of future mustard crops with low erucic acid content.

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GLC analysis of Indian rapeseed-mustard to study the variability of fatty acid composition

Biochemical Society Transactions ◽

10.1042/bst0280581 ◽

2000 ◽

Vol 28 (6) ◽

pp. 581-582 ◽

Cited By ~ 21

Author(s):

N. Kaushik ◽

A. Agnihotri

Keyword(s):

Fatty Acid Composition ◽

Oleic Acid ◽

Fatty Acid ◽

Erucic Acid ◽

Linolenic Acid ◽

Acid Composition ◽

Acid Content ◽

Erucic Acid Content ◽

High Erucic Acid ◽

Low Erucic Acid

Rapeseed-mustard is one of the most economically important oilseed crops in India. Speciality oils having high amounts of a specific fatty acid are of immense importance for both nutritional and industrial purposes. Oil high in oleic acid has demand in commercial food-service applications due to a long shelf-life and cholesterol-reducing properties. Both linoleic and linolenic acids are essential fatty acids; however, less than 3% linolenic acid is preferred for oil stability. High erucic acid content is beneficial for the polymer industry, whereas low erucic acid is recommended for food purposes. Therefore, it is important to undertake systematic characterization of the available gene pool for its variable fatty acid profile to be utilized for specific purposes. In the present study the Indian rapeseed-mustard germplasm and some newly developed low-erucic-acid strains were analysed by GLC to study the fatty acid composition in these lines. The GLC analysis revealed that the rapeseed-mustard varieties being commonly grown in India are characterized by high erucic acid content (30–51%) in the oil with low levels of oleic acid (13–23%). However, from among the recently developed low-erucic-acid strains, several lines were identified with comparatively high oleic acid (60–70%), moderate to high linoleic acid (13–40%) and low linolenic acid (< 10%) contents. Work is in progress at TERI (New Delhi, India) to utilize these lines for development of strains with particular fatty acid compositions for specific purposes.

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Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650362 ◽

1996 ◽

Vol 75 (05) ◽

pp. 757-759 ◽

Cited By ~ 11

Author(s):

Rainer Blasczyk ◽

Markus Ritter ◽

Christian Thiede ◽

Jenny Wehling ◽

Günter Hintz ◽

...

Keyword(s):

Direct Sequencing ◽

Activated Protein C ◽

Factor V Leiden ◽

Pcr Amplification ◽

Factor V ◽

Specific Pcr ◽

Pcr Rflp ◽

Allele Specific ◽

Specific Amplification ◽

Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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MUPrimer : a tool for finding allele specific PCR-primers for homologous gene sequences

10.32469/10355/6660 ◽

2009 ◽

Author(s):

M. Mursaleen Ahmad

Keyword(s):

Pcr Primers ◽

Homologous Gene ◽

Gene Sequences ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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FAE1 Sequence Characteristics and Its Relationship with Erucic Acid Content in Brassica juncea

ACTA AGRONOMICA SINICA ◽

10.3724/sp.j.1006.2010.00794 ◽

2010 ◽

Vol 36 (5) ◽

pp. 794-800 ◽

Cited By ~ 3

Author(s):

Ai-Xia XU ◽

Zhen HUANG ◽

Chao-Zhi MA ◽

En-Shi XIAO ◽

Xiu-Sen ZHANG ◽

...

Keyword(s):

Erucic Acid ◽

Brassica Juncea ◽

Acid Content ◽

Erucic Acid Content ◽

Sequence Characteristics

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Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

BMC Plant Biology ◽

10.1186/s12870-021-02932-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Zhengjie Chen ◽

Dengguo Tang ◽

Jixing Ni ◽

Peng Li ◽

Le Wang ◽

...

Keyword(s):

Marker Assisted Selection ◽

Inbred Lines ◽

Average Density ◽

Snp Markers ◽

Data Sets ◽

Rna Seq ◽

Specific Pcr ◽

Maize Inbred Lines ◽

Allele Specific ◽

Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

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Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Agronomy ◽

10.3390/agronomy11050982 ◽

2021 ◽

Vol 11 (5) ◽

pp. 982

Author(s):

Zhiliang Xiao ◽

Congcong Kong ◽

Fengqing Han ◽

Limei Yang ◽

Mu Zhuang ◽

...

Keyword(s):

Molecular Markers ◽

Brassica Oleracea ◽

Rapid Identification ◽

Inbred Lines ◽

Hybrid Lethality ◽

Specific Pcr ◽

Allele Specific ◽

User Friendly ◽

Allele Specific Pcr ◽

Kasp Marker

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

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