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Mapping of important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut (Arachis hypogaea L.).

ABSTRACT: A mapping population of recombinant inbred lines (RILs) derived from TMV 2 and its mutant, TMV 2-NLM was employed for mapping important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut. Single nucleotide polymorphism and copy number variation using RAD-Sequencing data indicated very limited polymorphism between TMV 2 and TMV 2-NLM. But phenotypically they differed significantly for many taxonomic and productivity traits. Also, the RIL population showed significant variation for a few additional agronomic traits. A genetic linkage map of 1,205.66 cM was constructed using 91 genic and non-genic Arachis hypogaea transposable element (AhTE) markers. Using single marker analysis and QTL analysis, the markers with high phenotypic variance explained (PVE) were identified for branching pattern (32.3%), number of primary and secondary branches (19.9% and 28.4%, respectively), protein content (26.4%), days to 50% flowering (22.0%), content of oleic acid (15.1%), test weight (13.6%) and pod width (12.0%). Three genic markers (AhTE0357, AhTE0391, AhTE0025) with Arachis hypogaea miniature inverted-repeat transposable element (AhMITE1) activity in the genes Araip.TG1BL (B02 chromosome), Aradu.7N61X (A09 chromosome) and Aradu.7065G (A07 chromosome), respectively showed strong linkage with these taxonomic, productivity and quality traits. Since TMV 2 and TMV 2-NLM differed subtly at DNA level, the background noise in detecting the marker-trait associations was minimum; therefore, the markers identified in this study for the taxonomic and productivity traits may be significant and useful in peanut molecular breeding.

SUBMITTER: Hake AA

PROVIDER: S-EPMC5645101 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Mapping of important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut (Arachis hypogaea L.).

Hake Anil A AA Shirasawa Kenta K Yadawad Arati A Sukruth M M Patil Malagouda M Nayak Spurthi N SN Lingaraju S S Patil P V PV Nadaf H L HL Gowda M V C MVC Bhat R S RS

PloS one 20171017 10

A mapping population of recombinant inbred lines (RILs) derived from TMV 2 and its mutant, TMV 2-NLM was employed for mapping important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut. Single nucleotide polymorphism and copy number variation using RAD-Sequencing data indicated very limited polymorphism between TMV 2 and TMV 2-NLM. But phenotypically they differed significantly for many taxonomic and productivity traits. Also, the RIL population ...[more]

PMID: 29040293

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Project description:BackgroundAllergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut.ResultsTwo similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme.ConclusionsThis work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.

Project description:BACKGROUND:Microsatellites, or simple sequence repeats (SSRs), represent important DNA variations that are widely distributed across the entire plant genome and can be used to develop SSR markers, which can then be used to conduct genetic analyses and molecular breeding. Cultivated peanut (A. hypogaea L.), an important oil crop worldwide, is an allotetraploid (AABB, 2n = 4× = 40) plant species. Because of its complex genome, genomic marker development has been very challenging. However, sequencing of cultivated peanut genome allowed us to develop genomic markers and construct a high-density physical map. RESULTS:A total of 8,329,496 SSRs were identified, including 3,772,653, 4,414,961, and 141,882 SSRs that were distributed in subgenome A, B, and nine scaffolds, respectively. Based on the flanking sequences of the identified SSRs, a total of 973,984 newly developed SSR markers were developed in subgenome A (462,267), B (489,394), and nine scaffolds (22,323), with an average density of 392.45 markers per Mb. In silico PCR evaluation showed that an average of 88.32% of the SSR markers generated only one in silico-specific product in two tetraploid A. hypogaea varieties, Tifrunner and Shitouqi. A total of 39,599 common SSR markers were identified among the two A. hypogaea varieties and two progenitors, A. duranensis and A. ipaensis. Additionally, an amplification effectiveness of 44.15% was observed by real PCR validation. Moreover, a total of 1276 public SSR loci were integrated with the newly developed SSR markers. Finally, a previously known leaf spot quantitative trait locus (QTL), qLLS_T13_A05_7, was determined to be in a 1.448-Mb region on chromosome A05. In this region, a total of 819 newly developed SSR markers were located and 108 candidate genes were detected. CONCLUSIONS:The availability of these newly developed and public SSR markers both provide a large number of molecular markers that could potentially be used to enhance the process of trait genetic analyses and improve molecular breeding strategies for cultivated peanut.

Project description:BACKGROUND: Lack of sufficient molecular markers hinders current genetic research in peanuts (Arachis hypogaea L.). It is necessary to develop more molecular markers for potential use in peanut genetic research. With the development of peanut EST projects, a vast amount of available EST sequence data has been generated. These data offered an opportunity to identify SSR in ESTs by data mining. RESULTS: In this study, we investigated 24,238 ESTs for the identification and development of SSR markers. In total, 881 SSRs were identified from 780 SSR-containing unique ESTs. On an average, one SSR was found per 7.3 kb of EST sequence with tri-nucleotide motifs (63.9%) being the most abundant followed by di- (32.7%), tetra- (1.7%), hexa- (1.0%) and penta-nucleotide (0.7%) repeat types. The top six motifs included AG/TC (27.7%), AAG/TTC (17.4%), AAT/TTA (11.9%), ACC/TGG (7.72%), ACT/TGA (7.26%) and AT/TA (6.3%). Based on the 780 SSR-containing ESTs, a total of 290 primer pairs were successfully designed and used for validation of the amplification and assessment of the polymorphism among 22 genotypes of cultivated peanuts and 16 accessions of wild species. The results showed that 251 primer pairs yielded amplification products, of which 26 and 221 primer pairs exhibited polymorphism among the cultivated and wild species examined, respectively. Two to four alleles were found in cultivated peanuts, while 3-8 alleles presented in wild species. The apparent broad polymorphism was further confirmed by cloning and sequencing of amplified alleles. Sequence analysis of selected amplified alleles revealed that allelic diversity could be attributed mainly to differences in repeat type and length in the microsatellite regions. In addition, a few single base mutations were observed in the microsatellite flanking regions. CONCLUSION: This study gives an insight into the frequency, type and distribution of peanut EST-SSRs and demonstrates successful development of EST-SSR markers in cultivated peanut. These EST-SSR markers could enrich the current resource of molecular markers for the peanut community and would be useful for qualitative and quantitative trait mapping, marker-assisted selection, and genetic diversity studies in cultivated peanut as well as related Arachis species. All of the 251 working primer pairs with names, motifs, repeat types, primer sequences, and alleles tested in cultivated and wild species are listed in Additional File 1.

Dataset Information

Mapping of important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut (Arachis hypogaea L.).

Publications

Mapping of important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut (Arachis hypogaea L.).

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