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Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae).

ABSTRACT: UNLABELLED: PREMISE OF THE STUDY:Novel markers were developed for pea (Pisum sativum) from pea expressed sequence tags (ESTs) having significant homology to Medicago truncatula gene sequences to investigate genetic diversity, linkage mapping, and cross-species transferability. • METHODS AND RESULTS:Seventy-seven EST-derived genic markers were developed through comparative mapping between M. truncatula and P. sativum in which 75 markers produced PCR products and 33 were polymorphic among 16 pea genotypes. • CONCLUSIONS:The novel markers described here will be useful for future genetic studies of P. sativum; their amplification in lentil (Lens culinaris) demonstrates their potential for use in closely related species.

SUBMITTER: Jain S

PROVIDER: S-EPMC4103456 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae).

Jain Shalu S McPhee Kevin E KE

Applications in plant sciences 20131028 11

<h4>Unlabelled</h4><h4>Premise of the study</h4>Novel markers were developed for pea (Pisum sativum) from pea expressed sequence tags (ESTs) having significant homology to Medicago truncatula gene sequences to investigate genetic diversity, linkage mapping, and cross-species transferability. •<h4>Methods and results</h4>Seventy-seven EST-derived genic markers were developed through comparative mapping between M. truncatula and P. sativum in which 75 markers produced PCR products and 33 were poly ...[more]

PMID: 25202494

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Project description:BackgroundField pea (Pisum sativum L.) is among the prominent crops in the world as food and feed. There are relatively few simple sequence repeat (SSR) markers developed from expressed sequence tags (ESTs) in P. sativum.ResultsIn the present study, 15 new EST-SSR markers were developed from publicly available ESTs. These markers have successfully amplified their target loci across seven Pisum sativum subsp. sativum accessions. Eleven (73%) of these SSRs were trinucleotide repeats, two (13%) dinucleotide and two (13%) were hexanucleotide repeats. Across-taxa transferability of these new markers was also tested on other subspecies of Pisum as well as on P. fulvum, Vicia faba and Lens culinaris. In Pisum sativum subsp. sativum, 13 of the 15 markers were polymorphic and 12 of them subsequently used for genetic diversity analysis. Forty six accessions, of which 43 were from Ethiopia, were subjected to genetic diversity analysis using these newly developed markers. All accessions were represented by 12 individuals except two (NGB103816 and 237508) that were represented by 9 and 11 individuals, respectively. A total of 37 alleles were detected across all accessions. PS10 was the most polymorphic locus with six alleles, and the average number of alleles per locus over the 12 polymorphic loci was 3.1. Several rare and private alleles were also revealed. The most distinct accession (32048) had private alleles at three loci with 100% frequency.ConclusionThese newly developed EST-SSR primer-pairs successfully amplified expected loci in P. sativum subsp. sativum as well as in other subspecies of the genus Pisum and related genera. High levels of genetic variation were detected in field pea accessions from Ethiopia using these markers. This result implies the potential of the Ethiopian field pea gene pool for improvement of field peas in various desirable traits. In addition, these markers could be a valuable asset in resolving the inconsistency in the taxonomic status of the different subspecies of genus Pisum as well as for characterization of field pea accessions in different gene banks around the world for breeding and conservation purposes.

Project description:BackgroundProanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied.ResultsPAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. 'Courier' and 'Solido' PAs were primarily prodelphinidin-types, whereas the PAs from 'LAN3017' were mainly the procyanidin-type. The mean degree of polymerization of 'LAN3017' PAs was also higher than those from 'Courier' and 'Solido'. Next-generation sequencing of 'Courier' seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis.ConclusionSignificant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in development to be used as substrates for polymerization into PAs. Biochemically competent recombinant PsANR and PsLAR activities were consistent with the pea seed coat PA profile composed of both cis- and trans-flavan-3-ols. Since the expression of PsLAR in Arabidopsis did not alter the PA subunit profile (which is only comprised of cis-flavan-3-ols), it necessitates further investigation of in planta metabolic flux through PsLAR.

Project description:BackgroundEndophytic bacteria reside inside healthy plant tissues and provide several benefits to their host, and help them to tolerate various stresses. Aminocyclopropane-1-carboxylate deaminase (ACCD) production is one of the mechanisms by which these bacteria help the plant to survive under ethylene stress.ObjectivesThe main focus of this study was to isolate endophytic bacteria and effectively screen them for ACCD production. The selected isolate was identified and assessed for plant growth-promoting potential under pot conditions.Materials and methodsEndophytic bacteria were isolated from root nodules of Pisum sativum plants, grown in northern India (Haryana state). ACCD activity was initially screened on DF minimal salt medium with ACC as a sole nitrogen source. To narrow down the number of the isolates, another screening method was adopted using a modified medium containing indicator dyes along with ACC. The strain producing ACCD as well as a significant amount of Indole 3 acetic acid (IAA) was identified using 16S rDNA gene sequencing and amplification of acdS gene. Its ability to promote plant growth was evaluated under pot culture conditions.ResultsTwenty-six endophytic bacteria were isolated from nodules of P. sativum plants. Sixteen isolates showed growth on DF minimal salts medium supplemented with ACC along with negative control. On the modified medium containing indicator dyes, two isolates, PJN13 and PJN17, showed zones of the color gradient. The ACC deaminase activity was further confirmed by enzymatic assay. The strains PJN13 and PJN17 produced 160 and 130 µM of ?-ketobutyrate m.g-1 protein h-1, respectively. The IAA production in the strain PJN13 (79.04 ± 0.78 µg.mL -1) was significantly more than that in the strain PJN17 (38.36 ± 1.89 µg.mL-1). It could enhance pea plant growth parameters, including root and shoot length and fresh and dry weight from 1 to 4 times compare to the control (untreated pea plants) under pot conditions. The results of 16S rDNA amplification and sequencing showed that PJN13 has maximum similarity to Bacillus mojavensis, and the sequence submitted to GenBank under accession number MH298523. Also, a band about 800 bp was amplified for the acdS gene.ConclusionsThough Bacillus is known as a predominant non-rhizobial endophytic genus, however in the present study, a B. mojavensis Bacillus mojavensis PRN2 (MH298523) was reported for the first time as an endophyte from the nodules of pea plants. The isolated strain possesses ACC deaminase activity along with IAA production capability, and high potentials as PGPE (Plant growth-promoting endophyte) for plant growth, so it has potential to be used as biofertilizers in pea fields.

Dataset Information

Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae).

Publications

Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae).

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