Development and Characterization of Simple Sequence Repeat (SSR) Markers Based on RNA-Sequencing of <i>Medicago sativa</i> and <i>In silico</i> Mapping onto the <i>M. truncatula</i> Genome

<div><p>Sufficient codominant genetic markers are needed for various genetic investigations in alfalfa since the species is an outcrossing autotetraploid. With the newly developed next generation sequencing technology, a large amount of transcribed sequences of alfalfa have been generated and are available for identifying SSR markers by data mining. A total of 54,278 alfalfa non-redundant unigenes were assembled through the Illumina HiSeqTM 2000 sequencing technology. Based on 3,903 unigene sequences, 4,493 SSRs were identified. Tri-nucleotide repeats (56.71%) were the most abundant motif class while AG/CT (21.7%), AGG/CCT (19.8%), AAC/GTT (10.3%), ATC/ATG (8.8%), and ACC/GGT (6.3%) were the subsequent top five nucleotide repeat motifs. Eight hundred and thirty- seven EST-SSR primer pairs were successfully designed. Of these, 527 (63%) primer pairs yielded clear and scored PCR products and 372 (70.6%) exhibited polymorphisms. High transferability was observed for ssp <i>falcata</i> at 99.2% (523) and 71.7% (378) in <i>M. truncatula</i>. In addition, 313 of 527 SSR marker sequences were <i>in silico</i> mapped onto the eight <i>M. truncatula</i> chromosomes. Thirty-six polymorphic SSR primer pairs were used in the genetic relatedness analysis of 30 Chinese alfalfa cultivated accessions generating a total of 199 scored alleles. The mean observed heterozygosity and polymorphic information content were 0.767 and 0.635, respectively. The codominant markers not only enriched the current resources of molecular markers in alfalfa, but also would facilitate targeted investigations in marker-trait association, QTL mapping, and genetic diversity analysis in alfalfa.</p></div

The evaluation of microsatellite primer pairs for different repeat classes.

<p>The evaluation of microsatellite primer pairs for different repeat classes.</p

Summary frequencies of different SSR repeat motif types related to variation of repeat unit numbers in alfalfa EST-SSR loci.

<p>Summary frequencies of different SSR repeat motif types related to variation of repeat unit numbers in alfalfa EST-SSR loci.</p

Summary of EST-SSR found in alfalfa.

<p>Summary of EST-SSR found in alfalfa.</p

Characterization of 30 alfalfa cultivars by 36 EST-SSR markers.

<p>Note: A (Allelic richness), Ho (Observed heterozygosity), He (Expected heterozygosity), PIC (Polymorphism information content.</p>b<p>expected heterozygosity assuming random mating and random chromosomal segregation (He), assuming random mating and non random chromatid segregation (He(ce)), i.e..</p>a<p>double reduction frequency of a = 1/7 (Wrike and Weber 1986) (He(cd)).</p

<i>β</i>-galactosidase activity of the P<i>exsA</i>-<i>lacZ</i> transcriptional fusion in the wide-type PAO1 strain, Δ<i>miaB</i>, and Δ<i>miaB</i> with <i>in trans</i> expression of <i>miaB</i>.

ns, not significant compared to the wild-type PAO1 strain based on Student’s t test. (TIF)</p

Disruption of the spermidine transporter by deleting <i>spuE</i> led to repressed expression of <i>miaB</i> and T3SS genes.

(A) Expression of spuE and miaB in the strains of ΔmiaB and Δvfr relative to that in the wild-type PAO1 strain was measured by RT-qPCR, respectively. (B) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, ΔspuE, and ΔspuE with in trans expression of miaB. (C) Productions of the intracellular ExsA and extracellular ExoS proteins measured by western blot in the wide-type PAO1 strain, ΔspuE, and ΔspuE with in trans expression of miaB. (D) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1, ΔmiaB, ΔspuE, and ΔmiaBΔspuE strains. (E) Productions of the intracellular ExsA and extracellular ExoS proteins measured by western blot in the wide-type PAO1, ΔmiaB, ΔspuE, and ΔmiaBΔspuE strains. ns, not significant; **, P P t test.</p

Relative expression of <i>gacS</i> and <i>rsmA</i> in the wild-type PAO1 and Δ<i>miaB</i> mutant measured by RT-qPCR.

ns, not significant compared to the wild-type PAO1 strain based on Student’s t test. (TIF)</p

A diagram showing the MiaB-mediated regulation of T3SS in <i>P</i>. <i>aeruginosa</i>.

The adenosine tRNA methylthiotransferase MiaB was upregulated by the cAMP-dependent regulator Vfr and the spermidine transporter-dependent pathway. MiaB independently repressed the expression of ladS, gacA, rsmY and rsmZ in the LadS-Gac/Rsm signaling pathway, which released RsmA to activate the transcription of exsCEBA and T3SS gene expression through the intrinsic regulator ExsA. Solid lines indicate direct regulations while dashed lines indicate probable indirect regulations. Arrows and T-shaped symbols indicate upregulation (activation) and repression, respectively.</p

LadS was involved in the MiaB-mediated regulation of T3SS gene expression.

(A) Relative fluorescence intensity of the pLadS-gfp transcriptional fusion measured in the wild-type PAO1 strain and the ΔmiaB mutant. (B) Relative fluorescence intensity of the pRetS-gfp transcriptional fusion measured in the wild-type PAO1 strain and the ΔmiaB mutant. (C) Relative expression of the ladS gene in the wild-type PAO1 and ΔmiaB mutant measured by RT-qPCR. (D) Relative fluorescence intensity of the pLadS-gfp transcriptional fusion measured in the wild-type PAO1 strain and the ΔmiaB mutant when cell growth at the OD600 of 0.5, 1.0, 1.5 and 2.0. (E) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion in the wide-type PAO1 strain, ΔmiaB, ΔladS and the ΔmiaBΔladS mutants as well as the ΔmiaBΔladS mutant with in trans expression of miaB and ladS. (F) Production of the ExoS protein measured by western blot in the wide-type PAO1 strain, ΔmiaB, ΔladS and the ΔmiaBΔladS mutants as well as the ΔmiaBΔladS mutant with in trans expression of miaB and ladS. ns, not significant; *, P P P t test.</p