Reference gene selection for qRT-PCR analysis of flower development in <i>Lagerstroemia indica</i> and <i>L</i>. <i>speciosa</i>

<div><p>Quantitative real-time polymerase chain reaction (qRT-PCR) is a prevalent method for gene expression analysis, depending on the stability of the reference genes for data normalization. <i>Lagerstroemia indica</i> and <i>L</i>. <i>speciosa</i> are popular ornamental plants which are famous for the long flowering period. However, no systematic studies on reference genes in <i>Lagerstroemia</i> have yet been conducted. In the present study, we selected nine candidate reference genes (<i>GAPDH</i>, <i>TUA</i>, <i>TUB</i>, <i>18S</i>, <i>RPII</i>, <i>EF-1α</i>, <i>ATC</i>, <i>EIF5A</i> and <i>CYP</i>) and evaluated their expression stability in different tissues during floral development of <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i> using four algorithms (geNorm, NormFinder, BestKeeper and, RefFinder). Results showed that <i>RPII</i> and <i>EF-1α</i> were the most stably expressed and suitable reference genes for both of <i>Lagerstroemia</i> species. Moreover, <i>ACT</i> exhibited high expression stability in <i>L</i>. <i>indica</i> and <i>GAPDH</i> was a suitable reference gene for <i>L</i>. <i>speciosa</i> in different flower development stages. <i>TUB</i> was an unsuitable reference gene for gene expression normalization due to significant variations in expression across all samples. Finally, we verified the reliability of the selected candidate reference genes by amplifying an <i>AGAMOUS</i> homolog (<i>LsAG1</i>) of <i>Arabidopsis thaliana</i>. This study provides a list of suitable reference genes, thereby broadening the genetic basis of the gene expression patterns in <i>Lagerstroemia</i> species.</p></div

Genes and primer sets used for qRT-PCR in <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i>.

<p>Genes and primer sets used for qRT-PCR in <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i>.</p

Relative expression level of <i>LsAG1</i>.

<p>(<b>A</b>) <i>L</i>. <i>indica</i>, (<b>B</b>) <i>L</i>. <i>speciosa</i>. Se = Sepal, Pe = Petal, Sta = Stamen, Pi = Pistil, S1 = Stage 1, S2 = Stage 2, S3 = Stage3, S4 = Stage 4.</p

Expression levels of candidate reference genes across all samples of <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i> at different flower development stages.

<p>The lines across the box are the medians, the boxes depicts the 25/75 percentiles, the whiskers represent the 95% confidence intervals, and the dots are outliers.</p

Expression stability of the reference gene calculated by Ref-finder for <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i>.

<p>Expression stability of the reference gene calculated by Ref-finder for <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i>.</p

Flower samples and developmental stage of <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i>.

<p><b>A</b> = <i>L</i>. <i>indica</i>; <b>B</b> = <i>L</i>. <i>speciosa</i>; Se = Sepal; Pe = Petal; Sta = Stamen; Pi = Pistil; S1 = Stage 1; S2 = Stage 2; S3 = Stage3; S4 = Stage 4.</p

Expression stability of the reference gene calculated by NormFinder for <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i>.

<p>Expression stability of the reference gene calculated by NormFinder for <i>L</i>. <i>indica</i> and <i>L</i>. <i>speciosa</i>.</p

Plant materials used in this study.

<p>(a) Female parent, (b) male parent, (c) non-dwarf phenotype in the F₁ population, (d) dwarf phenotype in the F₁ population, and (e) two contrasting phenotypes in the BC₁ population.</p

Testing the SNP markers in a set of 28 <i>Lagerstroemia</i> stocks with diverse plant architectures.

<p>Testing the SNP markers in a set of 28 <i>Lagerstroemia</i> stocks with diverse plant architectures.</p

Primer sequences of the AS-PCR and the SNP loci.

<p>Primer sequences of the AS-PCR and the SNP loci.</p