Galli Vanessa, Borowski Joyce Moura, Perin Ellen Cristina, Messias Rafael da Silva, Labonde Julia, Pereira Ivan dos Santos, Silva Sérgio Delmar Dos Anjos, Rombaldi Cesar Valmor
Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil; Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil.
Embrapa Clima Temperado, Rodovia BR 396, Km 78 Caixa Postal 403, CEP 96001-970 Pelotas, RS, Brazil; Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Caixa Postal 354, CEP 96010-900 Pelotas, RS, Brazil.
Gene. 2015 Jan 10;554(2):205-14. doi: 10.1016/j.gene.2014.10.049. Epub 2014 Oct 29.
The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts.
对草莓（Fragaria×ananassa Duch）果实需求的不断增加，主要与其感官特性和抗氧化化合物含量有关。然而，草莓生产因其对非生物胁迫敏感而受到阻碍。因此，了解突出应激反应的分子机制对于采用基因工程方法提高草莓耐受性至关重要。然而，研究草莓基因的表达需要使用合适的内参基因。在本研究中，利用RefFinder对七个传统和新型候选内参基因进行了评估，以用于十个草莓品种果实和两种非生物胁迫下的转录本标准化，RefFinder整合了目前可用的四个主要软件程序：geNorm、NormFinder、BestKeeper和比较ΔCt法。结果表明，表达稳定性取决于实验条件。候选内参基因DBP（DNA结合蛋白）被认为是在草莓品种样本和干旱胁迫条件下标准化表达数据最合适的基因，而候选内参基因HISTH4（组蛋白H4）在渗透胁迫和盐胁迫下最稳定。传统基因GAPDH（甘油醛-3-磷酸脱氢酶）和18S（18S核糖体RNA）在所有条件下被认为是最不稳定的基因。利用苯丙氨酸解氨酶（PAL）和9-顺式环氧类胡萝卜素双加氧酶（NCED1）基因的表达进一步证实了经过验证的候选内参基因，表明使用不合适的内参基因可能会导致错误结果。本研究是首次对草莓品种和渗透胁迫下内参基因的稳定性进行调查，并为未来育种工作获得更准确的RT-qPCR结果提供了指导。
