Identification and Analysis of Differentially Expressed Genes During Seed Development Using Suppression Subtractive Hybridization (SSH) in Phaseolus vulgaris

Abstract

Interspecific hybridization in the genus Phaseolus, conducted to introgress desired traits into common bean (Phaseolus vulgaris L.), leads to the abortion of immature embryos, usually at early developmental stages. Little is known about the physiological responses of embryo dysfunction in P. vulgaris during the early stages of embryogenesis and the genes that are involved in these responses. Identification of the genes involved in Phaseolus embryogenesis may provide information that will help to understand the molecular basis of Phaseolus embryo dysfunction. To investigate the genes expressed during Phaseolus seed development, we constructed a suppression subtractive hybridization (SSH) library using cDNA from abortive seed development as the driver and those from normal seed development as tester. The differentially expressed cDNA fragments were identified by differential screening. We identified 72 unique ESTs of which we selected 12 candidates on the basis of their redundancy. These candidates were subjected to a validation procedure based on the study of their expression level by real-time PCR. Sequence analysis revealed that most of the differentially expressed genes are related to metabolism and regulation such as protein synthesis. Some genes also encoded transcription factors. These genes showed high mRNA transcript levels in seed tissues and little or no expression in other tissues (root, stem, flower, leaf, and cotyledon). Seven genes were chosen and their expression profile during seed development in P. vulgaris was analyzed by real-time PCR using RNA preparations originating from different seed development stages. This study revealed hitherto unknown genes putatively involved in dicotyledonous embryogenesis, which serve as a starting point for understanding Phaseolus embryogenesis.

Differentially Expressed Genes during Flowering and Grain Filling in Common Bean (Phaseolus vulgaris) Grown under Drought Stress Conditions

“…The four cDNA libraries constituted a genetic sample that was representative of the two plant genotypes and their developmental stages, as demonstrated by the high number of redundant sequences (≥5 reads; 63 %), which was almost twice as high as the number of unique sequences (37 %); this level of efficiency is needed to ensure that differentially expressed sequences are represented, which might be used to suggest profiles of expression in silico (Yu et al 2003). The identification of responsive genes by the subtraction library strategy was previously demonstrated (Zhou et al 2011;Abid et al 2012). In the common bean, Kavar et al (2008) identified 32 differentially expressed genes in eight genotypes of diverse origins and from different gene pools of the common bean; 18.75 % of those sequences shared similar functions with the 67 contigs identified in the present study.…”

“…The phases of seed development have been well established for some legumes (Weber et al 2005) and, more recently, significant advances are being achieved in understanding the genetic mechanisms of pathways related to seed development (Abid et al 2012;Jones and Vodkin 2013). In the present study, it was observed that the tolerant genotype responded to drought stress modulating different set of genes during the grain-filling stage; in addition, many subcategories related to metabolic processes, carbohydrate and secondary metabolic processes were identified.…”

mQTL-seq delineates functionally relevant candidate gene harbouring a major QTL regulating pod number in chickpea

“… 9 – 13 , 22 , 23 A pod number-regulating PPR gene identified in our study, showing >80% sequence conservation with Arabidopsis gene orthologue ( At1g52620 ), is reportedly involved in controlling growth and development-related traits (pollination and embryogenesis) in multiple crop plants, including Arabidopsis and Phaseolus . 52 , 53 Specifically, PPR gene family proteins have definite functional role in regulating proper pollen development by higher accumulation of transcripts in the mature pollen and efficient nuclear-organelle interactions to produce higher fertile seeds (embryogenesis) and pods in diverse plant species. 54 – 62 Accordingly, we observed a higher expression of PPR gene transcripts in the mature pollen and two pod developmental stages of high pod number-containing parental accessions and homozygous mapping individuals vis-a-vis low pod number accessions and mapping individuals in chickpea.…”

Differential expression analysis of transcripts related to oil metabolism in maturing seeds of Jatropha curcas L.

“…Here we report a suppression subtractive hybridization (SSH) based approach to identify differentially expressing genes in different maturation stages of J. curcas seeds that correspond to active oil metabolism. SSH is a powerful technique that enriches differentially expressing genes (Diatchenko et al 1996) and has been used to study seed development related genes in Phaseolus vulgaris (Abid et al 2012). In Brassica napus, differentially expressing genes in seeds of two near-isogenic lines having different oil content were investigated using SSH.…”