Development of Molecular Marker Linked to Seed Hardness in Pomegranate Using Bulked Segregant Analysis.

The pomegranate (Punica granatum L.) is one of the fruit species with the oldest cultural history. There are many traits to determine the quality of pomegranate fruits. Among them, soft-seeded feature of pomegranate fruit is important trait for the market value of the fruit. For this reason, the demand for pomegranate varieties with soft seeds has been increasing, especially in recent years. In this study, molecular markers associated with seed hardness were developed to distinguish pomegranate cultivars with soft-seeded feature based on genomic DNA at the early stages of the pomegranate breeding process. For this purpose, pomegranate genotypes and/or cultivars from the population involved in reciprocal crosses of hard-seeded Ernar, medium-hard-seeded Hicaznar, and soft-seeded Fellahyemez cultivars were grouped as soft-seeded or hard-seeded. Further, leaf samples were collected from individuals belonging to each group. Then, the genomic DNA was isolated from each plant separately, and equal amount of genomic DNA from individuals with the similar seed hardness were mixed for bulked segregant analysis (BSA). The bulked genomic DNAs of opposite characters were analyzed by polymerase chain reaction (PCR) using random decamer primers to develop random amplified polymorphic DNA (RAPD) markers associated with soft-seeded or hard-seeded pomegranates. A total of three RAPD markers were determined to distinguish the individuals having soft- or hard-seeded pomegranate genotypes and/or cultivars. As a result of the comparison of the DNA sequences of these RAPD markers, insertion-deletions (inDels) primers were designed to developed and validate a PCR assay to distinguish the soft- and hard-seeded pomegranate genotypes/cultivars from each other. The molecular markers developed in this study will enable us to distinguish soft-seeded pomegranate types easily in a short time at the early stages of the pomegranate breeding programs.

Identification and Expression Analysis of Genes Induced in Response to Tomato chlorosis virus Infection in Tomato.

Tomato (Solanum lycopersicum) is one of the most widely grown and economically important vegetable crops in the world. Tomato chlorosis virus (ToCV) is one of the recently emerged viruses of tomato distributed worldwide. ToCV-tomato interaction was investigated at the molecular level for determining changes in the expression of tomato genes in response to ToCV infection in this study. A cDNA library enriched with genes induced in response to ToCV infection were constructed and 240 cDNAs were sequenced from this library. The macroarray analysis of 108 cDNAs revealed that the expression of 92 non-redundant tomato genes was induced by 1.5-fold or greater in response to ToCV infection. The majority of ToCV-induced genes identified in this study were associated with a variety of cellular functions including transcription, defense and defense signaling, metabolism, energy, transport facilitation, protein synthesis and fate and cellular biogenesis. Twenty ToCV-induced genes from different functional groups were selected and induction of 19 of these genes in response to ToCV infection was validated by RT-qPCR assay. Finally, the expression of 6 selected genes was analyzed in different stages of ToCV infection from 0 to 45 dpi. While the expression of three of these genes was only induced by ToCV infection, others were induced both by ToCV infection and wounding. The result showed that ToCV induced the basic defense response and activated the defense signaling in tomato plants at different stages of the infection. Functions of these defense related genes and their potential roles in disease development and resistance to ToCV are also discussed.

Development of a graft inoculation method and a real-time RT-PCR assay for monitoring Tomato chlorosis virus infection in tomato.

A graft inoculation method coupled with RT-qPCR was developed for monitoring ToCV infection in tomato plants. Ten seed-grown tomato seedlings were graft inoculated with phloem tissue-containing stem segments from a ToCV-infected tomato plants. Another group of tomato seedling were grafted with similar stem segments from a healthy tomato plant as mock inoculated control. The CP gene of ToCV was cloned under the control of T7 promoter and in vitro synthesized RNA was used as a standard for quantification. Total RNA was isolated from leaf samples of ToCV-inoculated and mock-inoculated control plants before the inoculation and 1-60 days post inoculation (dpi). The presence and the titer of ToCV were determined from all ToCV-inoculated or mock-inoculated control plants by RT-qPCR. After 15 dpi, ToCV was detected in 20-30% of graft-inoculated plants. The infection rate then increased progressively and reached to 70-80% by 60 dpi. Titer of ToCV was at the detectable level at 15 dpi and increased and reached to maximum level by 40 dpi and then started to decrease. The results showed that patch grafting is a simple and efficient method for experimental inoculation of ToCV and can be used as an alternative and/or complementary to vector transmission in the laboratories. The patch grafting could be combined with RT-qPCR and used for infecting and quantitative monitoring of ToCV or other phloem-limited viruses in tomato or in other plants.

Identification and expression analysis of early cold-induced genes from cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is one of the most economically important fruit crops growing in subtropical and tropical regions. Most commercially important Citrus varieties are susceptible to cold; therefore, low and freezing temperatures are the main limiting factors for citrus production in subtropical areas. Since Poncirus trifoliata (L.) Raf. is a cold-hardy, interfertile Citrus relative, it serves as a genetic resource for improving cold tolerance in cold sensitive commercial Citrus species. While gene induced in response to long-term cold acclimation was previously identified in Poncirus, early response of Poncirus to cold has not been explored in detail. To identify early cold-responsive genes, a subtractive cDNA library was constructed using 4-h cold-treated and untreated control Poncirus seedlings in this study. A total of 210 randomly picked clones from the subtracted library with cold-induced genes were sequenced. The sequences obtained from the majority of these clones shared homology with previously identified cold-induced and/or environmental stress-regulated genes in other plants. Reverse northern blot analysis of the expression of these cDNAs with cold-treated and untreated control probes revealed that expression of 64 cDNAs was increased two to 11 fold in response to 4-h cold treatment. While the majority of these genes were related with cell rescue, defense, cell death and aging, transcription, metabolism, protein fate, energy, cellular communication and signal transduction, transport facilitation and development, some of them did not show homology with genes with known functions. Individual expression analysis of nine selected genes by semi-quantitative RT-PCR using mRNA from cold-treated and untreated control plants confirmed that the expression of selected cDNAs was all induced in response to cold. The results demonstrated that although a few genes were commonly induced in response to both short and long-term cold acclimation in Poncirus, majority of early cold-responsive genes were different from previously identified late cold-responsive genes in Poncirus.

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as -26 degrees C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.

Two AP2 domain containing genes isolated from the cold-hardy Citrus relative Poncirus trifoliata are induced in response to cold.

Poncirus trifoliata (L.) Raf. is a cold-hardy, interfertile Citrus relative able to tolerate temperatures as low as -26°C when cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus varieties. A cold-induced cDNA library was constructed by subtractive hybridisation of non-acclimated and 2-d cold-acclimated P. trifoliata seedlings and many genes induced in response to cold were identified. Two of these cDNAs, PI-B05 and PI-C10, were selected from this library for further characterisation. Full-length cDNA sequences of these genes were obtained by 5' and 3' rapid amplification of cDNA ends (RACE). Sequence analysis revealed that PI-B05 contained an apetala2 / ethylene response factor (AP2 / ERF) domain and showed homology with ERF proteins from other plants, some of which are involved in environmental stress-induced gene expression. PI-C10 contained both AP2 / ERF and B3 DNA binding domains and showed homology with other plant proteins in the RAV subfamily of the AP2 / ERF transcription factors, some of which are induced in response to cold and other environmental stresses. Expression patterns of these genes in cold-tolerant P. trifoliata and cold-sensitive pummelo [Citrus grandis (L.) Osb.] in response to cold and drought at different time points were analysed by northern blots. Expression analysis showed that both genes were induced in response to cold, but not under drought conditions in cold-hardy P. trifoliata. However, little or no expression of these genes was detected by northern analysis in cold-sensitive pummelo under cold or drought conditions. The sequence analysis and expression data indicated that these genes may play a role in cold-responsive gene expression in cold-hardy P. trifoliata and could possibly be used for improving cold tolerance in cold-sensitive citrus cultivars.