Genetic relationships of some Citrus genotypes based on the candidate iron chlorosis genes

Iron is one of the most important elements in plant mineral nutrition. Fe deficiency is a critical abiotic stress factor for Mediterranean citriculture; the development of marker-assisted selection for this trait would greatly enhance rootstock breeding. In this study, DNA sequencing and single-stranded conformation polymorphism (SSCP) analyses were performed to determine the allelic diversity of genes associated with tolerance to iron chlorosis in citrus. Two candidate iron chlorosis tolerance genes were selected from existing Citrus EST databases and Arabidopsis thaliana genome databases. Ferritin-3 chloroplast precursor and putative membrane transporter candidate gene sequences were used to define primers in conserved regions. Six citrus genotypes from the basic taxon of Citrus were used to identify polymorphic regions in the genes. Direct sequencing of the amplified DNA fragments from the candidate genes was performed, and single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified after sequence alignment. Based on the DNA sequencing analysis, a total of 6840 nucleotides of DNA were sequenced to identify SNPs and indels. In total, 263 SNPs and 15 indels were identified for both genes. We detected 38.45 SNPs and 2.19 indels for each 1000 b on average from the DNA sequencing results. New primers were designed in conserved areas flanking polymorphic ones for SSCP analysis. SSCP-PCR analysis was performed with 25 citrus genotypes. The neighbor-joining method was used for cluster analysis. Trifoliate genotypes and their hybrids (known to be sensitive to iron chlorosis) clustered together, whereas genotypes tolerant to iron chlorosis were more spread out on the dendrogram. Mandarins also showed high diversity for both genes according to SSCP results. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis. (Résumé d'auteur

Investigation of self incompatibility mechanism by using cDNA AFLP in clementine (Citrus clementina hort. ex tan).

TEZ8406Tez (Yüksek Lisans) -- Çukurova Üniversitesi, Adana, 2011.Kaynakça (s.59-64) var.xiii, 65 s. : res., rnk. ; 29 cm.In this study pollen-pistil interaction was investigated to understand which genes are involved in self-incompatibility mechanism of Clementine mandarins. Pollen tube growth of a Clementine variety, 'Algerian Tangerine Ranch Selection' (self-incompatible) was analyzed by using complementary DNA-amplified fragment length polymorphism technique to identify genes putatively involved selfincompatibility. The transcript profiles of unpollinated and self-pollinated (for 1, 2, 3, 4, 5, 6, 7th days) styles and stigmas of the Algerian Tangerine cultivar were ompared. DNA profiles showing transcriptional differences between the days of pollination were excised from the gels and directly sequenced. DNA sequencens were investigated by doing BLAST analysis on NCBI database. Some unigenes were determined from the results of directly DNA sequencing. Selected gene tags showed transcriptional differences between unpollinated and self-pollinated styles and stigmas during pollen germination and pollen tube elongation.Klemantin mandarinlerinde kendine uyuşmazlık mekanizmasında rol oynayan genleri tespit etmek amacıyla yapılan bu çalışmada cDNA AFLP yöntemi kullanılmıştır. Çalışmada kendine uyuşmaz olduğu bilinen 'Algerian Tangerine Ranch Selection' Klemantin mandarinine ait pistillerin stigma ve stil kısımları bitkisel materyal olarak kullanılmıştır. İlk olarak Klemantin mandarinine ait balon aşamasındaki çiçekler toplanmış ve çiçeklere ait erkek organlar 1 gün oda sıcaklığında ışık altında bekletilmiştir. Tozlanma için kullanılmaya hazır hale gelen polenler ile kendilemeler yapılmış ve ilgili pistiller yabancı tozlanma ve zararlanmalara karşı keseler ile kapatılmıştır. Kendilemelerden sonra 1, 2, 3, 4, 5, 6, ve 7. günlere ait pistil örnekleri ve endileme yapılmamış pistil örnekleri olmak üzere toplamda 8 farklı güne ait pistil örneklerinin stigma ve stil kısımlarından toplam RNA izolasyonları gerçekleştirilmiştir. Daha sonra bu RNA'lardan mRNA sentezi gerçekleştirilerek çift zincir cDNA sentezi yapılmıştır. Sentezlenen cDNA'lar, AFLP analizlerinde kullanılmıştır. cDNA-AFLP analizlerinde toplam 55 primer kombinasyonu denenmiş ve PCR ürünleri poliakrilamid jel içerisinde koşulmuştur. Jel üzerinde kendine uyuşmayan farklı seviyelerde ifadesi gerçekleşen DNA bant profilleri poliakrilamid jelden kesilmiş ve bu profillerin DNA dizi analizleri yapılmıştır. DNA dizileri elde edilen profillerin BLAST analizleri NCBI web sitesinde gerçekleştirilmiş ve kendine uyuşmazlık mekanizmasında rol oynayan olası aday genlerin varlığı tespit edilmiştir. Elde edilen aday genlere karşılık gelen proteinler ve bu proteinlerin fonksiyonları tartışılmıştır.Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi tarafından desteklenmiştir. Proje No: ZF2009YL69, TÜBİTAK Proje No:108O702

Anther culture studies in W. Murcott mandarin genotype

Obtaining haploid plants is one of the important tool that shorten the plant breeding process. Anther culture is one of the method used for obtaining haploid plants. Anther culture studies are carried out for several plant species. In the present study, flowers at different sizes of W. Murcott mandarin were collected at the blooming period. Stage of mononuclear microspore of anthers was determined according to method of acetocarmine. Mononuclear microspore stage of anthers was observed in flowers 4-5 mm in diameter. Anther culture experiments were carried out with 4-5 mm in diameter anthers. In this study, temperature pre-treatment was performed at 4˚C and 25˚C for two days. Surface sterilization was performed in flowers and anthers were cultured in nutrient medium N6. Anther culture assays were performed with the three different concentrations of TDZ (0 mg l-1 TDZ, 0.1 mg l-1 TDZ, 0.5 mg l-1 TDZ). Half of anthers were cultured in dark condition while the other half in 16 hours light, 8 hours dark climate room conditions. Anthers containing mononuclear microspore stage were cultured and, changes occurring in the anther explants were discussed in the article

Genetic relationships of some Citrus genotypes based on the candidate iron chlorosis genes

Iron is one of the most important elements in plant mineral nutrition. Fe deficiency is a critical abiotic stress factor for Mediterranean citriculture; the development of marker-assisted selection for this trait would greatly enhance rootstock breeding. In this study, DNA sequencing and single-stranded conformation polymorphism (SSCP) analyses were performed to determine the allelic diversity of genes associated with tolerance to iron chlorosis in citrus. Two candidate iron chlorosis tolerance genes were selected from existing Citrus EST databases and Arabidopsis thaliana genome databases. Ferritin-3 chloroplast precursor and putative membrane transporter candidate gene sequences were used to define primers in conserved regions. Six citrus genotypes from the basic taxon of Citrus were used to identify polymorphic regions in the genes. Direct sequencing of the amplified DNA fragments from the candidate genes was performed, and single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified after sequence alignment. Based on the DNA sequencing analysis, a total of 6840 nucleotides of DNA were sequenced to identify SNPs and indels. In total, 263 SNPs and 15 indels were identified for both genes. We detected 38.45 SNPs and 2.19 indels for each 1000 b on average from the DNA sequencing results. New primers were designed in conserved areas flanking polymorphic ones for SSCP analysis. SSCP-PCR analysis was performed with 25 citrus genotypes. The neighbor-joining method was used for cluster analysis. Trifoliate genotypes and their hybrids (known to be sensitive to iron chlorosis) clustered together, whereas genotypes tolerant to iron chlorosis were more spread out on the dendrogram. Mandarins also showed high diversity for both genes according to SSCP results. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis.Iron is one of the most important elements in plant mineral nutrition. Fe deficiency is a critical abiotic stress factor for Mediterranean citriculture; the development of marker-assisted selection for this trait would greatly enhance rootstock breeding. In this study, DNA sequencing and single-stranded conformation polymorphism (SSCP) analyses were performed to determine the allelic diversity of genes associated with tolerance to iron chlorosis in citrus. Two candidate iron chlorosis tolerance genes were selected from existing Citrus EST databases and Arabidopsis thaliana genome databases. Ferritin-3 chloroplast precursor and putative membrane transporter candidate gene sequences were used to define primers in conserved regions. Six citrus genotypes from the basic taxon of Citrus were used to identify polymorphic regions in the genes. Direct sequencing of the amplified DNA fragments from the candidate genes was performed, and single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified after sequence alignment. Based on the DNA sequencing analysis, a total of 6840 nucleotides of DNA were sequenced to identify SNPs and indels. In total, 263 SNPs and 15 indels were identified for both genes. We detected 38.45 SNPs and 2.19 indels for each 1000 b on average from the DNA sequencing results. New primers were designed in conserved areas flanking polymorphic ones for SSCP analysis. SSCP-PCR analysis was performed with 25 citrus genotypes. The neighbor-joining method was used for cluster analysis. Trifoliate genotypes and their hybrids (known to be sensitive to iron chlorosis) clustered together, whereas genotypes tolerant to iron chlorosis were more spread out on the dendrogram. Mandarins also showed high diversity for both genes according to SSCP results. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis

Anther culture studies in W. Murcott mandarin genotype

Haploid bitkilerin elde edilmesi, bitki ıslahında, ıslah sürecini kısaltan önemli yöntemlerden bir tanesidir. Haploid bitkilerin elde edilmesinde kullanılan yöntemlerden biri olan anter kültürü çalışmaları birçok bitki türünde yürütülmektedir. Bu çalışmada anterlerden haploid bitki elde etmek amacıyla W. Murcott mandarin çeşidinde, çiçeklenme dönemlerinde farklı boyutlardaki çiçekler toplanmış ve asetokarmin yöntemine göre anterler içerisindeki tek çekirdekli mikrospor aşaması belirlenmiştir. Çalışma sonucunda tek çekirdekli mikrospor aşamasının olduğu dönem çiçeklerin 4-5 mm çapında oldukları dönem olarak belirlenmiş ve bu boyuttaki çiçekler temin edilerek anter kültürü denemesi kurulmuştur. Çalışmada çiçeklere 25 ?C ve 4 ?C’de iki gün bekletilmesi şeklinde ön uygulama gerçekleştirilmiş ve ardından çiçeklerde yüzey sterilizasyonu uygulanarak anterler N6 besin ortamı içerisinde kültüre alınmışlardır. Anter kültürü denemelerinde 0 mg l-1 TDZ (Thidiazuron), 0.1 mg l -1 TDZ ve 0.5 mg l-1 TDZ olmak üzere TDZ’nin üç farklı konsantrasyonu denenmiş ve anterlerin yarısı karanlıkta, diğer yarısı ise 16 saat aydınlık, 8 saat karanlık iklim odası koşullarında kültüre alınmıştır. W. Murcott çeşidinde tek çekirdekli mikrospor aşamasında olan anterlerin kültüre alınması sonucunda yapılan incelemelerde anter eksplantlarında meydana gelen değişimler gözlemlenerek makalede tartışılmıştır.Obtaining haploid plants is one of the important tool that shorten the plant breeding process. Anther culture is one of the method used for obtaining haploid plants. Anther culture studies are carried out for several plant species. In the present study, flowers at different sizes of W. Murcott mandarin were collected at the blooming period. Stage of mononuclear microspore of anthers was determined according to method of acetocarmine. Mononuclear microspore stage of anthers was observed in flowers 4-5 mm in diameter. Anther culture experiments were carried out with 4-5 mm in diameter anthers. In this study, temperature pre-treatment was performed at 4 ̊C and 25 ̊C for two days. Surface sterilization was performed in flowers and anthers were cultured in nutrient medium N6. Anther culture assays were performed with the three different concentrations of TDZ (0 mg l-1 TDZ, 0.1 mg l-1 TDZ, 0.5 mg l-1 TDZ). Half of anthers were cultured in dark condition while the other half in 16 hours light, 8 hours dark climate room conditions. Anthers containing mononuclear microspore stage were cultured and, changes occurring in the anther explants were discussed in the article