Cytogenetics Of Citrus Somatic Hybrids And Fish Mapping Of CTV Resistance And Other Genes In Poncirus Trifoliata

Citrus allotetraploid somatic hybrids are valuable resources for rootstock and cultivar improvement. In situ hybridization (ISH) technique plays an impressive role in molecular cytogentics research for differentiating parental genome of hybrids and physical mapping of genes on chromosome. In this study, the meiotic chromosomal behavior in pollen mother cells (PMCs) of citrus somatic hybrids and their corresponding fusion parents was investigated, and their pollen viability was evaluated. The experimental procedure of GISH was optimized in citrus to analyse the nuclear genome of intergeneric sexual and somatic hybrids. Meanwhile, CTV resistance gene, two rDNA genes, retrotransposons copia-like gene and polygalacturcaase (PG) gene were mapped on chromosomes of trifoliate orange respectively using FISH method. The main results were as follows:1 Microsporogenesis of six citrus interspecific and intergeneric somatic hybrids was examined and compared with their corresponding five diploid fusion parents. Meiotic analysis revealed that the chromosome behavior of meiosis was more complicated in most citrus allotetraploid somatic hybrid plants than their diploid parents. The conformations of chromosome pairing were multiplicate with high frequency of univalents and multi-valents, and abnormal chromosome associations, such as chromosomes orientated away from the equatorial plate at metaphase Ⅰ or metaphase Ⅱ, chromosome bridges and lagging chromosomes at anaphase Ⅰ or anaphase Ⅱ, were frequently observed in somatic hybrids. Whereas, meiosis of allotetraploid somatic hybrid between 'Cleopatra' mandarin and Argentine trifoliate (Citrus reticulata + Poncirus trifoliata) was normal with few meiotic abnormalities. Bivalent was the main conformation of homologous chromosome pairing, which was similar with chromosomal behavior of diploid. In addition, more meiotic abnormalities occurred in diploid cybrid between 'Page' tangelo and Rough lemon [(C. reticulata × C. paradisi) + C. jambhiri] than in their parents.2 Pollen fertility of eight citrus somatic hybrids and six parents was analysed through investigating the frequencies of normal tetrads, micropollen, pollen stainability and pollengerminability. The results showed that the micropollen percentage of citrus allotetraploid somatic hybrid was higher than that of their parents evidently and pollen germinability was intermediate between their corresponding fusion parents. Pollen germinability of allohexaploid between'Jincheng' orange + HR [C. sinensis -*- (C. sinensis + C. jambhiri)] and diploid cybrid 'Page' tangelo + Rough lemon [(C reticulata * C. paradisi) + C. jambhiri] was 3.7% and 6.8% respectively, lower than their parents.3 GISH technique was modified and optimized for determination of parental chromosome number and position of intergeneric hybrids in citrus. Flame drying slide after enzyme digestion of cell wall is used for Citrus chromosome slide preparation and resulted in clear, well-dispersed chromosomes with less debris. With probe DNA labeled by nick translation, dot blotting to evaluate the labeling efficiency, blocking DNA broken by boiling and being 50 times the amount of probe DNA, two citrus intergeneric sexual hybrids were successfully identified by GISH method.4 With GISH method, homology degree of trifoliate orange with sweet orange was analysed by using genomic DNA of trifoliate orange (P. trifoliata) as the probe to hybridize the metaphase chromosomes of sweet orange (C. sinensis). The results indicated that five pair chromosomes of sweet orange were highly homologous to the genome of trifoliate orange.5 Four intergeneric citrus allotetraploid somatic hybrids were analysed by GISH technique. GISH confirmed that most symmetric somatic hybrid plant have both parents' chromosomes, half to half. Moreover, chromosome translocations were observed in one plant of somatic hybrid 'Goutou' sour orange + trifoliate orange (C. aurantium + P. trifoliata), which revealed that parental genome rearrangement behavior had occurred after somatic hybridization. In addition, a few triploid somatic cells were observed in somatic hybrid between 'Hongju' tangerine and trifoliate orange (C. reticulata + P. trifoliata); GISH method detected that a set of chromosomes from 'Hongju' tangerine was lost, as resulted in triploid cells.6 Cot—1 DNA was extracted successfully from trifoliate orange for blocking in BAC-FISH technique. The distribution of Cot— 1 DNA on genome of trifoliate orange was studied via using Cot—1 DNA as the probe to hybridize trifoliate orange metaphase chromosomes. The results illuminated that Cot—1 DNA located on all the chromosomes throughout, and mainly on the terminal region of chromosomes; as implied that sequence repeats in heterochromatic zone were dominant components of Cot— 1 DNA.7 Chromosomal mapping of CTV resistance gene in trifoliate orange was performed with TAC, BAC clone containing CTV resistance candidate gene and plasmid pt8 as probes respectively. FISH results showed that CTV resistance gene was mapped on the long arm of chromosome 3, and the percentage distance from centromere tc signal site was about 87.8 + 2.2. The detection rate of signals by FISH with TAC or BAC clone containing large insert as probe was much higher than that with plasmid containing small insert. The detection rate of mix-labeled TAC and BAC clone was the highest. At the same time, the use of Cot— 1 DNA as blocking is necessary in FISH technique when TAC or BAC clone is used as the probe.8 Two fragments of rDNA gene including 45 S and 5 S rDNA were amplified by PCR from trifoliate orange genomic DNA. They were used as probes to detect and locate rDNA loci on trifoliate orange metaphase chromosomes by FISH. Six sites of 45 S rDNA were found in trifoliate orange displaying three pair nucleolus organization regions (NORs). 5 S rDNA gene was located at two sites on the short arms of homologous chromosome 4.9 Physical mapping of retrotransposons copia-like gene on trifoliate orange chromosomes by FISH suggested that retrotransposons, copia-like gene presented clustering distribution on telomere regions of 11 chromosomes. Out of them, copy number of retrotransposons copia-like gene was greatly high on telomere regions on six chromosomes of the genome, and moderate on other three ones, and the least at the rest two.10 From Cara Cara navel orange (C. sinensis), two cDNA fragments and two genomic DNA fragments of polygalacturonase (PG) gene were amplified by PCR respectively, and sequenced. The cDNA fragments were 574 bp and 650 bp, and corresponding genomic DNA fragments were 775 bp and 1, 818 bp. The amino sequences coded by cDNA sequences of PG gene were 80-93% homologous to that of the correspongding regions in other plants. FISH mapping of PG gene on trifoliate orange chromosomes displayed that this gene was located on the long arm of chromosome 5, and the percentage distance from centromere to signal site was about 66.7 + 2.2.The reasons of abnormal chromosome behavior during meiosis in citrus somatic hybrids, the utilization of pollen fertility on citrus genetic improvement, the potential of CISH method for citrus somatic hybrid verification, and the limitation of FISH technique in citrus research were also discussed in the paper.