1 Research Centre of Characteristic Fruit Tree / College of Horticulture and Forestry, Xinjiang Agricultural University, Urumqi 830052, China;
2 Turpan Institute of Agricultural Sciences, Xinjiang Academy of Agricultural Sciences, Turpan 838000, China;
3 Luntai National Fruit Germplasm Resources Garden, Xinjiang Academy of Agricultural Sciences, Luntai 841600, China
Abstract：Optimizing a flow cytometry method technical scheme for determining genome size and ploidy of local pear (Pyrus) varieties in Xinjiang can detect and identify the genome size and ploidy more efficiently. Different source materials (dry method to store new shoot tender leaves, annual branch hydroponic tender leaves, autumn shoots tender leaves, seed cultivation cotyledon and seed cultivation tender leaves) of local pear varieties in Xinjiang, different mechanical dissociation methods (blade chopped method, grinding method), membrane filtration times (500 mesh filter once and filter twice) and propidium iodide (PI) dyeing liquid dosage (200 and 300 μL) were compared based on the detection results that taken diploid 'Dangshansuli' as reference. The results showed that the genomic size estimation and chromosome ploidy identification of 29 local pear varieties in Xinjiang were carried out by using the optimized detection method. The optimization method as follow: Young leaves which from annual branches hydroponic tender leaves in spring were collected 0.1 g, washed with distilled water and deionized water for 2 to 3 times, respectively. Then wipe it dry and put into a culture dish which had been added with 1 mL precooled woody plant buffer (WPB) dissociation solution, and chopped up quickly with a sharp blade. After chopping, add 1 mL WPB dissociation solution, mix and stand for 2~5 min. The mixture liquid was filtered with 500 mesh membrane into 2.0 mL centrifuge tube and incubated at 4 ℃ for 5 min. The single cell nuclear suspension were got from the treated mixture liquid by centrifuge at 4 ℃ at 1 000 r/min for 5 min. Retain 0.1 mL supernate at bottom and add 200 μL precooled WPB, 200 μL precooled PI, mix well. Place it in refrigerator at 4 ℃ and dye for 10 min without light. After staining, detect it with flow cytometer, and collect 5 000 mononuclear particle granules. Using this method, the results showed that the mean genome size of local pear varieties in Xinjiang detected was (566.5±125.17) Mb. The genome size of 'Yilikamut' was (666.80±154.56) Mb, and it was defined chimera for its chromosome peaks of diploid and triploid appeared simultaneously. The genome sizes of 'Kotoamut', 'Aiwenqieke', and 'Heisuanli' were (721.12±20.87) Mb, (791.33±36.84) Mb and (725.16±76.40) Mb, respectively, which were triploid. The genome size of 'Sha01' was (1032.61±49.41) Mb, which was tetraploid. Among the 24 diploid local pear varieties in Xinjiang, the genome size ranged from (480.95±16.24) to (599.14±38.36) Mb. Among the 3 triploid, the genome size ranged from (721.12±20.87) to (791.33±36.84) Mb. In this study, flow cytometry was optimized to estimate the genomic size and identify the ploidy of local pear varieties in Xinjiang. It overcame the disadvantages of the traditional dyeing method, such as difficulty and time consuming, and the defect was that more detailed chromosome characteristic information could not be obtained. The results of this study can provide technical support for ploidy breeding and genomics research of pear in the future.
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