Distinguishing Zanthoxylum Armatum Cultivars And Varieties Based On Morphology,oligo-FISH And SSR Fluorescent Mark

Zanthoxylum armatum DC is a Zanthoxylum L plant belonging to the Rutaceae Juss family,which has edible and medicinal value.Sichuan area retains a large number of highquality Z.armatum resources,different varieties and cultivars are difficult to distinguish due to phenotype similarity.In this study,different varieties and cultivars of Z.armatum were selected and innovatively combined with oligo-FISH and SSR fluorescent markers for intraspecies differentiation.1)To compare the morphological characteristics of flower buds and fruits among different varieties and cultivars of Z.armatum,eight materials of Z.armatum at the same management level were selected from the Zigong variety gardens for morphological observation.The eight materials included three fine varieties of Z.armatum ’Tengjiao’,Z.armatum ’Zaosujiuyeqing’ and Z.armatum var.novemfolius,and five excellent cultivars of Z.armatum.The research results show that there was a significant difference in the lateral diameter of the flower buds of eight Z.armatum,while there was no significant difference in the vertical diameter;The difference in fruit hundred-grain weight very significance difference,with significant differences in vertical,which no significant differences in horizontal diameters.The fruit and flower bud phenotypes of eight materials were similar and difficult to distinguish.2)It was difficult to distinguish eight different varieties and cultivars of Z.armatum in Zigong variety gardens based on morphological observation,so fluorescence in situ hybridization was used to further distinguish from the perspective of exploring chromosome differences.However,Z.armatum had many and small chromosomes,making it difficult to obtain clear pictures of mitosis metaphase chromosomes.Among these eight materials,only five seedling materials achieved good results.Five seedlings’ materials include two superior varieties of Z.armatum ’Tengjiao’ and Z.armatum var.novemfolius,and three superior cultivars of Z.armatum.This result indicates that the chromosome data of these five materials were different,indicating that there were differences within the Z.armatum species.The number of chromosomes in five materials was inconsistent.The number of chromosomes in Z.armatum ’Tengjiao’ was 98,while the number of chromosomes in Z.armatum ’Youkangtengjiao’,Z.armatum var.novemfolius,Z.armatum ’YT-03’,and Z.armatum ’YT-06’ were all 136.The oligonucleotide signals of five materials were inconsistent.There were two pairs of 5S rDNA signals and five pairs of(GAA)6 signals on the chromosomes of Z.armatum ’Tengjiao’,three pairs of 5S rDNA signals and 17 pairs of(GAA)6 signals on the chromosomes of Z.armatum’Youkangtengjiao’,two pairs of 5S rDNA signals and eight pairs of(GAA)6 signals on the chromosomes of Z.armatum var.novemfolius,three pairs of 5S rDNA signals and nine pairs of(GAA)6 signals on the Z.armatum ’YT-03’ chromosome,and three pairs of 5S r DNA signals and two pairs of(GAA)6 signals on the Z.armatum ’YT-06’ chromosome.The karyotype data of five materials were inconsistent.The karyotype formula of Z.armatum ’Tengjiao’ was 2n= 2x= 98 m,the karyotype type was 1C,and the arm ratio was4.3237;The karyotype formula of Z.armatum ’Youkangtengjiao’ was 2n= 2x= 128m+ 8sm,the karyotype type was 2B,and the arm ratio was 3.5336;The karyotype formula of Z.armatum var.novemfolius was 2n= 2x= 134m+ 2sm,the karyotype type was 1C,and the arm ratio was 5.5224;The karyotype formula of Z.armatum ’YT-03’ was 2n= 2x= 2M+128m+ 4sm+ 2st,the karyotype type was 2C,and the arm ratio was 4.1829;The karyotype formula of Z.armatum ’YT-06’ was 2n= 2x= 126m+ 10 sm,the cytotype was 2B,and the arm ratio was 3.3011.Among the five varieties and cultivars of Z.armatum,the chromosomal symmetry of Z.armatum ’Tengjiao’ was high and relatively primitive;the chromosomal symmetry of Z.armatum ’YT-03’ was low and were relatively evolutionary,with the karyotypes of five materials showing a trend towards polyploid evolution.The differences in oligonucleotide signals and karyotype data indicate that there were differences in genetic relationships among the five varieties of Z.armatum.The oligoFISH method can be used to distinguish different varieties and cultivars of Z.armatum.3)Due to the similarity in morphology and difficulty in obtaining chromosome data from all materials of eight Z.armatum materials in the Zigong Variety Garden,SSR fluorescent markers were selected for further differentiation.At the same time,in order to explore varieties and cultivars of Z.armatum,the genetic relationships,an additional ten Z.armatum materials were added for research.The 18 materials included five excellent varieties,including Z.armatum ’Tengjiao’,Z.armatum var.novemfolius,Z.armatum’Zaosujiuyeqing’,Z.armatum ’Jinyang’ and Z.armatum ’Putaoqing’,and others were excellent cultivars of Z.armatum.Using 25 pairs of primers for SSR analysis,the research results indicate that three primers(ZY14,ZY18,ZY24)can accurately distinguish different materials within the Z.armatum species.When the genetic similarity coefficient was 0.36,18 Z.armatum materials can be divided into two categories based on regional environment.The first category includes 16 materials,all of which are produced in Sichuan;The second category contains two materials,all from Yunnan.The allelic size and phylogenetic relationship of Z.armatum intraspecific materials were different,and SSR can achieve the goal of intra specific differentiation of Z.armatum.In summary,based on oligo FISH and SSR analysis,effectively distinguishing these five different Z.armatum materials.The research results are conducive to providing data for the differentiation of varieties and cultivars of Z.armatum,and can provide reference for the intra species differentiation of other species with similar morphology.