| Sweet orange [Citrus sinensis(L.)Osbeck] is widely cultivated citrus varieties in the world,and it is also an important parent in citrus breeding.‘Licheng’ is a variation of ‘Jin cheng’.It is a unique sweet orange variety in China.It has the characteristics of producing single embryo seeds and is an ideal parent for citrus cross breeding.However,up to now,the study on the genome and genetic characteristics of ‘Licheng’ has not been systematically reported.In this study,SSR markers,InDel markers and genome resequencing were used to study the genetic characteristics of ‘Licheng’ genome and analyze its genetic law,so as to provide reference for citrus cross breeding using ‘Licheng’.The main results are as follows:1、From the reported SSR molecular markers,18 heterozygous SSR loci of ‘Licheng’were obtained,and the progeny were amplified,and the segregation rules of the markers were statistically analyzed.The results showed that a total of 11 pairs of 18 pairs of primers were deviated(p < 0.05),accounting for 61.11% of the total number of markers,of which 9 pairs were highly significant(p < 0.01),accounting for 50% of the total number of markers.The proportion of segregation distortion markers is large,which indicates that there is a phenomenon of segregation distortion in ‘Licheng’.2、Taking sweet orange genome as a reference,the resequencing data of ‘Licheng’genome was analyzed.7173 heterozygous InDel loci were detected and screened in the whole genome,and a total of 735 pairs of primers were designed with insertion or deletion sequence length at or above 10 bp loci.The results showed that 192 pairs of primers could amplify the target band,of which 105 pairs of primers could detect allelic variation,and the size of the amplification product was consistent with the predicted size.From the above 10 pairs of polymorphic InDel primers,30 pairs of primers were randomly selected to analyze the mono-embryo materials in the progeny of 59 ‘Licheng’.All the 30 loci were separated in the progeny,which confirmed that these materials were derived from sexual hybridization.The phenomenon of segregation distortion of these polymorphic markers was analyzed.Chi-square test showed that there were 26 pairs of segregation distortion markers(p < 0.05)in30 pairs of primers,accounting for 86.67% of the total number of markers.Among them,25 pairs were significantly segregation distortion(p < 0.01),Accounted for 83.33% of the total number of markers.3、In order to verify the segregation distortion of ’Licheng’ genome and preliminarily clarify the segregation distribution region,‘Licheng’ and its progeny in the resource garden of Chongqing Academy of Agricultural Sciences were re-sequenced in individual plant and mixed pool,the InDel loci in the genome were detected,and the segregation of heterozygous loci was analyzed.The results of single plant re-sequencing analysis showed that the segregation distortion region of chromosome 1 were distributed in 600-800 Mbp and2200-2868 Mbp.Among them,there is a tendency of variant genotypes in the regions of2158-800 Mbp and 2158-2540 Mbp,and a tendency of non-variant genotypes in the region of2545-2868 Mbp.Chromosome 4 shows an obvious segregation trend as a whole,and the more significant regions are mainly distributed in 1-1284 Mbp,in which there are non-variant genotypes in the 1-90 Mbp region and variant genotypes in the 91-1284 Mbp region.Chromosome 5 showed an obvious trend of segregation as a whole,and deviated toward variant genotypes as a whole.The segregation distribution region of chromosome 7 were distributed in 2500-2800 Mbp.And chromosome 9 showed an obvious trend of segregation distribution as a whole,it deviated toward non-variant genotypes.The results of mixed pool re-sequencing also showed that the result of mixed pool sequencing was the same as that of individual plant sequencing.This shows that mixed pool sequencing can accurately detect the segregation distribution region of ‘Licheng’ genome.The developed InDel molecular markers were compared with the corresponding sites in the results of re-sequencing(individual plant).The chi-square test results showed that there was no significant difference between 29 of the30 sites,and the accuracy was as high as 96.67%,which proved that the results of InDel markers were consistent with the results of re-sequencing.4、In order to fully understand the segregation distortion of ‘Licheng’ genome,we took‘Licheng’ and its progenies from two orchards in Yubei and Yongchuan to establish a mixed pool for genome re sequencing,and analyzed the distribution of segregation distortion sites in the genome.The results showed that segregation distortion also existed in the progenies of‘Licheng’ from these two sites,and there were many similarities with the segregation distortion regions of ‘Licheng’ genome from Chongqing Academy of Agricultural Sciences,including: the 600-800 Mbp region of chromosome 1 tends to variant genotypes,the2545-2868 Mbp region deviated toward non-variant genotypes,and the 1500-2600 Mbp region of chromosome 2 deviated toward non-variant genotypes.The 1-90 Mbp region of chromosome 4 deviated toward non-variant genotype,91-1000 Mbp region deviated toward non-variant genotype,while chromosome 9 shows an obvious segregation trend as a whole,and deviated toward non-variant genotype as a whole.It can be seen that the segregation distortion phenomenon in the ‘Licheng’ genome is highly consistent in the results of mixed pool sequencing in different locations.In conclusion,this study combined SSR markers,InDel Markers and genome re-sequencing,and found that there was segregation distortion in the genome of ‘Licheng’.The segregation distortion region of ‘Licheng’ genome was verified and analyzed by genome re sequencing.This study provides an important reference for the study of Citrus genetics and breeding with ‘Licheng’ as material. |
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