Screening Of SSR Molecular Marker Primers In Identification Of Blueberry Hybrid Authenticity

Blueberry is a perennial deciduous shrub or small shrub in the Cyanococcus group of the Vaccinium L.genus in the Ericaceae family.In the process of hybrid breeding,the large number of hybrid seedlings can lead to heavy management work.Therefore,early determination of the authenticity of offspring can not only prevent or reduce the interference of false hybrids on the population,improve breeding speed,accelerate breeding process,but also provide materials for the later selection of new varieties and reduce breeding costs.This has important reference value for the development of blueberry breeding and industrialization in China.In this experiment,parents of 15 blueberry hybrid combinations and 225 hybrid progeny were selected as the research objects,and the parental specific primers of 15 hybrid combinations were screened by polyacrylamide gel electrophoresis technology;Subsequently,two SSR molecular marker techniques,PAGE and capillary electrophoresis,were used to identify the authenticity of each hybrid combination’s offspring.Differences in DNA levels were revealed between the F1 hybrid and its parents,in order to clarify the genetic relationship between the hybrid and its parents.The main results obtained in this study are as follows:（1）Using 15 parents of blueberry hybrid combinations as templates,PCR amplification of72 pairs of blueberry specific primers using PAGE technology.Five pairs of SSR primers with specific amplification in 11 parents of hybrid combinations were screened,and then the F₁generation of 11 hybrid combinations and their parents were used as templates to amplify again using the screened primers.The analysis results indicate that,the primer RX-26 amplified parent-specific bands in five hybrid combinations,which were‘Chandler’בDraper’（1908）,‘Duke’בBluetta’（1803）,‘Liberty’בBluecrop’（1830）,‘Draper’בBluecrop’（1831）,‘Liberty’בChandler’（1833）.The primer RX-56 amplified parent-specific bands in three hybrid combinations,which were‘Blueheaven’בDuke’（1823）,‘Draper’בSpartan’（1828）,‘Sweetheart’בDraper’（1612）.The primer RX-17 amplified parent-specific bands in hybrid combination of‘Liberty’בDraper’（1907）,and primer RX-22 amplified parent-specific bands in hybrid combination of‘Liberty’בLateblue’（1837）,and the primer RX-31 amplified parent-specific bands in hybrid combination of‘Legacy’בDraper’（1909）,all amplification bands are clear,stable,and have good repeatability,the other four hybrid combinations did not find parental specific primers.（2）The research results of capillary electrophoresis indicate that in hybrid combinations1908,1803,1833,and 1830,primer RX-26 belongs to paternal specific primers;In the 1837hybrid combination,primer RX-22 belongs to the paternal specific primer.The results of other primer types were consistent with those of polyacrylamide gel electrophoresis.（3）Based on the detection results of two molecular marker techniques,primers RX-26,RX-56,RX-17,and RX-31 showed relatively ideal identification results in hybrid combinations1908,1612,1907,and 1909,with effective identification rates higher than 80.0%.This indicates that these primers have high efficiency in identifying the authenticity of the offspring of the selected hybrid combinations.These four hybrid combinations can produce true hybrid offspring,with a high probability of true hybrid and strong compatibility.（4）The authenticity results of 11 hybrid combinations identified by PAGE technology and capillary electrophoresis indicate that the two molecular detection results of 7 hybrid combinations were inconsistent.Based on the existing 72 pairs of blueberry specific primers,the following conclusions were drawn through analysis,Polyacrylamide gel electrophoresis is more suitable than capillary electrophoresis for screening and identification of SSR primers with few repeat base units or small variation sites.