| The vast majority of plants in the genus Actinidia are functional dioecious,and their seedlings have a long childhood of 5 to 7 years.The varieties cultivated in early production are mainly from A.chinensis Planch..A.arguta has been developing rapidly in recent years and has become a main commercial species.At present,the economic value and demand of female plants in the industry are significantly higher than that of male plants,and the breeding work is still mainly concentrated on the breeding of female elite varieties.Therefore,using techniques or markers for early gender identification of seedlings in the breeding process and removing excess male plants early will save a lot of breeding costs and accelerate the breeding process.This study firstly used PCR amplification technology to verify the versatility of the previous sex-linked molecular markers developed in A.chinensis or its related populations in the materials used in this study;further study developed two molecular markers in A.arguta by using the whole genome resequencing method for sex identification of A.arguta seedlings.The versatility of the two markers in A.chinensis was also verified.The main results are as follows:1.Verification of the versatility of previous sex molecular markers.In this study,we tested the versatility of two groups of previous kiwifruit sex molecular markers,SmX and SmY1,A001,A002 and A003.64 plants of A.chinensis Planch.and 64 plants of A.arguta with known genders were used as research materials.The results showed that:(1)In the 64 samples of A.chinensis used in this study,SmX did not show gender specificity,while SmY1 could well identify the sex of A.chinensis samples.Its sex identification accuracy was 96.55% in the male plants,100% in the females,which meant it could be widely applied to the sex identification of the original A.chinensis variety;A001,A002 and A003 did not show gender specificity.(2)For the 64 samples of A.arguta used in this study,the two SCAR markers SmX and SmY1 could not effectively identify their gender,and the three SSR markers A001,A002 and A003 were not gender specific.(3)Since the two groups of markers were not universal in A.arguta samples,it was necessary to develop molecular markers that suitable for early sex identification of A.arguta seedlings.2.Development and validation of sex-linked molecular markers of A.arguta.In this study,15 male and female individuals of the hybrid population of '11-17'(?)and 'Rubystar'(?)were used as materials for genome resequencing and BSA analysis to developing gender identification molecular markers.The genome of 'Hongyang' kiwifruit was used as reference genome for bioinformatics analysis.The results were as follows:(1)Analysis aimed at gender specific fragments obtained 9 gender-different sequences,g11.t2,g1.t1,g11.t3,g2.t2,g5.t1,g3.t1,g6.t1,g4.t1,and g11.t1.Primers were designed based on these 9 sequences to test their reliability by PCR experiments.Eventually,two pairs of gender-specific primers,Primer11 and Primer51,were tested to be gender-specific.After being tested in 128 samples from both the hybrid populations and wild germplasm resources,the two markers respectively presented an accuracy of 97.89% and 97.03%.(2)Products of Primer 11 and Primer 51,L11 and L51,were sequenced.Blast analysis of L11 and L51 in the kiwifruit genome database and Gene-bank showed that the two fragments may be located in the male-specific region of the kiwifruit sex chromosome,but further localization was still required.(3)After verifying the versatility of the two markers in 48(16 males,32 females)A.chinensis plants,it was found that the accuracy of Primer11 was 100% in males,68.8% in females,indicating that Primer11 had a certain accuracy in the gender identification of A.chinensis,but further validation work was still needed.Primer51 did not amplify clear bands in these samples,indicating it only applicable to the sex identification of A.arguta. |
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