Construction Of A Molecular Karyotype Analysis Method For Decteting Loquat(Eriobotrya Japonica)Aneuploids Based On SSR Markers And Quantitative PCR

Loquat(Eriobotrya japonica Lindl.)(2n=2x=34)is a Rosaceae fruit species and is one of the main fruit trees in Southern China.Loquat aneuploids are important for the genetics and breeding of loquat.A delicate and sensitive balance between gene dosages occurs in some organisms.Generally,euploid does not alter chromosome types or the relative dosage of genes.Aneuploid,a condition caused by an imbalance between the relative dosages of chromosomes,generally produces a novel phenotype specific to the molecular karyotype.Few techniques are currently available for detecting the molecular karyotypes of aneuploids in plants,and some technologies are being used at higher cost and higher assay complexity.Therefore,it is necessary to develop a rapid,convenient method for detecting molecular karyotypes of loquat aneuploids.Based on the imbalance between the relative dosages of the aneuploid chromosomes,a new approach(referred to as ‘SSR-qPCR')combining simple sequence repeat(SSR)markers and quantitative real-time PCR(qPCR)has been developed and utilized to detect some aneuploids from triploid loquat.This method provides technical support for rapid detection of loquat aneuploids and the construction of the molecular karyotypes.The main results are as follows: 1.Screening of qPCR primers(1)The genomic DNA of 23 different ploidy loquat strains(varieties)(including 22 euploids and 1 aneuploid H39)were used as the PCR templates to amplify 209 pairs of SSR primers.17 pairs of SSR primers with no polymorphism and stable amplification were screened out,which were sequentially located in 17 loquat linkage groups according to a known high-density genetic linkage map in loquat.(2)After qPCR detection,17 pairs of SSR primers showed the same amplification efficiency in 22 euploid loquat strains.Each of the dissolution curves of the 17 pairs of SSR primers showed a single peak.The CT values of the 17 pairs of SSR primers were between 15 and 25,and the negative control had no CT value,which indicated that 17 pairs of SSR primers were all specific.2.The preliminary construction of a molecular karyotype analysis method for detecting loquat aneuploids based on SSR-qPCRThe genomic DNA of 3 euploid loquat strains(‘Ruantiaobaisha'(2x),‘Wuheguoyu'(3x)and H424(4x))and 1 aneuploid loquat strains H39(2n=39)were used as the PCR templates to amplify 17 pairs of SSR primers screened previously.The PCR product amounts(?Rn values)with the cycle threshold(CT)values between 16 and 24 were detected in the exponential phase of qPCR.The results showed that the relative intensities of ?Rn values of the 17 SSR markers were similar among ‘Ruantiaobaisha',‘Wuheguoyu' and H424,whereas the ?Rn values of H39 were altered in five LGs.The ?Rn values obtained for H39 in LG3,LG8,LG10,LG16 and LG17 were approximately 41.8% higher than those obtained for the euploids.The results implied that LG3,LG8,LG10,LG16 and LG17 have one more chromosome in H39 than in the diploid ‘Ruantiaobaisha'.In other words,H39 might have five more chromosomes than the diploid ‘Ruantiaobaisha'.This result was consistent with the result obtained with conventional cytological methods.This result indicated that the molecular karyotype of H39 can be detected using qPCR based on these 17 pairs of SSR primers.3.Karyotype identification of other materialsSome aneuploid plants can be derived from triploids;therefore,the offspring of triploid loquat strains(varieties)were selected as materials to verify and improve the above method in this study.(1)Chromosome countsChromosome counts were performed on some open-pollination progeny of triploid and hybrids of triploid and diploid by chromosome preparation.Nine hybrid offspring of Q24(3x)× ‘Huabai No.1'(2x),7 open-pollination progeny of the triploid loquat A313 and 9 open-pollination progeny of the triploid loquat A322 were detected by chromosome preparation,and these included a total of 22 aneuploids and 3 euploids.(2)The SSR-qPCR detection(1)The offspring of Q24(3x)× ‘Huabai No.1'(2x)To quantify the agreement rate between the SSR-qPCR and conventional cytological methods,the SSR-qPCR method was used to estimate and describe the level of aneuploidy in the 9 hybrid offspring of Q24 × ‘Huabai No.1'.We included some known euploid strains,such as ‘Huabai No.1'(2x),‘Changbai No.1'(2x),Q24(3x)and ‘Huayuwuhe No.1'(3x)as controls in the SSR-qPCR analysis of the detection of aneuploid molecular karyotype variation in the offspring of Q24 × ‘Huabai No.1'.The detection results showed that the ?Rn values of the control strains were between 0.84 and-1.08,and 88.9% of the SSR-qPCR results for the 9 hybrids,including 8 trisomic samples,were consistent with the results obtained using conventional cytological methods.Furthermore,the LGs of the added chromosomes in 8 loquat aneuploids were located.Eight trisomic samples were detected to have trisomy mainly in LG1,LG2,LG7,LG10,LG13,and LG14.Moreover,we found a new model of aneuploidization in loquat,as trisomies were found in 17 LGs.(2)The open-pollination progeny of triploid loquat strains(A313 and A322)The SSR-qPCR method was also used to estimate and describe the level of aneuploidy in 16 open-pollination progeny of triploid loquat strains(A313 and A322).In the SSR-qPCR analysis of the detection of aneuploid molecular karyotype variation in the offspring of A313 and A322,we included 22 known euploid loquat strains as controls,such as ‘Dawuxing'(2x),A313(3x),A322(3x)and so on.The detection results showed that the ?Rn values of the control strains were between 0.81 and-1.15,and 62.5% of the SSR-qPCR results for 16 open-pollination progeny,including 2 tetraploid samples,3 trisomic samples and 5 pentasomic samples,were consistent with the results obtained using conventional cytological methods.Three trisomic individuals with trisomy mainly in LG1,LG4,LG7,LG9,LG10,LG11,LG12,LG14.Five pentasomic individuals with pentasomy mainly in LG1,LG3,LG4,LG5,LG7,LG8,LG9,LG12,LG13,LG14,LG17.In summary,we screened 17 specific and no polymorphic SSR markers covering all loquat linkage groups and redesigned 6 pairs of primers for SSR markers that can detect loquat chromosome aneuploidies.The SSR-qPCR detection results obtained for hybrid progeny and open-pollination progeny of triploid loquat showed diagnostic accuracies of 88.9% and 62.5%,respectively,compared with the chromosome preparation results.We conclude that the SSR-qPCR can be used to construct the entire molecular karyotypes of loquat aneuploids.It is a novel method for detecting plant aneuploidy;however,it is necessary to improve its accuracy.Detection accuracy may be related to the polymorphism of SSR markers.Therefore,It is necessary to continually screen more non-polymorphic molecular markers to improve this method.