基于小麦SNP芯片对簇毛麦6V#2和6V#4染色体及其与小麦6A、6D染色体的多态性分析

Polymorphism Analysis Among Chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and Wheat 6A and 6D Based on Wheat SNP Chip

【目的】利用大数据比较2条不同的簇毛麦6V(#2和#4)染色体及其与小麦6A、6D染色体间DNA水平上的差异,为小麦-簇毛麦靶向易位的精准设计育种提供依据。【方法】以6V#4(6D)异代换系RW15为父本和6V#2(6A)异代换系南87-88为母本进行杂交,获得F2分离群体,利用6V#4S/6V#2S/6AS/6DS/6VL特异分子标记检测F2植株,筛选新类型的代换系,并用分子标记结合基因组原位杂交(genomic in situ hybridization,GISH)对新类型代换系进行确认,再利用小麦55K芯片中的6A、6D探针,对新代换系及其双亲南87-88和RW15进行分析;结合660K芯片6A、6D探针对2份簇毛麦的SNP分析结果,筛选6V特异SNP。【结果】GISH分析表明,19EL124和19EL134的体细胞染色体数2n=42,分别携带2条完整的外源染色体;分子标记鉴定结果表明,19EL124含有6V#4S/6DS特异标记带,缺失了6V#2S/6AS特异带,而19EL134含有6V#2S/6AS特异标记带,缺失了6V#4S/6DS特异带;19EL124和19EL134都含有6VL的特异带,证明19EL124为6V#4(6A)异代换系,19EL134为6V#2(6D)异代换系。55K芯片检测结果表明,异代换系中关键染色体探针的检测效率显著低于其他染色体,且对同类型异代换不同系的检测效率也有所不同。1177个6A探针中,63.21%不能对6A代换系南87-88分型,68.90%不能对6A异代换系19EL124分型,22.51%检测到6V#2和6V#4间的多态性,其中88个只能检测到6V#4染色体,而155个只能检测到6V#2染色体;479个6D探针中,49.48%不能对6D异代换系RW15分型,53.44%不能对6D代换系19EL134分型,16.70%检测到6V#2和6V#4间的多态性,其中23个只能检测到6V#2染色体,42个只能检测到6V#4染色体。整合55K和660K芯片的共有探针,分别从395个6A、231个6D探针筛选获得簇毛麦6V特异的SNP标记22个和15个,其中3个可在6V#2和6V#4染色体间显示多态性。【结论】小麦染色体的缺失与外源染色体的替换,使相应染色体探针的检测效率大幅降低,NA分型比例极大增加,且多数NA分型在2条不同的外源染色体间显示多态;相同探针对2条外源染色体的检测效率不同,小麦6A探针可以更好地检测6V#2,而小麦6D探针可更好检测6V#4;在簇毛麦与异代换系6V染色体的一致性分型中,筛选获得簇毛麦6V特异的SNP标记37个。

【Objective】The polymorphism among the chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and their wheat homeologous 6A and 6D in the DNA level was compared in this study based on big data to provide a theoretical basis for precision designing breeding of wheat-Dasypyrom villosum targeted chromosome translocation.【Method】A 6V#4(6D)alien disomic substitution line RW15 was crossed with a 6V#2(6A)alien disomic substitution line Nan 87-88,and their F2 plants were detected using 6V#4S/6V#2S/6AS/6DS/6VL specific molecular markers,and the new types of substitution lines were confirmed in F3 employing the above-mentioned markers again as well as genomic in situ hybridization(GISH)technique.Subsequently,polymorphism among the targeted chromosomes in the new types of the substitution lines and their parents Nan 87-88 and RW15 were detected by the probes of 6A and 6D-specific in the wheat 55K chip,and combined with the results of SNP analysis of two accessions of D.villosum with 6A and 6D probes in 660K chip to screen 6V specific SNPs.【Result】GISH analysis showed that 19EL124 and 19EL134 had 42 chromosomes including two complete foreign chromosomes in the somatic cells of root tips.Identification using molecular markers showed that 19EL124 had 6V#4S/6DS and missed 6V#2S/6AS specifically amplified bands,while 19EL134 displayed 6V#2S/6AS distinctive bands and lacked 6V#4S-specific bands;both 19EL124 and 19EL134 contained 6VL specific amplified bands,which confirmed that 19EL124 is a 6V#4(6A)disomic substitution line,and 19EL134 is a 6V#2(6D)disomic substitution line.The results of 55K chip showed that the detected efficiency of the key chromosome probes in the alien substitution lines was significantly lower than that of other chromosome probes,and the detected efficiency of different lines in the same type of substitution was also different.Among the 1177 probes of 6A,the 6A substitution line Nan87-88 and 19EL124 could not be genotyped by 63.21%and 68.90%of the probes,respectively.And the polymorphism between 6V#2 and 6V#4 was detected by 22.51%of the probes.Among the 479 probes of 6D,49.48%of the probes could not identify the 6D substitution line RW15,and 53.44%could not genotype the 6D substitution line 19EL134.The polymorphism between 6V#2 and 6V#4 was indicated by 16.70%of the 6D probes,there were 23 and 42 probes that could only detect 6V#2 and 6V#4 chromosome,respectively.Twenty-two and fifteen 6V specific SNPs were screened from 3956A and 2316D probes through integrating the same genotypes detected by probes which were shared by 55K and 660K chips in this study,and three of them showed polymorphism between 6V#2 and 6V#4 chromosomes.【Conclusion】The detection efficiency of the wheat chromosome probes was greatly reduced when the target wheat chromosome was replaced by the alien chromosome,and the proportion of NA genotyping was greatly increased,and most of the NA genotypes showed polymorphism between the two different alien chromosomes in the same type of substitution.The detection efficiency of probe for two alien chromosomes in the same type of alien substitution lines was different,wheat 6A probe could detect 6V#2 better,and wheat 6D probe could detect 6V#4 better.Thirty-seven SNP markers of 6V specific were obtained base on the association analysis of 6V genotyping between D.villosum and the alien substitution lines.