122份栽培桃品种(系)黄白肉性状的分子标记辅助鉴定

Molecular Marker-Assisted Identification of Yellow/White Flesh Trait for 122 Peach Cultivars(Lines)

【目的】类胡萝卜素裂解双脱氧酶基因(Carotenoid Cleavage Dioxygenases 4,CCD4)控制桃果肉颜色(白/黄),CCD4存在3种等位基因。本研究利用Indel、SSR荧光标记毛细管电泳及SNP鉴定等基因分型技术分析我国主要桃黄白肉品种(系)中CCD4等位基因的差异,为主要黄/白肉品种(系)的基因型鉴定、亲本选配和选择相应的分子标记对不同来源子代的果肉颜色进行鉴定奠定基础。【方法】利用已经报道的桃不同果肉颜色中CCD4等位基因3种突变类型,合成不同引物进行PCR扩增,LTR反转录转座子插入突变经1%的琼脂糖凝胶电泳检测,CT单元重复的PCR产物在ABI3730XL测序仪上进行SSR荧光标记毛细管电泳检测,SNP标记经Sanger测序后利用ContigExpress软件分析CCD4等位基因的碱基替换(A→T)。综合以上结果,统计每份材料中CCD4等位基因的突变类型与果肉颜色的一致性。【结果】通过对不同来源的122份桃品种(系)材料进行基因型分析,发现CCD4发生LTR反转录转座子插入突变材料的基因型共有31份,占总材料的25.4%,其中纯合插入突变材料的片段扩增长度为729 bp,共有8份,占总突变的25.8%;CCD4发生微卫星重复序列突变材料存在2 bp的插入,扩增片段长度为179 bp,该类型共有68份,占总材料的55.7%,其中纯合插入材料25份,占总突变的36.8%;CCD4发生A→T碱基替换突变的材料较少,仅有1份,占总材料的0.82%,实际应用中可以不考虑该种类型。CT和LTR插入的两种突变类型的黄肉品种(系)有7份,占总材料的5.7%。研究结果表明,LTR反转录转座子插入突变和微卫星序列重复突变是黄肉桃中CCD4等位基因的主要突变类型。其中CCD4发生一种纯合突变或两种杂合突变桃品种(系)为黄肉类型,分子标记鉴定结果与调查的122份桃品种(系)黄白肉表型性状完全一致,准确率为100%。【结论】采用分子标记明确了122个桃品种(系)黄/白肉性状的基因型,为不同亲本组合子代表型鉴定的标记类型选择提供了技术支撑,为建立桃种质材料黄/白性状的分子辅助育种体系和黄肉桃的选育奠定了基础。

【Objective】It shows that peach flesh color(yellow/white)is controlled by the gene CCD 4(carotenoid cleavage dioxygenase 4).Based on three types of CCD4 allele variations,molecular markers of indel,SSR-CE,and Sanger sequence for SNP were used to analyze the genotypes of 122 peach cultivars(lines),with the aim to determine the correlation between flesh color and genetic variation.This result could provide information for parental matching and the selection of corresponding molecular markers for phenotyping in their offspring.【Method】Three types of variations were detected via PCR.LTR transposable element insertion was detected by 1%agarose gel electrophoresis,and SSR repeat numbers were detected using CE-SSR in ABI3730 XL.Nucleotide substitution was detected using the Sanger sequence and analyzed with the ContigExpress software.In total,the genotypes of 122 cultivars(lines)were analyzed,and the correlation between phenotype and genetic variation was determined.【Result】After genotyping of the 122 cultivars,it was found that 31 accessions were LTR transposable element insertions with 729-bp amplified fragments,accounting for 25.4%of the total accessions,of which eight accessions(25.8%)were homozygous.Sixty-eight cultivars(lines)were SSR repeat number variations,accounting for 55.7%of the total accessions and including 25(36.8%)homozygous types with 2-bp insertions.Of the 122 cultivars(lines),only one cultivar(Fertilia Morettini)was caused by nucleotide substitution and SSR repeat number variation,accounting for 0.82%,which was not widely used in the breeding program.LTR transposable element insertion and SSR repeat number variation were the key types affecting flesh color.Of the 122 cultivars(lines),seven yellow-flesh cultivars(lines)were caused by SSR repeat number variation and LTR transposable element insertion,accounting for 5.7%.One homozygous or two heterozygous sequence variations were both responsible for yellow flesh.The results showed that genotypes were identical with phenotypes of the 122 accessions,with 100%accuracy.【Conclusion】The genotypes of 122 peach cultivars(lines)(white and yellow flesh color)were identified using molecular markers,which could be applied for parental selection,offspring identification in breeding programs,and flesh color selection(white or yellow)using molecular marker-assisted selection.