技术领域technical field
本发明涉及农业生物技术领域。具体的说，本发明涉及一种与甜瓜果实甜味、酸味性状相关基因功能性分子标记及其应用的技术领域。The invention relates to the field of agricultural biotechnology. Specifically, the invention relates to the technical field of functional molecular markers of genes related to the sweetness and sourness of melon fruit and their application.
背景技术Background technique
甜瓜(Cucumis melo L.)属葫芦科甜瓜属， 是一种重要的园艺作物，我国甜瓜的种植面积和产量均居世界第一。甜瓜是一种高多态性物种，含有各种不同果实风味的基因型。成熟甜瓜果实糖、酸组分及其含量对果实内在品质有着重要的影响，是决定果实风味的重要指标。甜、酸等风味的变化是一个复杂的过程，受一系列相关基因的影响和调控。定位甜瓜甜味、酸味性状相关基因并开发功能性标记，为提高甜瓜果实品质性状选育效率及甜瓜分子育种提供思路。Melon (Cucumis melo L. ) belongs to Cucurbitaceae and is an important horticultural crop. The planting area and output of melon in China rank first in the world. Melon is a highly polymorphic species containing a variety of genotypes with different fruit flavors. The sugar and acid components and their content of ripe melon fruit have an important influence on the internal quality of the fruit, and are important indicators to determine the flavor of the fruit. The change of sweet, sour and other flavors is a complicated process, which is affected and regulated by a series of related genes. Locate genes related to sweetness and sourness traits of melon and develop functional markers to provide ideas for improving the efficiency of selection of melon fruit quality traits and molecular breeding of melon.
在现有甜瓜育种实践中，对甜味、酸味等风味性状大多是通过表现型间接对基因型进行选择，致使育种周期长，效率低，制约着甜瓜品种改良的进程。标记辅助选择,尤其是对与目标性状关联的功能性分子标记的选择可极大提高选择的效率，加速遗传改良。In the current breeding practice of melon, most of the flavor traits such as sweetness and sourness are indirectly selected through phenotype, which leads to long breeding cycle and low efficiency, which restricts the progress of melon variety improvement. Marker-assisted selection, especially the selection of functional molecular markers associated with target traits, can greatly improve the efficiency of selection and accelerate genetic improvement.
现有技术中，功能性分子标记作为与表型相关的功能基因基序中功能性单核苷酸多态性（SNP）位点开发而成的分子标记。功能标记的开发必须具备以下两个条件：(1)有确定功能的候选基因并已知等位基因的序列信息；(2)在多个材料中对目标性状进行调查，对目标基因进行序列分析，结合性状和基因序列信息进行基于连锁不平衡(LD)的关联分析。功能基因定位是功能性分子标记发掘的基础，目前功能基因定位常用的方法有基于传统遗传图谱定位与集群分离分析法（BSA）。Javier M等（2009）基于传统遗传图谱对甜瓜糖、酸性状进行了QTL定位，但该方法的周期长、密度低、成本高；而基于传统集群分离分析法（BSA）可快速、有效寻找与目标基因紧密连锁的分子标记，但混池个数有限，一般不超过10个，关联分析假阳性几率大，标记密度低。近年来发展的选择性基因型鉴定和集群分离分析（Super BSA），利用SLAF-seq（Specific Location Amplified Fragments seq，特定位置扩增片段测序），选择均匀分布在整个基因组、且避开重复序列区域的特异片段进行高深度测序，通过比较SNP标记的不同基因型在两个混池中出现频率的差异，确定与性状紧密相关的分子标记。Super BSA分子标记密度高，30-200个体的混池，保证了基因定位的功效和准确性。同时，由于SLAF标签中的多态性位点已经定位在基因组上，可以根据基因组的位置使用引物设计软件进行引物设计，开发功能标记。这些近几年发展起来的分子标记技术，通过分子标记辅助选择可以将传统的表型选择转变为直接选择基因型都具有重要的意义，但是至今未见开展甜瓜甜味酸味性状相关基因功能性分子标记方面的研究和报道。In the prior art, functional molecular markers are molecular markers developed as functional single nucleotide polymorphism (SNP) sites in functional gene motifs related to phenotype. The development of functional markers must meet the following two conditions: (1) Candidate genes with definite functions and known allele sequence information; (2) Investigate the target traits in multiple materials and perform sequence analysis on the target genes , combining trait and gene sequence information for linkage disequilibrium (LD)-based association analysis. Functional gene mapping is the basis for the discovery of functional molecular markers. At present, the commonly used methods for functional gene mapping are based on traditional genetic map mapping and cluster separation analysis (BSA).Javier M et al. (2009) conducted QTL mapping for melon sugar and acidity traits based on traditional genetic maps, but this method has a long cycle, low density, and high cost; while based on traditional cluster segregation analysis (BSA), it can quickly and effectively search for QTLs related to sugar and acidity. Molecular markers closely linked to target genes, but the number of mixed pools is limited, generally no more than 10, the probability of false positives in association analysis is high, and the marker density is low. The selective genotype identification and cluster separation analysis (Super BSA) developed in recent years usesSLAF-seq (Specific Location Amplified Fragments seq , specific location amplified fragment sequencing) to select evenly distributed regions throughout the genome and avoid repetitive sequences High-depth sequencing of the specific fragments of the specific fragments, and the molecular markers closely related to the traits were determined by comparing the frequency difference of the different genotypes of the SNP markers in the two mixed pools.The Super BSA molecular marker density is high, and the mixed pool of 30-200 individuals ensures the efficacy and accuracy of gene mapping. At the same time, since the polymorphic site in the SLAF tag has been located on the genome, primer design software can be used to design primers and develop functional markers according to the position of the genome. These molecular marker technologies developed in recent years are of great significance for transforming traditional phenotypic selection into direct selection of genotypes through molecular marker-assisted selection. Research and reporting on labeling.
发明内容Contents of the invention
本发明的目的旨在针对现有技术的不足，利用Super-BAS技术，提供一种甜瓜甜味酸味性状相关基因功能性分子标记，确定了风味甜瓜甜味、酸味性状相关基因序列，并在此基础上成功开发了出一个甜味性状相关基因功能性分子标记SLAF18745-S01和三个个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04，并通过甜味酸味标记辅助选择将传统的表型选择转变为直接选择基因型的应用对于甜瓜甜味、酸味性状的改良和改善具有重要的应用价值和意义。The object of the present invention aims to address the deficiencies in the prior art, and utilizeSuper -BAS technology to provide a functional molecular marker of genes related to the sweetness and sourness of melons, determine the gene sequences related to the sweetness and sourness of melons, and hereby Based on this, a functional molecular marker SLAF18745-S01 of a gene related to sweet taste traits and three functional molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 of genes related to sour taste traits were successfully developed, and the sweet and sour taste markers were assisted The application of selection to transform traditional phenotypic selection into direct selection of genotype has important application value and significance for the improvement and improvement of sweet and sour traits of melon.
本发明通过以下技术方案实现的：通过对甜味、酸味性状极端群体进行基于简化基因组测序（SLAF-seq）的选择性基因型鉴定和集群分离分析（Super-BAS)，结合生物信息学方法定位与甜味、酸味性状关联紧密的候选区域，获得性状相关候选基因，针对该候选基因开发出甜瓜甜味、酸味性状基因的功能性分子标记，达到选育甜酸味品系、缩短育种周期、提高选择准确率良好的技术效果。The present invention is achieved through the following technical solutions: through selective genotype identification and cluster separation analysis (Super -BAS) based on simplified genome sequencing (SLAF -seq) for extreme populations of sweet and sour traits, combined with bioinformatics methods for positioning Candidate regions closely related to sweet and sour traits were obtained to obtain trait-related candidate genes, and functional molecular markers for sweet and sour trait genes of melons were developed for the candidate genes, so as to achieve the purpose of breeding sweet and sour strains, shortening the breeding cycle, and improving selection. Good technical effect of accuracy.
本发明具体提供了一种甜瓜甜味酸味性状相关基因功能性分子标记，包括以下步骤获得。The present invention specifically provides a functional molecular marker of a gene related to sweet and sour taste traits of melon, which comprises the following steps of obtaining.
（1）鉴定“风味四号”甜瓜的父本、母本、F1及F2群体单株果实风味。(1) Identify the fruit flavor of the single plant of "Fangwei No. 4" muskmelon in the male parent, female parent, F1 and F2 populations.
（2）对“风味四号”甜瓜父本、母本、F2群体中只甜不酸、只酸不甜、既不酸又不甜单株DNA分别等量混池建库，进行简化基因组测序，获得SLAF标签，并对SLAF标签进行单核苷酸（SNP）多态性分析。(2) The DNA from the male parent, female parent, andF2 population of "Flavour No. 4" muskmelon were mixed with equal amounts of DNA from only sweet but not sour, only sour but not sweet, and neither sour nor sweet, and the simplified genome was constructed. Sequencing, obtaining SLAF tags, and performing single nucleotide (SNP) polymorphism analysis on the SLAF tags.
（3）根据多态性标记与甜味、酸味性状的关联性定位甜味、酸味性状侯选区域，并对候选区域基因进行功能注释。(3) According to the correlation between polymorphic markers and sweet and sour traits, the candidate regions of sweet and sour traits were located, and the genes in the candidate regions were functionally annotated.
（4）根据定位的功能基因的SLAF标签序列，设计引物，开发甜味、酸味性状功能性标记。(4) According to the SLAF tag sequence of the mapped functional gene, design primers and develop functional markers for sweetness and sourness.
进一步，本发明详细提供了一种甜瓜甜味酸味性状相关基因功能性分子标记，包括以下步骤获得。Further, the present invention providesin detail a functional molecular marker of a gene related to sweet and sour taste traits of melon, which comprises the following steps to obtain.
（1）对“风味四号”甜瓜的父本、母本、F1及F2代每个单株果实的甜酸味通过仪器测量结合品尝鉴定，获得只酸不甜、只甜不酸、既酸又甜、既不酸又不甜4种风味。(1) The sweet and sour taste of each single fruit of the male parent, female parent, F1 and F2 generations of "Fangwei No. 4" muskmelon was measured by instruments combined with tasting identification, and the sour but not sweet, only sweet but not sour, and both sweet and sour were obtained. Sour and sweet, neither sour nor sweet 4 flavors.
（2）通过CTAB法提取父本、母本、F1及F2代每个单株基因组DNA，分别等量混合父本、母本、只酸不甜、只甜不酸、既不酸又不甜每个单株基因组DNA，形成5个混池，分别对5个混池基因组进行酶切，每个混池各单株同一位点处的DNA片段序列，经过PCR扩增、测序，Blat比对软件进聚类、Cap snp软件检测单核苷酸多态性，获得多态性的SLAF标签。(2) Extract the genomic DNA of each individual plant of the male parent, female parent, F1 and F2 generations by CTAB method, and mix the male parent and female parent in equal amounts, sour but not sweet, sweet but not sour, neither sour nor sour The genomic DNA of each individual plant of Butian forms 5 mixed pools, and the genomes of the 5 mixed pools are digested respectively. The DNA fragment sequence at the same site of each individual plant in each mixed pool is amplified by PCR and sequenced. Blat Clustering was carried out by comparison software, single nucleotide polymorphisms were detected by Cap snp software, and SLAF tags of polymorphisms were obtained.
（3）根据多态性标记与甜味、酸味性状的关联性使用比值关联法进行定位。分别计算群体既不酸又不甜的ac与甜不酸的ab之间和既不酸又不甜的ac与酸不甜的aa之间来源于不同基因型的差异比值，得到差异标记。将与甜味、酸味性状关联的差异标记通过blat软件比对，在双亲基因组上定位，根据标记物理位置提取出标记所在的基因，与Swissprot及Kegg数据库比对，得到与甜味性状、酸味性状相关的基因功能注释，挑选出处于功能基因外显子的SLAF标签。(3) According to the correlation between polymorphic markers and sweet and sour taste traits, use the ratio correlation method to locate. Differential markers were obtained by calculating the difference ratios between ac that is neither sour nor sweet and ab that is neither sour nor sweet, and between ac that is neither sour nor sweet and aa that is neither sour nor sweet, respectively. The differential markers associated with sweetness and sourness traits were compared by blat software, located on the parent genome, and the genes where the markers were located were extracted according to the physical position of the markers, compared with the Swissprot and Kegg databases, and sweetness traits and sourness traits were obtained. For related gene function annotations, SLAF tags in exons of functional genes were selected.
（4）根据定位的并处于外显子的一个甜味性状相关功能基因MELO3C011944T1所在的标签SLAF18745的多态性序列、3个酸味性状相关功能基因MELO3C020002T1、MELO3C025794T1、MELO3C026251T1所在标签SLAF36334、SLAF50072、SLAF51212的多态性序列，利用Primer Primer5.0软件设计引物，以父本、母本、F1、F2只甜不酸、只酸不甜、既不酸又不甜材料为材料，进行PCR扩增，发展出一个甜味性状相关基因功能性分子标记SLAF18745-S01和三个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04。(4) According to the polymorphic sequence of the label SLAF18745 of a sweet taste trait-related functional gene MELO3C011944T1 located in the exon, and the tags SLAF36334, SLAF50072, and SLAF51212 of the three sour taste trait-related functional genes MELO3C020002T1, MELO3C025794T1, and MELO3C026251T1 For the polymorphic sequence, use the Primer Primer5.0 software to design primers, and use the paternal parent, female parent, F1 , F2 materials that are sweet but not sour, only sour but not sweet, neither sour nor sweet, for PCR amplification , developed a sweetness trait-related gene functional molecular marker SLAF18745-S01 and three sour taste trait-related gene functional molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04.
一个甜味、三个酸味性状相关基因的功能性分子标记相应的DNA序列分别为。The corresponding DNA sequences of the functional molecular markers of one sweet taste and three sour taste trait-related genes are respectively.
SLAF18745-S01的DNA序列DNA sequence of SLAF18745-S01
tgggaccagttcctcattctttctcagcagttcacttgccatcgtatctgctggaaaatgcagaagactacaaattccttttgcctgggaattgcatacgagagagtggctgatctctaactttggcgtctacctccctgtgaaggttagttgcgatgtagcttgtctgacacctcgtggaccttccatctcgatagttgttgaccaatatgcagtcaaccgtggtggtcttttgcctccagataagcttgggaccagttcctcattctttctcagcagttcacttgccatcgtatctgctggaaaatgcagaagactacaaattccttttgcctgggaattgcatacgagagagtggctgatctctaactttggcgtctacctccctgtgaaggttagttgcgatgtagcttgtctgacacctcgtggaccttccatctcgatagttgttgaccaatatgcagtcaaccgtggtggtcttttgcctccagataagct
SLAF18745-S02的DNA序列DNA sequence of SLAF18745-S02
catagctggtgtctggatatctctaatgtatatgttatttatgtgtgaggttggattttgagatcacatagctggtgtctggatatctctaatgtatatgttatttatgtgtgaggttggattttgagatca
SLAF18745-S03的DNA序列DNA sequence of SLAF18745-S03
caaagccgctggactactcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggggtcagcttggacctgaaattgcaagtttgactcaattgcgtactattgatttgacaaccaacgatttctctggtgaaattccttatgggattggtaactGtacccatttagagttcttggatctctctttcaaccgatttggtgcaaagccgctggactactcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggggtcagcttggacctgaaattgcaagtttgactcaattgcgtactattgatttgacaaccaacgatttctctggtgaaattccttatgggattggtaactGtacccatttagagttcttggatctctctttcaaccgatttggtg
SLAF18745-S04SLAF18745-S04
cacccttcaacctaatcttgttccacaaggagtaatacatgattcatttgtactatgtatgcttcgaaagaagattttgaaacaacacaaaagagcaccatgtaccagaaaagagcatctaattcctaaacgcaaaagagtggaaggacaaatttccattgatagagttgaaaacgaatgtttcgtcaatacgagacagttttcagaactacttttgaatggatgcgatttggaagaacaatctcaacttctttttcaggaaagcggtcatcaacacccttcaacctaatcttgttccacaaggagtaatacatgattcatttgtactatgtatgcttcgaaagaagattttgaaacaacacaaaagagcaccatgtaccagaaaagagcatctaattcctaaacgcaaaagagtggaaggacaaatttccattgatagagttgaaaacgaatgtttcgtcaatacgagacagttttcagaactacttttgaatggatgcgatttggaagaacaatctcaacttctttttcaggaaagcggtcatcaa
本发明中，一个甜味性状相关基因功能性分子标记SLAF18745-S01中，特异引物名称为SLAF_No.1，序列为。In the present invention, in a functional molecular marker SLAF18745-S01 of a gene related to sweetness traits, the name of the specific primer is SLAF_No.1, and the sequence is .
5'TGGGACCAGTTCCTCATT 3'5'TGGGACCAGTTCCTCATT 3'
5'AGCTTATCTGGAGGCAAA 3'5'AGCTTATCTGGAGGCAAA 3'
本发明中，三个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04中，特异引物名称分别为。In the present invention, the names of specific primers for the three functional molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 of genes related to sour taste traits are respectively .
SLAF_No.2特异序列为，The specific sequence of SLAF_No.2 is,
5'CATAGCTGGTGTCTGGAT 3'5'CATAGCTGGTGTCTGGAT 3'
5' GATCTCAAAATCCAACCT 3'5'GATCTCAAAATCCAACCT 3'
SLAF_No.3特异序列为，The specific sequence of SLAF_No.3 is,
5'CAAAGCCGCTGGACTACTCAC 3'5'CAAAGCCGCTGGACTACTCAC 3'
5' CACCAAATCGGTTGAAAG 3'5' CACCAAATCGGTTGAAAG 3'
SLAF_No.4，序列分别为：SLAF_No.4, the sequences are:
5'ACACCCTTCAACCTAATC 3'5'ACACCCTTCAACCTAATC 3'
5' TTGATGACCGCTTTCCTG 3'。5' TTGATGACCGCTTTCCTG 3'.
同时，本发明提供甜瓜甜味性状相关基因功能性分子标记的应用，即将甜瓜甜味性状相关基因功能性分子标记SLAF18745-S01用于甜瓜品种或品系的甜味基因型检测中的应用，具体包括以下步骤。At the same time, the present invention provides the application of functional molecular markers of genes related to sweetness traits of melons, that is, the application of the functional molecular markers SLAF18745-S01 of genes related to sweetness traits of melons in the detection of sweetness genotypes of muskmelon varieties or lines, specifically including The following steps.
（1）以甜瓜自交系“寿星”与其他甜瓜杂交并繁衍至F2代以上。(1) The melon inbred line "Shouxing" was crossed with other melons and multiplied to more thanF2 generation.
（2）对通过步骤（1）获得的甜瓜单个植株，提取基因组 DNA ，检测提取基因组的 DNA 中是否存在与甜味性状相关功能基因MELO3C011944T1相连锁的分子标记；如有甜味性状功能性分子标记SLAF36334-S01出现，预测甜瓜植株是否具有甜味。(2) For the single melon plant obtained through step (1), extract genomic DNA, and detect whether there is a molecular marker linked to the functional gene MELO3C011944T1 related to sweetness traits in the extracted genomic DNA; if there is a functional molecular marker for sweetness traits SLAF36334-S01 appeared to predict whether the melon plants had sweet taste.
进一步，本发明提供甜瓜酸味性状相关基因功能性分子标记的应用，即酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04用于甜瓜品种或品系的酸味基因型检测中的应用，具体包括以下步骤。Further, the present invention provides the application of functional molecular markers of genes related to sourness traits of melons, that is, the functional molecular markers of genes related to sourness traits SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 are used in the detection of sourness genotypes of melon varieties or lines The application specifically includes the following steps.
（1）以甜瓜自交系“酸甜瓜”与其他甜瓜杂交并繁衍至F2代以上。(1) The melon inbred line "Sour Melon" was crossed with other melons and propagated to more thanF2 generation.
（2）对通过步骤（1）获得的甜瓜单个植株，提取基因组 DNA ，检测提取基因组的 DNA 中是否存在与酸味性状相关功能基因MELO3C020002T1、MELO3C025794T1、MELO3C026251T1相连锁的分子标记；如有酸味性状功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04出现，预测甜瓜植株是否具有酸味。(2) Extract genomic DNA from the single melon plant obtained in step (1), and detect whether there are molecular markers linked to functional genes MELO3C020002T1, MELO3C025794T1, and MELO3C026251T1 related to sourness traits in the extracted genomic DNA; Molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 appeared to predict whether the melon plants had sour taste.
通过实施本发明具体的发明内容，可以达到以下效果。By implementing the specific invention content of the present invention, the following effects can be achieved.
（1）本发明利用super-BAS技术仅用四个月的时间，即获得甜瓜甜味、酸味性状的功能性分子标记和精细定位区域，周期短、成本低、效率高，可为在其它物种中开发功能性分子标记提供重要的成功案例，也为分子育种、系统进化、种质资源鉴定中分子标记提供了一种重要的开发途径。(1) The present invention uses the super-BAS technology to obtain the functional molecular markers and fine-positioned areas of the sweet and sour traits of melon in only four months, with short cycle, low cost and high efficiency, which can be used in other species It provides an important successful case in the development of functional molecular markers, and also provides an important development approach for molecular markers in molecular breeding, phylogenetic evolution, and identification of germplasm resources.
（2）本发明首次获得一个甜味性状相关基因功能性分子标记SLAF18745-S01和三个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04，为甜瓜品质性状基因的分子标记辅助选择提供了重要的标记基础。通过应用甜瓜果实甜味、酸味性状相关基因功能性分子标记，检测与甜味、酸味性状连锁的分子标记，来预测甜瓜果实的甜味、酸味，快速筛选出甜酸味的品种或品系用于甜瓜品质育种。(2) The present invention obtained for the first time a functional molecular marker SLAF18745-S01 of a gene related to sweet taste traits and three functional molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 of genes related to sour taste traits, which are the molecular markers of melon quality trait genes Marker-assisted selection provides an important marker basis. By applying the functional molecular markers of the genes related to the sweetness and sourness traits of melon fruits, and detecting the molecular markers linked to the sweetness and sourness traits, the sweetness and sourness of melon fruits can be predicted, and the sweet and sour varieties or lines can be quickly screened for use in melons Quality breeding.
附图说明 Description of drawings
图1显示为甜味性状相关基因功能性分子标记SLAF18745-S01对双亲、只甜不酸、不甜不酸材料中的扩增图。图中，M为500bp Ladder DNA、1为“寿星”、2为“酸甜瓜”、5、6、7、9、10为甜不酸单株、3、4、8为既不甜又不酸单株。Figure 1 shows the amplification diagram of the functional molecular marker SLAF18745-S01 of the gene related to sweet taste traits in the parents, only sweet but not sour, and neither sweet nor sour materials. In the figure, M is 500bp Ladder DNA, 1 is "Shouxing", 2 is "Sour Melon", 5, 6, 7, 9, 10 are sweet and not sour individual plants, 3, 4, 8 are neither sweet nor sour single plant.
图2显示为酸味性状相关基因功能性分子标记SLAF36334-S02对双亲、只酸不甜、不甜不酸材料中的扩增图。图中，M为500bp Ladder DNA、1为“寿星”、2为“酸甜瓜”、4、5、7、8、10为只酸不甜单株、3、6、9为既不甜又不酸单株。Figure 2 shows the amplification diagram of the gene functional molecular marker SLAF36334-S02 related to sourness traits in the parents, only sour but not sweet, and neither sweet nor sour materials. In the figure, M is 500bp Ladder DNA, 1 is "Shouxing", 2 is "sour melon", 4, 5, 7, 8, 10 are only sour and not sweet single plant, 3, 6, 9 are neither sweet nor sweet Sour single plant.
图3显示为酸味性状相关基因功能性分子标记SLAF50072-S03对双亲、只酸不甜、不甜不酸材料中的扩增图。图中，M为500bp Ladder DNA、1为“寿星”、2为“酸甜瓜”、4、7、8、9、10为只酸不甜单株、3、5、6为既不甜又不酸单株。Figure 3 shows the amplification diagram of the gene functional molecular marker SLAF50072-S03 related to sourness traits in the parents, only sour but not sweet, and neither sweet nor sour materials. In the figure, M is 500bp Ladder DNA, 1 is "Shouxing", 2 is "Sour Melon", 4, 7, 8, 9, 10 are only sour and not sweet single plant, 3, 5, 6 are neither sweet nor sweet Sour single plant.
图4显示为酸味性状相关基因功能性分子标记SLAF31212-S04对双亲、只酸不甜、不甜不酸材料中的扩增图。图中，M为500bp Ladder DNA、1为“寿星”、2为“酸甜瓜”、4、5、7、8、9、10为只酸不甜单株、3、5为既不甜又不酸单株。Fig. 4 shows the amplification diagram of the gene functional molecular marker SLAF31212-S04 related to sourness traits in the parents, only sour but not sweet, and neither sweet nor sour materials. In the figure, M is 500bp Ladder DNA, 1 is "Shouxing", 2 is "Sour Melon", 4, 5, 7, 8, 9, 10 are only sour and not sweet single plant, 3 and 5 are neither sweet nor sweet Sour single plant.
图5显示为甜味性状相关基因功能性分子标记SLAF18745-S01在F1、F2单株的稳定性图。图中，M为500bp Ladder DNA，1、2、3、4为既不甜又不酸单株，5、6为只甜不酸单株，7、8为F1单株，9为只酸不甜单株，10为F2中既酸又甜单株。Fig. 5 shows the stability diagram of SLAF18745-S01, a functional molecular marker of genes related to sweetness traits, in F1 and F2 individual plants. In the figure, M is 500bp Ladder DNA, 1, 2, 3, 4 are neither sweet nor sour individual plants, 5, 6 are only sweet but not sour individual plants, 7, 8 are F1 individual plants, 9 is only sour Not sweet single plant, 10 is both sour and sweet single plant inF2 .
图6显示为酸味性状相关基因功能性分子标记SLAF36334-S02在F1、F2单株的稳定性图。图中，M为500bp Ladder DNA，1、2为既不甜又不酸单株，3、4只甜不酸单株，5、6为只酸不甜单株，7、8为F1单株，9、10为F2中既酸又甜单株。Fig. 6 shows the stability diagram of the functional molecular marker SLAF36334-S02 in F1 and F2 individual plants of sour taste traits-related genes. In the figure, M is 500bp Ladder DNA, 1 and 2 are neither sweet nor sour single plants, 3 and 4 are sweet and not sour single plants, 5 and 6 are only sour and not sweet single plants, 7 and 8 are F1 single plants 9 and 10 are both sour and sweet single plants inF2 .
图7显示为酸味性状相关基因功能性分子标记SLAF50072-S03在F1、F2单株的稳定性图。图中，M为500bp Ladder DNA，1为F1单株，2为只甜不酸单株，3、4为F2中既酸又甜单株，5为既不甜又不酸单株，6、7、8、9、10为酸不甜单株。Fig. 7 shows the stability graph of the functional molecular marker SLAF50072-S03 in F1 and F2 individual plants of sour taste traits-related genes. In the figure, M is 500bp Ladder DNA, 1 is F1 single plant, 2 is sweet but not sour single plant, 3 and 4 are both sour and sweet single plants in F2 , 5 is neither sweet nor sour single plant, 6, 7, 8, 9, and 10 are sour but not sweet single plants.
图8显示为酸味性状相关基因功能性分子标记SLAF31212-S04在在F1、F2单株的稳定性图。图中，M为500bp Ladder DNA，1为F1单株，2为甜不酸单株，3、4、5为F2中既酸又甜单株，6为甜不酸单株，7、8、10为酸不甜单株，9为既不甜又不酸单株。Fig. 8 shows the stability graph of the functional molecular marker SLAF31212-S04 in F1 and F2 individual plants of sour taste traits-related genes. In the figure, M is 500bp ladder DNA, 1 is F1 single plant, 2 is sweet but not sour single plant, 3, 4, 5 are both sour and sweet single plants in F2 , 6 is sweet but not sour single plant, 7, 8 and 10 are single plants that are not sweet and sour, and 9 are single plants that are neither sweet nor sour.
具体实施方式Detailed ways
下面，举实施例说明本发明，但是，本发明并不限于下述的实施例。Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.
本发明中设备、试剂和信息分析软件有：Equipment, reagent and information analysis software among the present invention have:
PAL-1糖度测定仪（日本ATATO公司）、STARTER300便携式PH计(奥豪斯仪器（上海）有限公司)、TC-512型PCR扩增仪（英国Techne公司）、JY3000型电泳仪（北京君意东方电泳设备有限公司）、JY-SPE型电泳槽（北京君意东方电泳设备有限公司）、JY04S-3C型数码凝胶成像仪（北京君意东方电泳设备有限公司）。XhoI、 MseI核酸内切酶、酶切缓冲液4及建库试剂（美国New Englang Biolabs 公司），纯化试剂盒（德国QIAGEN公司）。特异引物开发所用Taq酶、dNTPs均购于上海生工生物工程技术服务有限公司。Primer Primer5.0引物设计软件、blat短序列比对软件、blast比对软件、Cap snp 检测软件，可通过一般生物网站、生物技术公司获得。PAL-1 Sugar Meter (ATATO Japan), STARTER300 Portable PH Meter (Ohaus Instruments (Shanghai) Co., Ltd.), TC-512 PCR Amplifier (Techne, UK), JY3000 Electrophoresis Instrument (Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.), JY-SPE electrophoresis tank (Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.), JY04S-3C digital gel imager (Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.). XhoI, MseI endonuclease, digestion buffer 4, library construction reagents (New England Biolabs, USA), and purification kits (QIAGEN, Germany). Taq enzymes and dNTPs used in the development of specific primers were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. Primer Primer5.0 primer design software, blat short sequence alignment software, blast alignment software, and Cap snp detection software can be obtained through general biological websites and biotechnology companies.
一个甜味性状相关基因功能性分子标记SLAF18745-S01和三个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04特异引物利用Primer Primer5.0软件设计。One functional molecular marker SLAF18745-S01 of a gene related to sweetness traits and three specific primers of functional molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 were designed using Primer Primer5.0 software.
本发明中选用的所有试剂、仪器和数据信息分析软件都为本领域熟知选用的，但不限制本发明的实施。All reagents, instruments and data information analysis software selected in the present invention are well known and selected in the art, but do not limit the implementation of the present invention.
实施例一：甜瓜甜味酸味性状相关基因功能性分子标记的获得Example 1: Acquisition of functional molecular markers for genes related to sweet and sour taste traits of melon
甜瓜甜味酸味性状相关基因功能性分子标记获得包括以下步骤：Obtaining functional molecular markers of genes related to sweet and sour taste traits of melons includes the following steps:
（1）将高糖甜瓜自交系“寿星”为母本与甜瓜自交系“酸甜瓜”为父本进行杂交，得到杂种F1即“风味4号”；由杂种F1自花授粉获得F2代群体，对父母本、F1及每个F2代单株果实的甜酸味通过仪器测量结合品尝进行鉴定，获得只酸不甜、只甜不酸、既酸又甜、既不酸又不甜4种类型。(1) The high-sugar melon inbred line "Shouxing" was used as the female parent and the melon inbred line "Sour Melon" was used as the male parent to obtain the hybrid F1 , namely "Flavour 4", which was obtained by self-pollination of the hybrid F1 For the F2 generation group, the sweet and sour taste of the parents, F1 and each F2 generation individual fruit was identified by instrumental measurement and tasting, and the results were only sour but not sweet, only sweet but not sour, both sour and sweet, and neither sour There are 4 types that are not sweet.
（2）通过CTAB法提取（1）中每份材料的基因组DNA，对5株父本、5株母本及只酸不甜、只甜不酸、既不酸又不甜各50个单株的基因组DNA等量混合，形成5个混池,分别对5个混池基因组进行酶切，混池中各个样品同一位点处的DNA片段序列，经过PCR扩增、测序，Blat比对软件对测序read进行聚类、Cap snp 软件检测单核苷酸多态性，获得多态性的SLAF标签。(2) Extract the genomic DNA of each material in (1) by CTAB method, 50 individual plants for 5 male parents, 5 female parents, only sour but not sweet, only sweet but not sour, neither sour nor sweet The genomic DNA of the mixed pools was mixed in equal amounts to form 5 mixed pools, and the genomes of the 5 mixed pools were digested respectively. The DNA fragment sequences at the same site of each sample in the mixed pools were amplified and sequenced by PCR, and compared with Blat software. Sequencing reads were clustered, Cap snp software was used to detect single nucleotide polymorphisms, and polymorphic SLAF tags were obtained.
（3）根据多态性标记与甜味、酸味性状的关联性使用比值关联法进行定位。选择不酸不甜混池中a/b>3或者b/B>3的纯和标记进行关联；分别计算群体既不酸又不甜的ac与甜不酸的ab之间和既不酸又不甜的ac与酸不甜的aa之间来源于不同基因型的差异比值，得到差异标记。将与甜味、酸味性状关联的差异标记通过blat软件比对，在双亲基因组上定位，根据标记物理位置提取出标记所在的基因，与Swissprot及Kegg数据库比对，得到与甜味性状、酸味性状相关的基因功能注释，挑选出处于功能基因外显子的SLAF标签。(3) According to the correlation between polymorphic markers and sweet and sour taste traits, use the ratio correlation method to locate. Select the pure sum mark of a/b>3 or b/B>3 in the mixed pool of neither sour nor sweet for association; respectively calculate the sum between the ac which is neither sour nor sweet and the ab which is neither sour nor sweet Differential markers were obtained from the difference ratios between the non-sweet ac and the sour-but-sweet aa derived from different genotypes. The differential markers associated with sweetness and sourness traits were compared by blat software, located on the parent genome, and the genes where the markers were located were extracted according to the physical position of the markers, compared with the Swissprot and Kegg databases, and sweetness traits and sourness traits were obtained. For related gene function annotations, SLAF tags in exons of functional genes were selected.
（4）根据定位的并处于外显子的一个甜味性状相关功能基因MELO3C011944T1所在的标签SLAF18745的多态性序列、3个酸味性状相关功能基因MELO3C020002T1、MELO3C025794T1、MELO3C026251T1的所在标签SLAF36334、SLAF50072、SLAF51212的多态性序列，利用Primer Primer5.0软件设计引物，对只甜不酸、只酸不甜、既不酸又不甜材料基因组DNA，进行PCR扩增，发展出一个甜味性状相关基因功能性分子标记SLAF18745-S01和三个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04。(4) According to the polymorphic sequence of the label SLAF18745 of a sweet taste trait-related functional gene MELO3C011944T1 located in the exon, and the tags SLAF36334, SLAF50072, and SLAF51212 of the three sour taste trait-related functional genes MELO3C020002T1, MELO3C025794T1, and MELO3C026251T1 Using the Primer Primer5.0 software to design primers for the polymorphic sequence of the polymorphic sequence, perform PCR amplification on the genomic DNA of only sweet but not sour, only sour and not sweet, neither sour nor sweet, and develop a function of a gene related to sweetness traits Sexual molecular marker SLAF18745-S01 and three functional molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 of genes related to sour taste traits.
实施例二：基于简化基因组测序（SLAF-Seq）的Super-BSAExample 2: Super-BSA based on simplified genome sequencing (SLAF-Seq)
（1）通过CTAB法提取5株父本、5株母本、F2群体只甜不酸、只酸不甜、既不酸又不甜各50个单株每个单株基因组DNA，分别等量混合形成5个混池。(1) Extract the genomic DNA of 5 male and 5 female parents, F2 populations that are only sweet but not sour, only sour but not sweet, neither sour nor sweet, and 50 individual plants, respectively, by CTAB method. Mix together to form 5 pools.
（2）SLAF-seq文库构建及测序。(2)SLAF -seq library construction and sequencing.
一是简化设计及基因组DNA酶切：利用酶切预测软件分别对步骤（1）5个基因组DNA混池进行GC含量、重复序列和基因特点分析后，利用XhoI+ MseI核酸内切酶将混合的基因组DNA切成450-500bp片段；酶切体系为基因组DNA 500ng，New Englang Biolabs 公司缓冲液 41L，XhoI、MseI各0.12L，补ddH2O至50L，试剂配好后混匀，37℃ 15h，QIAGEB公司试剂盒纯化。The first is to simplify the design and genomic DNA digestion: use the enzyme digestion prediction software to analyze the GC content, repeat sequence and gene characteristics of the five genomic DNA pools in step (1), and use XhoI+ MseI endonuclease to digest the mixed genomic DNA. Cut the DNA into 450-500bp fragments; the enzyme digestion system is 500ng of genomic DNA, 41L of buffer solution from New England Biolabs, 0.12L each of XhoI and MseI, supplemented with ddH2 O to 50L, mixed well after the reagents are prepared, 37°C for 15h, QIAGEB Company kit purification.
二是将5'末端修复：反应体系为纯化样本DNA30L，含10 mM ATP的T4 DNA连接缓冲液10L、10 mM dNTP 混合液4L、T4 DNA 聚合酶5L、Klenow酶1 L、T4 PNK5L、补ddH2O至45L，试剂配好后混匀，20℃ 30min，反应结束后用QIAGEN试剂盒纯化，33L EB回溶。The second is to repair the 5' end: the reaction system is 30L of purified sample DNA, 10L of T4 DNA ligation buffer containing 10 mM ATP, 4L of 10 mM dNTP mixture, 5L of T4 DNA polymerase, 1 L of Klenow enzyme, T4 PNK5L, supplemented with ddH2 O to 45L. After the reagents are prepared, mix them evenly and incubate at 20°C for 30 minutes. After the reaction is completed, use the QIAGEN kit to purify and redissolve in 33L EB.
三是将3'端加A：反应体系为上述步骤中纯化样本DNA 32L、Klenow 缓冲液 5L、1 mM dATP 10L、Klenow Exo - 3L、补ddH2O至50L，37℃ 30min。The third is to add A to the 3' end: the reaction system is 32L of purified sample DNA in the above steps, 5L of Klenow buffer, 10L of 1 mM dATP, 3L of Klenow Exo-3L, supplemented with ddH2 O to 50L, and 37°C for 30min.
四是连接solexa测序接头:反应体系为上述步骤中纯化样品DNA 10L、2×DNA连接缓冲液25L、solexa测序接头10L、DNA连接酶5L、补ddH2O至50L，反应结束后用QIAGEN试剂盒纯化，30L EB回溶。The fourth is to connect the solexa sequencing adapter: the reaction system is 10L of purified sample DNA in the above steps, 25L of 2×DNA ligation buffer, 10L of solexa sequencing adapter, 5L of DNA ligase, and supplemented with ddH2 O to 50L. After the reaction, use the QIAGEN kit Purification, 30L EB redissolved.
五是采用电泳切胶：提前配好2%低熔点琼脂糖并铺好胶块；将上步纯化后产物加入6 x Loading buffer 6L混匀后加入配好胶块中，加入10L ladder，120V 60min。QIAGEN胶回收试剂盒回收目的片段。The fifth is to use electrophoresis to cut the gel: prepare 2% low-melting point agarose in advance and lay the gel block; add the product purified in the previous step to 6 x Loading buffer 6L and mix well, then add it to the prepared gel block, add 10L ladder, 120V 60min . QIAGEN Gel Extraction Kit was used to recover the target fragments.
六是经PCR反应：增大起始模板量，达到上机测序所需量；反应步骤为步骤5）纯化样本DNA8L, PCR primer PE 2.0、PCR primer PE 1.0各1.5L、Phusion DNA 聚合酶20L、ddH2O 9L；反应程序为98℃预变性30s；98℃变性40s，65℃退火30s，72℃延伸30s，10-12个循环；72℃延伸5min；反应结束后用QIAGEN试剂盒纯化，30L EB回溶。Sixth, by PCR reaction: increase the amount of initial template to reach the amount required for sequencing on the machine; the reaction step is step 5) Purify sample DNA 8L, PCR primer PE 2.0, PCR primer PE 1.0 each 1.5L, Phusion DNA polymerase 20L, ddH2 O 9L; the reaction program is pre-denaturation at 98°C for 30s; denaturation at 98°C for 40s, annealing at 65°C for 30s, extension at 72°C for 30s, 10-12 cycles; extension at 72°C for 5min; after the reaction, purify with a QIAGEN kit, 30L EB dissolves back.
七是上机测序：将前步处理好的样品进行精确定量(Qubit)，然后在芯片(flow cell)表面进行桥式PCR，使DNA片段扩增为单分子DNA簇；单分子DNA簇长好后将flow cell移入Hi-Seq中，进行测序。Seventh, on-machine sequencing: accurately quantify (Qubit) the samples processed in the previous step, and then perform bridge PCR on the surface of the flow cell to amplify the DNA fragments into single-molecule DNA clusters; the single-molecule DNA clusters grow well Then move the flow cell into Hi-Seq for sequencing.
（3）SLAF标签获得：基因组经酶切处理打断为多个小片段，每个片段为一个标记位点，同一位点reads序列使用Blat软件进行相似性聚类，选出深度大于10x以上的基因型为主要基因型,最终获得46,087个SLAF标签，整体平均深度达161.81x。(3) SLAF tag acquisition: the genome is broken into multiple small fragments by enzyme digestion, each fragment is a marker site, and the reads sequences at the same site are similarly clustered using Blat software, and the reads with a depth greater than 10x are selected The genotype is the main genotype, and finally 46,087 SLAF tags are obtained, with an overall average depth of 161.81x.
（4）多态性分析：对得到的SLAF标签，根据等位基因数和基因序列之间的差异，先用blat软件比对，再用call snp软件寻找多态性位点，共得到4480个多态性标记。(4) Polymorphism analysis: For the obtained SLAF tags, according to the difference between the number of alleles and the gene sequence, first use the blat software to compare, and then use the call snp software to find polymorphic sites, and a total of 4480 were obtained polymorphic markers.
实施例三：甜瓜甜味、酸味性状相关基因挖掘Example 3: Mining of genes related to sweetness and sourness traits of melon
（1）通过对果实风味进行仪器测定结合品尝鉴定，母本为只甜不酸、父本为只酸不甜、F1为既酸又甜、F2群体中只甜不酸130株、只酸不甜77株、既不酸又不甜60株，既酸又甜233株，其中，甜：不甜=2.65：1，酸：不酸=1.63：1。结合亲本性状和F1性状及F2性状分离比，甜和酸均为显性性状，甜味偏向质量性状，酸味偏向数量性状,设计亲本为AAbbxaaBB的杂交模型。(1) Through the instrumental measurement of fruit flavor combined with tasting identification, the female parent is only sweet but not sour, the male parent is only sour but not sweet, F1 is both sour and sweet, and in the F2 population, there are 130 plants with only sweet but not sour. There were 77 sour and not sweet plants, 60 neither sour nor sweet plants, and 233 sour and sweet plants. Among them, sweet:not sweet=2.65:1, sour:not sour=1.63:1. Combining the parental traits and the segregation ratio of F1 traits and F2 traits, sweetness and sourness are both dominant traits, sweetness is biased towards quality traits, and sourness is biased toward quantitative traits, and a cross model with AAbbxaaBB as the parent is designed.
（2）对甜味、酸味性状使用比值关联法进行定位。选择既不酸又不甜混池中a/b>3或者b/B>3的纯和标记进行关联。分别计算群体ac（既不酸又不甜）与ab（只甜不酸）之间和ac（既不酸又不甜）与aa（只酸不甜）之间来源于不同基因型的差异比值，分别得到与甜味性状相关的114个、酸味性状相关215个差异标记；其中，连续3个以上满足分离比例的标记所在的区域即为关联区域，得到6个甜味性状相关候选区域，区域内共有13个差异标记，见表1，23个酸味性状相关候选区域，区域内共有48个差异标记，见表2。(2) Use the ratio correlation method to locate the sweetness and sourness traits. Select the pure sum mark with a/b>3 or b/B>3 in neither sour nor sweet mixed pool for association. Calculate the difference ratios from different genotypes between groups ac (neither sour nor sweet) and ab (only sweet but not sour) and between ac (neither sour nor sweet) and aa (only sour but not sweet) , and 114 different markers related to sweetness traits and 215 difference markers related to sourness traits were obtained respectively; among them, the region where three or more consecutive markers satisfying the separation ratio were located was the associated region, and 6 candidate regions related to sweetness traits were obtained. There are 13 differential markers in total, see Table 1, 23 candidate regions related to sourness traits, and there are 48 differential markers in the area, see Table 2.
表1：甜味性状候选区域及区域内差异标记 。Table 1: Candidate regions for sweetness traits and differential markers within regions.
表2：酸味性状候选区域及区域内差异标记。Table 2: Candidate regions for sour taste traits and differential markers within regions.
（3）将与甜味、酸味性状关联的差异标记通过软件blat比对，根据标记物理位置将标记所在的基因提取出来，与Swissprot及Kegg数据库比对，13个甜味性状差异标记中1个被定位在双亲基因组上、并处在功能基因的外显子中；48个酸味性状差异标记中3个定位在双亲基因组上、并处在功能基因的外显子中，见表3。(3) The differential markers associated with sweetness and sourness traits were compared through the software blat, and the genes where the markers were located were extracted according to the physical position of the markers, and compared with the Swissprot and Kegg databases, 1 of the 13 sweetness traits differential markers They were located on the parental genome and located in the exons of the functional genes; 3 of the 48 differential markers for sour taste traits were located on the parental genomes and located in the exons of the functional genes, as shown in Table 3.
表3：定位的甜味、酸味性状相关功能基因。Table 3: Mapped functional genes related to sweetness and sourness traits.
实施例四：甜瓜甜味、酸味性状相关基因功能性分子标记的开发Example 4: Development of functional molecular markers for genes related to sweetness and sourness traits of melon
通过对实施例三中步骤（3）得到 的1个甜味性状相关功能基因及3个酸味性状相关功能基因的SLAF标签的多态性序列，利用Primer Primer5.0软件各设计引物1对，引物序列见表4，以父本、母本、只甜不酸、只酸不甜、既不酸又不甜单株为材料，进行PCR扩增，以发展甜味、酸味性状功能性标记。PCR体系25L, 含100 ngμL–1模板DNA 1μL、10×buffer 2.5μL、2.5 mmol L–1 dNTP 2μL、10μmol L–1正向和反向引物各1 μL、5 U μL–1Taq DNA酶0.3 L、ddH2O 17.2μL。PCR反应程序为94℃预变性5 min；94℃变性50s，45-60℃退火1 min，72℃延伸1.5 min，35个循环；72℃延伸10min。用6%PAGE胶银染检测扩增产物；将只在母本、只甜不酸及父本、只酸不甜单株中有扩增产物，片段分别为249bp、66bp、283bp、274bp，见表5,而在既不酸又不甜单株中没有扩增产物的标记分别作为甜味、酸味性状功能性标记。结果成功发展了一个甜味性状相关功能性分子标记SLAF18745-S01参见附图1，三个个酸味性状相关基因功能性分子标记SLAF36334-S02参见附图2、SLAF50072-S03参见附图3和SLAF31212-S04参见附图4。By using the Primer Primer5.0 software to design a pair of primers for each of the polymorphic sequences of the SLAF tags of one sweetness trait-related functional gene and three sourness traits-related functional genes obtained in step (3) of Example 3, the primers The sequence is shown in Table 4. The male parent, female parent, only sweet but not sour, only sour but not sweet, neither sour nor sweet individual plants were used as materials for PCR amplification to develop functional markers for sweet and sour traits. PCR system 25L, containing 100 ngμL–1 template DNA 1μL, 10×buffer 2.5μL, 2.5 mmol L–1 dNTP 2μL, 10μmol L–1 forward and reverse primers 1 μL each, 5 U μL–1 Taq DNase 0.3 L. ddH2 O 17.2 μL. The PCR reaction program was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 50 s, annealing at 45-60°C for 1 min, extension at 72°C for 1.5 min, and 35 cycles; extension at 72°C for 10 min. Use 6% PAGE colloidal silver staining to detect the amplification products; there will be amplification products only in the female parent, only sweet but not sour and male parent, only sour but not sweet, the fragments are 249bp, 66bp, 283bp, 274bp, see Table 5. The markers with no amplified products in neither sour nor sweet individual plants were used as functional markers for sweet and sour traits, respectively. As a result, a functional molecular marker SLAF18745-S01 related to sweetness traits was successfully developed, see Figure 1, three functional molecular markers SLAF36334-S02 related to genes related to sourness traits, see Figure 2, SLAF50072-S03, see Figure 3 and SLAF31212- S04 see accompanying drawing 4.
实施例五：甜味、酸味性状相关基因功能性标记对FExample 5: Functional marker pairs of genes related to sweetness and sourness traits F11、F, F22单株检测Single plant detection
进一步对F1、F2单株材料进行PCR扩增，验证获得的甜味、酸味性状相关基因功能性分子标记的稳定性。甜味性状相关基因功能性分子标记SLAF18745-S01在F1及F2中只甜不酸、既酸又甜材料中扩增出249bp条带，而F2中既不酸又不甜、只酸不甜材料中没有出现，参见附图5；酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03、SLAF31212-S04只是在F1及F2中只酸不甜、既酸又甜材料中扩增出66bp、283bp、274bp条带，而F2中既不酸又不甜、只甜不酸材料中没有出现，参见附图6、附图7、附图8，说明4个功能性分子标记是稳定的。PCR amplification was further performed on F1 and F2 individual plant materials to verify the stability of the obtained functional molecular markers related to sweetness and sourness traits. The functional molecular marker SLAF18745-S01 of the gene related to sweet taste traits amplified a 249bp band in F1 and F2 which was only sweet but not sour, and both sour and sweet, while F2 was neither sour nor sweet and only sour It does not appear in non-sweet materials, see Figure 5; the functional molecular markers SLAF36334-S02, SLAF50072-S03, and SLAF31212-S04 of genes related to sourness traits are only sour but not sweet, and both sour and sweet materials in F1 and F2 66bp, 283bp, and 274bp bands were amplified, but neither sour nor sweet, only sweet but not sour materials did not appear in F2 , see attached drawings 6, 7, and 8 to illustrate the four functional molecules Markup is stable.
实施例六：甜味、酸味性状相关基因功能性标记的应用Example 6: Application of functional markers of genes related to sweet taste and sour taste traits
以自交系“寿星”、“酸甜瓜”为亲本的F2、F3后代群体播种后，取植株子叶或叶片提取基因组DNA，用引物SLAF_No.1、SLAF_No.2、SLAF_No.3、SLAF_No.4对DNA进行PCR扩增和电泳检测，扩增和检测方法为同上，具有甜酸味的植株能够分别扩增出249bp、66bp、283bp、274bp条带，而既不甜又不酸的植株在同一位置不能扩增出条带。从提取基因组DNA到得到鉴定结果，在实验室内只需要1-2个工作日即可完成。After sowing the F2 and F3 progeny populations of the inbred lines "Shouxing" and "Sour Melon" as parents, the cotyledons or leaves of the plants were taken to extract genomic DNA, and primers SLAF_No.1, SLAF_No.2, SLAF_No.3, SLAF_No. 4 Perform PCR amplification and electrophoresis detection on the DNA. The amplification and detection methods are the same as above. The plants with sweet and sour taste can amplify 249bp, 66bp, 283bp, and 274bp bands respectively, while the plants that are neither sweet nor sour can be amplified in the same The position cannot amplify a band. From the extraction of genomic DNA to the identification results, it only takes 1-2 working days in the laboratory.
在现有甜瓜品质育种实践中，首先要收集具有不同果实风味基因的亲本，进行一系列杂交回交建立群体，对甜味、酸味等风味性状主要是等果实成熟后，逐个进行鉴定，从播种到果实成熟，至少需要65天以上，致使选育周期长，效率低，成本高。通过一个甜味性状相关基因功能性分子标记SLAF18745-S01和三个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03、SLAF31212-S04检测甜瓜甜酸味，可以在实验室内对苗期单株进行甜酸味的基因型鉴定，既节约生产成本而且极大提高选择的效率，加速甜瓜品质性状的遗传改良。In the existing practice of muskmelon quality breeding, it is first necessary to collect parents with different fruit flavor genes, and carry out a series of cross-crossing backcrosses to establish populations. The sweetness, sourness and other flavor traits are mainly identified one by one after the fruit matures, and the It takes at least 65 days until the fruit matures, resulting in long breeding cycle, low efficiency and high cost. One functional molecular marker SLAF18745-S01 of a gene related to sweetness traits and three functional molecular markers SLAF36334-S02, SLAF50072-S03, and SLAF31212-S04 of genes related to sourness traits can be used to detect the sweet and sour taste of melons, which can be tested in the laboratory for single The genotype identification of sweet and sour taste can not only save production cost but also greatly improve the efficiency of selection, and accelerate the genetic improvement of melon quality traits.
表4：1个甜味、3个酸味功能性分子标记的引物序列Table 4: Primer sequences for 1 sweet and 3 sour functional molecular markers
表5：1个甜味、3个酸味功能性分子标记的DNA序列Table 5: DNA sequences of 1 sweet and 3 sour functional molecular markers
综上所述，本发明利用super-BAS技术仅用四个月的时间，即获得与甜瓜甜味、酸味性状功能性分子标记和精细定位区域，周期短、成本低、效率高，可为在其它物种中开发功能性分子标记提供重要的成功案例，也为分子育种、系统进化、种质资源鉴定中分子标记提供了一种重要的开发途径。In summary, the present invention uses the super-BAS technology to obtain functional molecular markers and fine-positioned regions for sweet and sour properties of melons in only four months, with a short period, low cost, and high efficiency. The development of functional molecular markers in other species provides important successful cases, and also provides an important development approach for molecular markers in molecular breeding, phylogenetic evolution, and identification of germplasm resources.
通过上述各实施例的系列试验，证实了本发明首次获得一个甜味性状相关基因功能性分子标记SLAF18745-S01和三个酸味性状相关基因功能性分子标记SLAF36334-S02、SLAF50072-S03和SLAF31212-S04，通过应用甜瓜果实甜味、酸味性状相关基因功能性分子标记，在不同世代间重复性好、操作简便，为甜瓜品质性状基因的分子标记辅助选择提供了重要的标记基础，可简便、快速的应用于育种实践。Through the above-mentioned series of experiments in each example, it is confirmed that the present invention obtains for the first time a functional molecular marker SLAF18745-S01 of a gene related to sweet taste traits and three functional molecular markers SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 of genes related to sour taste traits , through the application of functional molecular markers for genes related to sweetness and sourness traits of melon fruit, the reproducibility and easy operation between different generations provide an important marker basis for molecular marker-assisted selection of melon quality trait genes, which can be easily and quickly used in breeding practice.
Organization ApplicantOrganization Applicant
Street : 南昌路403号 Street : No. 403, Nanchang Road
City : 乌鲁木齐市 City : Urumqi
State : 新疆 State : Xinjiang
Country : 中国Country : China
PostalCode : 830091 PostalCode : 830091
PhoneNumber : 0991-4539658 PhoneNumber : 0991-4539658
FaxNumber : 0991-4539658FaxNumber : 0991-4539658
<110> OrganizationName : 新疆农业科学院哈密瓜研究中心<110> OrganizationName : Hami Melon Research Center of Xinjiang Academy of Agricultural Sciences
Application ProjectApplication Project
<120> Title : 一种甜瓜甜味酸味性状相关基因功能性分子标记及应用<120> Title : A functional molecular marker and application of a gene related to sweet and sour taste traits of melon
<130> AppFileReference : Jordi Garcia-Masa.The genome of melon (Cucumis melo L.).PNAS, 2012, 109(29): 11872-11877.<130> AppFileReference : Jordi Garcia-Masa. The genome of melon (Cucumis melo L.). PNAS, 2012, 109(29): 11872-11877.
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