WO2019128461A1 - 与南瓜光周期不敏感性状紧密连锁的Indel分子标记及其应用 - Google Patents

与南瓜光周期不敏感性状紧密连锁的Indel分子标记及其应用 Download PDF

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WO2019128461A1
WO2019128461A1 PCT/CN2018/113460 CN2018113460W WO2019128461A1 WO 2019128461 A1 WO2019128461 A1 WO 2019128461A1 CN 2018113460 W CN2018113460 W CN 2018113460W WO 2019128461 A1 WO2019128461 A1 WO 2019128461A1
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pumpkin
photoperiod
indel
seq7593
molecular marker
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French (fr)
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钟玉娟
黄河勋
李俊星
罗文龙
吴廷全
王瑞
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广东省农业科学院蔬菜研究所
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Priority to US16/497,863 priority Critical patent/US11492668B2/en
Publication of WO2019128461A1 publication Critical patent/WO2019128461A1/zh

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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention belongs to the technical field of molecular detection, and particularly relates to an Indel molecular marker closely linked to the photoperiod insensitivity of pumpkin and its application.
  • Chinese pumpkin is one of the three major cultivars of pumpkin, and its yield and planting area rank first among the three cultivars.
  • Chinese pumpkins are short-lived plants of the same sex and are similar to light and temperature sensitivity.
  • the suitable sowing period in south China is short (1-3 months), and the female flower differentiation in April is reduced to lead to severe production reduction.
  • Domestic Chinese pumpkin The main varieties have planting area restrictions.
  • honey pumpkins and derivatives are not suitable for planting in the area north of the Yellow River. It can be seen that the light-temperature sensitive characteristics make Chinese pumpkins greatly limited in planting time and geographical distribution.
  • the flowering of plants originating from tropical and subtropical regions is mainly affected by the photoperiod. Therefore, the selection of photoperiod-insensitive Chinese pumpkin varieties has broader application prospects.
  • the inventors conducted QTL mapping on the photoperiod-insensitive traits of pumpkin, and obtained a molecular marker closely linked to it, named SEQ7593.
  • SEQ7593 a molecular marker closely linked to it.
  • sequence deletion fragment TCATA shown in SEQ ID NO. 1 is found, that is, the base fragment of the sequence of SEQ ID NO. 1 from the 129th to the 133th position from 5' to 5 bp in size is deleted, and the size is 275 bp as SEQ ID.
  • the sequence shown in NO. 2 shows that the corresponding pumpkin exhibits light sensitivity.
  • Indel molecular marker SEQ7593 which is closely linked to the photoperiod insensitivity of pumpkin, is located on chromosome 10 of Chinese pumpkin, and has a size of 280 bp. Its nucleotide sequence is shown in SEQ ID NO. 1; its corresponding Indel sequence lacks SEQ. A base fragment of 5 bp in size from position 129 to position 133 of ID NO.1.
  • a primer pair for amplifying the Indel molecular marker SEQ7593 of claim 1 and/or its corresponding Indel sequence A primer pair for amplifying the above Indel molecule marker SEQ7593.
  • nucleotide sequence of the primer pair is as follows:
  • F1 5'-TTTCAGCTCTTACCCTATTCTTC-3' (SEQ ID NO. 3),
  • R1 5'-CACATTTTACTCCTTAACTTGGAC-3' (SEQ ID NO. 4).
  • a kit for pumpkin photoperiod-insensitive assisted breeding comprising an agent for detecting the presence of a TCATA deletion in the Indel molecular marker SEQ7593 of claim 1.
  • the reagent comprises a primer pair that amplifies the Indel molecular marker SEQ7593 and/or its corresponding Indel sequence.
  • nucleotide sequence of the primer pair is as follows:
  • F1 5'-TTTCAGCTCTTACCCTATTCTTC-3' (SEQ ID NO. 3),
  • R1 5'-CACATTTTACTCCTTAACTTGGAC-3' (SEQ ID NO. 4).
  • a method for assisting the breeding of pumpkin photoperiod insensitive comprising the following steps:
  • the photoperiod sensitivity of pumpkin is judged. Among them, if there is a TCATA deletion, it indicates that the pumpkin has light sensitivity; if there is no TCATA deletion, it indicates that the pumpkin has light insensitivity.
  • a method for photoperiod-insensitive breeding of pumpkin comprising knocking out a base fragment of 5 bp in size from position 129 to position 133 of Indel molecule marker SEQ7593 SEQ ID NO.
  • the present invention performs QTL mapping on the photoperiod insensitivity of pumpkin, and obtains the molecular marker SEQ7593 closely linked thereto, which has a high contribution rate and a phenotypic probability of 30%, and can be used for molecular markers of photoperiod insensitivity.
  • Establishment of an auxiliary breeding system PCR amplification primers designed according to Indel molecular markers can be applied to improved molecular-assisted breeding of pumpkin varieties in a simple, rapid and high-throughput manner, providing molecular breeding for reducing the dependence of pumpkin on long-day exposure and breaking planting time and planting area.
  • Technical support while greatly reducing the time of traditional gene mapping.
  • Figure 1 is a photoperiod insensitive parent (PPIS) and a photoperiod sensitive parent (PPS);
  • Figure 2 is a preliminary map of the photoperiod insensitivity of pumpkin in the high-density genetic linkage map: the abscissa indicates the position of the linkage group, and the ordinate indicates the LOD value; the threshold of the black line marker is the correlation threshold representing p ⁇ 0.001, indicating Relevance is extremely reliable;
  • Figure 3 is the result of PCR amplification of Indel molecular marker SEQ7593:
  • P1 and P2 are the product band types of photoperiod-insensitive parent and photoperiod-sensitive male parent, respectively, wherein the length of P1 amplified fragment is 280 bp; There are 280 bp and 275 bp fragments, and there are three types of P1, P2 and F1 in the random plant of the F2 population.
  • the molecular biology experimental techniques used in the following examples include DNA extraction, PCR amplification, PAGE gel electrophoresis, etc., unless otherwise specified, usually according to conventional methods, see the "Molecular Cloning Experiment Guide” (third Edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitang et al., 2002, Beijing: Science Press), or in accordance with the conditions recommended by the manufacturer.
  • Photoperiod insensitive and photoperiod sensitive materials are high-generation inbred lines obtained locally in Guangdong, named PPIS and PPS, respectively, as shown in Figure 1. Hybridization of PPIS and PPS yielded F1, and F1 self-crossed to obtain F2, which was used as a genetic analysis and localization population.
  • the pumpkin parent and 160 F2 population genomic DNA were extracted by CTAB method, and the extracted single DNA was used for library construction.
  • a total of 52,246 polymorphic SLAF tags were developed, and 4,655 high-quality SLAF tags were selected. Filters with other MLOD values below 5 were filtered out, and the linkage group was used as the unit. The high-software software was used to obtain the linearity of the Marker in the linkage group. Arrange and estimate the genetic distance between adjacent markers, and finally obtain a genetic map with a total map distance of 2,502.01 cM, which is divided into 20 linkage groups and obtain 8,051 upper SNP markers.
  • the MapQTL software was used to analyze the phenotypic data and genetic map information of the population by using the composite interval mapping method (MQM) to obtain the trait-related QTL.
  • the number of replacement tests was set to 1000, and the QTL criterion was: when the p value was less than 0.01
  • the LOD value is used as the threshold for screening, and is indicated by a black line in the figure.
  • the threshold value is expressed as a linkage location of a gene.
  • the LOD has a LOD value of 5.6.
  • One group represents a linkage group, and the Ppd gene is located in the 6th linkage group (Fig. 2), ranging from 35.00 cM to 38.30 cM.
  • the genetic distance of the marker was 3.30 cM (Fig. 2).
  • the markers at both ends of the interval were compared to the Chinese pumpkin genome for a total of 229.3 Kb.
  • the polymorphic marker of Indel was developed near the peak marker, and the whole genome was re-sequenced by the parental parent.
  • Interval Indel markers were used to detect small fragment insertion and deletion sites of less than 50 bp in length using SAMTOOLS software, and designed according to the sequence of 200 bp upstream and downstream.
  • the Indel tag SEQ7593 primer sequence is: F1: 5'-TTTCAGCTCTTACCCTATTCTTC-3' (SEQ ID NO. 3), R1: 5'-CACATTTTACTCCTTAACTTGGAC-3' (SEQ ID NO. 4).
  • the PCR amplification system used a 20 ⁇ L amplification system containing 1 U Taq enzyme, 1 ⁇ L of template DNA, 1 ⁇ L of dNTP, 1.5 ⁇ L of primer, 2 ⁇ L of 10 ⁇ PCR buffer, and ddH 2 O to 20 ⁇ L.
  • the PCR amplification procedure was: 94 ° C for 3 min, the cycle was 94 ° C for 30 s, annealing for 30 s, 72 ° C for 1 min, 30 cycles, and finally extended at 72 ° C for 10 min.
  • the annealing temperature was 54 °C.
  • Indel molecular marker SEQ7593 is a tightly linked marker of pumpkin photoperiod insensitivity, which is located on chromosome 10 of Chinese pumpkin, with a size of 280 bp. The sequence is shown in SEQ ID NO.
  • sequence of SEQ ID NO. 1 is deleted, the fragment of SEQ ID NO. 1 is deleted, and the base fragment of 5 bp in size from position 129 to position 133 from 5' is deleted, and the size is 275 bp as SEQ ID NO.
  • the sequence of .2, the corresponding pumpkin showed light sensitivity.
  • FIG. 3 shows the results of PCR amplification of Indel molecular marker SEQ7593:
  • P1 and P2 are the product bands of photoperiod-insensitive maternal and photoperiod sensitive paternal, respectively, wherein P1 amplified fragment length is 280 bp; P2 amplified fragment length It is 275 bp.
  • F1 there are two types of F1, there are 280 bp and 275 bp fragments, and there are three types of P1, P2 and F1 in the random group of F2 population.
  • the phenotype of photoperiod sensitivity found in combination with the phenotype of the individual was consistent with the results of PCR.
  • the above Indel molecular markers can separate photoperiod sensitive and insensitive individual plants.

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Abstract

提供与南瓜光周期不敏感性状紧密连锁的Indel分子标记，所述Indel分子标记SEQ7593定位于中国南瓜第10号染色体上，其核苷酸序列如SEQ ID NO.1所示，其对应的Indel序列缺少SEQ ID NO.1的第129位至第133位碱基片段。还提供用于南瓜光周期不敏感辅助育种的试剂盒和南瓜光周期不敏感辅助育种的方法。

Description

与南瓜光周期不敏感性状紧密连锁的Indel分子标记及其应用 技术领域
本发明属于分子检测技术领域，具体涉及与南瓜光周期不敏感性状紧密连锁的Indel分子标记及其应用。
背景技术
中国南瓜是南瓜的三大栽培种之一，其产量和种植面积位居三个栽培种之首。然而中国南瓜属雌雄异花同株的短日植物，大都具有光温敏感性，春季华南地区的适宜播种期短(1-3月)，延至4月播种雌花分化减少导致严重减产，国内中国南瓜主栽品种存在种植地域限制，例如，蜜本南瓜及衍生品种不适宜在黄河以北地区种植，由此可见，光温敏感特性使中国南瓜在播种时间和地理分布受到极大限制。此外，起源于热带和亚热带地区植物的开花主要受光周期的影响，因此，选育光周期不敏感中国南瓜品种具有更广阔的应用前景。
传统育种方式选育光周期不敏感品种困难，一般要在长日照下筛选，且耗时费力，大大影响我国的南瓜育种事业。但随着高通量测序技术的成熟，特别是开发大量的SNP(单碱基扩增多态性)标记和应用高密度遗传图谱结合方法开展植物性状精细定位成为挖掘植物基因的热点之一。进而基于南瓜的全基因组信息，开发性状连锁的Indel分子标记进行品种的初期筛选，达到分子辅助育种的目的，可大大缩短育种的周期提高育种效率。因此，进行南瓜光周期不敏感的基因定位，筛选紧密连锁的分子标记，建立早期辅助选择技术体系，对光周期不敏感的遗传改良具有重要意义。
发明内容
本发明的目的在于提供与南瓜光周期不敏感性状紧密连锁的Indel分子标记及其应用。
本发明所采取的技术方案是：
发明人通过研究，对南瓜光周期不敏感性状进行了QTL定位，筛选获得了与之紧密连锁的分子标记，命名为SEQ7593。通过测序，如果发现这段序列如SEQ ID NO.1所示，则相应的南瓜表现出光不敏感性状。如果发现SEQ ID NO.1所示序列缺失片段TCATA，即缺失SEQ ID NO.1所示序列自5’起第129位至第133位共5bp大小的碱基片段，获得大小为275bp如SEQ ID NO.2所示序列，则相应的南瓜表现出光敏感性状。
Figure PCTCN2018113460-appb-000001
Figure PCTCN2018113460-appb-000002
Figure PCTCN2018113460-appb-000003
与南瓜光周期不敏感性状紧密连锁的Indel分子标记SEQ7593，定位于中国南瓜第10号染色体上，大小为280bp，其核苷酸序列如SEQ ID NO.1所示；其对应的Indel序列缺少SEQ ID NO.1的第129位至第133位共5bp大小的碱基片段。
用于扩增权利要求1所述的Indel分子标记SEQ7593和/或其对应的Indel序列的引物对。用于扩增上述Indel分子标记SEQ7593的引物对。
优选的，所述的引物对的核苷酸序列如下所示：
F1：5’-TTTCAGCTCTTACCCTATTCTTC-3’(SEQ ID NO.3)，
R1：5’-CACATTTTACTCCTTAACTTGGAC-3’(SEQ ID NO.4)。
一种用于南瓜光周期不敏感辅助育种的试剂盒，包括用于检测权利要求1所述Indel分子标记SEQ7593是否存在 TCATA缺失的试剂。
进一步的，试剂包括扩增Indel分子标记SEQ7593和/或其对应的Indel序列的引物对。
进一步的，引物对的核苷酸序列如下所示：
F1：5’-TTTCAGCTCTTACCCTATTCTTC-3’(SEQ ID NO.3)，
R1：5’-CACATTTTACTCCTTAACTTGGAC-3’(SEQ ID NO.4)。
一种南瓜光周期不敏感辅助育种的方法，包括如下步骤：
1)提取待测南瓜基因组DNA，检测其Indel分子标记SEQ7593是否存在 TCATA缺失；
2)根据检测结果，判断南瓜的光周期敏感性，其中，如存在 TCATA缺失，表明南瓜具有光敏感性状；如不存在 TCATA缺失，表明南瓜具有光不敏感性状。
用于检测Indel分子标记SEQ7593是否存在 TCATA缺失的试剂在南瓜光周期不敏感辅助育种中的应用。
一种南瓜光周期不敏感育种的方法，包括敲除权利要求1所述Indel分子标记SEQ7593 SEQ ID NO.1的第129位至第133位共5bp大小的碱基片段。
本发明的有益效果是：
本发明对南瓜光周期不敏感性状进行了QTL定位，筛选获得了与之紧密连锁的分子标记 所SEQ7593，其贡献率高，解释表型概率达30％，可用于光周期不敏感性状的分子标记辅助育种体系的建立。根据Indel分子标记设计的PCR扩增引物可以简便、快速、高通量地应用于南瓜品种改良分子辅助育种，为减少南瓜对长日照依赖性和打破种植时间和种植地域的限制有关的分子育种提供技术支持，同时大大缩短了传统基因定位的时间。
附图说明
图1为光周期不敏感母本(PPIS)和光周期敏感父本(PPS)；
图2为南瓜光周期不敏感性状在高密度遗传连锁图谱的初步定位结果图：横坐标表示连锁群的位置，纵坐标表示LOD值；黑线标记的阈值是代表p<0.001的关联阈值，表示关联性极可靠；
图3为Indel分子标记SEQ7593的PCR扩增结果：P1、P2分别为光周期不敏感母本和光周期敏感父本的产物带型，其中P1扩增的片段长度为280bp；F1两种带型，存在280bp和275bp的片段，F2群体的随机单株中存在P1，P2和F1的三种类型。
具体实施方式
下面结合实施例对本发明作进一步的说明，但并不局限于此。
以下实施例中所采用的分子生物学实验技术包括DNA提取、PCR扩增、PAGE凝胶电泳等实验，如无特殊说明，通常按照常规方法操作，具体可参见《分子克隆实验指南》(第三版)(Sambrook J,Russell DW，Janssen K,Argentine J.黄培堂等译，2002，北京：科学出版社)，或按照制造厂商所建议的条件。
实施例1
一、遗传群体的构建及遗传分析
1、供试材料植物材料：光周期不敏感和光周期敏感材料均是广东本地获得的高代自交系，分别命名为PPIS和PPS，见图1。PPIS和PPS杂交获得F1，F1自交得F2，用作遗传分析和定位群体。
2、供试材料光周期不敏感性的确定和遗传规律分析。
对162株F2群体单株于4月播种，开花期间为长日照，调查第一雌花的开花节位，用Excel 2016处理数据，检测是否服从正态分布。
二、南瓜遗传图谱构建和果皮颜色的初步定位
1、南瓜基因组DNA的提取
使用CTAB法提取南瓜亲本及160株F2群体基因组DNA，提取的单株DNA用于文库构建。
2、遗传图谱构建
本研究前期委托北京百迈克生物科技有限公司利用SLAF-Seq技术进行高通量测序，一共162个样本，采用HaeIII和Hpy166II进行酶切，对得到的酶切片段(SLAF标签)进行3′端加A处理、连接Dual-index测序接头、PCR扩增、纯化、混样、切胶选取目的片段，文库质检合格后用IlluminaHiSeq进行PE125bp测序。插入片段长度为500bp；测序类型为PE125；去掉用于区分样品的标签序列，实际Read长度为2×100bp。对初始SNP集进行过滤，可得到较为可靠的基因型数据。共开发多态性SLAF标签52,246个，选取4,655个高质量SLAF标签，过滤掉与其他SLAF标签的MLOD值均低于5的标签，连锁群为单位，采用HighMap软件分析获得连锁群内Marker的线性排列，并估算相邻Marker间的遗传距离，最终得到图谱总图距为2,502.01cM的遗传图谱图谱，共分为20个连锁群，获得8,051个上图SNP标记。
3、光周期不敏感基因Ppd的定位
利用MapQTL软件采用复合区间作图法(MQM)对群体的表型数据和遗传图谱信息进行分析计算以获得性状相关的QTL，置换检验次数设为1000，QTL判断标准为：p值小于0.01时对应的LOD值作为筛选的阈值，图中以黑线表示。超过阈值表示为一个基因的连锁定位区间，黑线的LOD值为5.6，一个group代表一个连锁群，将Ppd基因定位在6号连锁群(图2)，区间从35.00cM到38.30cM，两个标记的遗传距离为3.30cM(图2)，将该区间两头的标记比对中国南瓜基因组，共229.3Kb，在峰值标记附近开发Indel的多态标记，利用双亲本的全基因组重测序获得这段区间的Indel标记，利用SAMTOOLS软件检测长度小于50bp的小片段插入与缺失位点，并根据该位点上下游200bp的序列，用premier 5.0软件设计而成。Indel标记SEQ7593引物序列为：F1：5’-TTTCAGCTCTTACCCTATTCTTC-3’(SEQ ID NO.3)，R1：5’-CACATTTTACTCCTTAACTTGGAC-3’(SEQ ID NO.4)。PCR扩增体系使用20μL的扩增体系，包含1U Taq酶，1μL模板DNA，1μL的dNTP，1.5μL引物，2μL的10×PCR buffer，加ddH 2O至20μL。PCR扩增程序为：94℃3min，循环过程为94℃30s、退火30s、72℃1min、30个循环，最后72℃延伸10min。退火温度为54℃。
对中国南瓜PPIS、PPS个体表型与基因型的比较分析，确定Indel分子标记SEQ7593为南瓜光周期不敏感性状紧密连锁标记，定位于中国南瓜第10号染色体上，大小为280bp，其核苷酸序列如SEQ ID NO.1所示。
Figure PCTCN2018113460-appb-000004
SEQ ID NO.1所述序列如果缺失片段TCATA，即缺失SEQ ID NO.1所示序列自5’起第129位至第133位共5bp大小的碱基片段，获得大小为275bp如SEQ ID NO.2所述序列，则相应的南瓜表现出光敏感性状。
Figure PCTCN2018113460-appb-000005
实施例2
采用实施例1的方法在两亲本间进行PCR扩增，使用聚丙烯酰胺凝胶电泳检测，亲本间表现特异，鉴定F2群体单株33个，结果见图3。
图3为Indel分子标记SEQ7593的PCR扩增结果：P1、P2分别为光周期不敏感母本和光周期敏感父本的产物带型，其中P1扩增的片段长度为280bp；P2扩增的片段长度为275bp。F1两种带型，存在280bp和275bp的片段，F2群体的随机单株中存在P1，P2和F1的三种类型。
结合单株表型发现光周期敏感性的表型与PCR的结果一致。上述Indel分子标记可以将光周期敏感和不敏感的单株分开。
以上实施例仅为介绍本发明的优选案例，对于本领域技术人员来说，在不背离本发明精神的范围内所进行的任何显而易见的变化和改进，都应被视为本发明的一部分。

Claims (9)

  1. 与南瓜光周期不敏感性状紧密连锁的Indel分子标记SEQ7593，其特征在于：所述Indel分子标记SEQ7593定位于中国南瓜第10号染色体上，大小为280bp，其核苷酸序列如SEQ ID NO.1所示；其对应的Indel序列缺少SEQ ID NO.1的第129位至第133位共5bp大小的碱基片段。
  2. 用于扩增权利要求1所述的Indel分子标记SEQ7593和/或其对应的Indel序列的引物对。
  3. 根据权利要求2所述的引物对，其特征在于，其核苷酸序列如下所示：
    F1：5’-TTTCAGCTCTTACCCTATTCTTC-3’，
    R1：5’-CACATTTTACTCCTTAACTTGGAC-3’。
  4. 一种用于南瓜光周期不敏感辅助育种的试剂盒，其特征在于：包括用于检测权利要求1所述Indel分子标记SEQ7593是否存在 TCATA缺失的试剂。
  5. 根据权利要求4所述的试剂盒，其特征在于：所述试剂包括扩增Indel分子标记SEQ7593和/或其对应的Indel序列的引物对。
  6. 根据权利要求5所述的试剂盒，其特征在于：扩增Indel分子标记SEQ7593的引物对如下：
    F1：5’-TTTCAGCTCTTACCCTATTCTTC-3’，
    R1：5’-CACATTTTACTCCTTAACTTGGAC-3’。
  7. 一种南瓜光周期不敏感辅助育种的方法，包括如下步骤：
    1)提取待测南瓜基因组DNA，检测其Indel分子标记SEQ7593是否存在 TCATA缺失；
    2)根据检测结果，判断南瓜的光周期敏感性，其中，如存在 TCATA缺失，表明南瓜具有光敏感性状；如不存在 TCATA缺失，表明南瓜具有光不敏感性状。
  8. 用于检测权利要求1所述Indel分子标记SEQ7593是否存在 TCATA缺失的试剂在南瓜光周期不敏感辅助育种中的应用。
  9. 一种南瓜光周期不敏感育种的方法，包括敲除权利要求1所述Indel分子标记SEQ7593SEQ ID NO.1的第129位至第133位共5bp大小的碱基片段。
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