CN114182041B - PARMS molecular marker related to number of ovules of rape per ovary and application - Google Patents
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Abstract
Description
技术领域technical field
本发明属于分子生物学及遗传育种技术领域,具体涉及一种与甘蓝型油菜每子房胚珠数显著关联的分子标记及其应用。The invention belongs to the technical field of molecular biology and genetic breeding, and specifically relates to a molecular marker significantly related to the number of ovules per ovary of Brassica napus and its application.
背景技术Background technique
菜籽油是国产食用植物油的第一大来源,占比为我国植物油生产总量的55%以上(王汉中,2018)。然而我国国产植物油自给率不足35%,严重威胁其供给安全。为了保障我国国产植物油的有效供给,在种植面积增长乏力的情况下,持续提高单位面积产油量(=单产×含油量)是唯一出路。近年来,我国油菜高油分育种取得突破(傅廷栋,2014)而单产仍然低于世界平均水平(http://apps.fas.usda.gov/psdonline/)且增加十分缓慢,这严重影响了油菜种植效益和油菜产业的国际竞争力。因此,我国油菜单产亟待提高(殷艳和王汉中,2012)。Rapeseed oil is the largest source of domestic edible vegetable oil, accounting for more than 55% of my country's total vegetable oil production (Wang Hanzhong, 2018). However, the self-sufficiency rate of domestic vegetable oil in my country is less than 35%, which seriously threatens its supply security. In order to ensure the effective supply of domestic vegetable oil in our country, in the case of sluggish planting area growth, the only way out is to continuously increase the oil production per unit area (=unit yield×oil content). In recent years, breakthroughs have been made in the breeding of rapeseed with high oil content in my country (Fu Tingdong, 2014), but the yield per unit area is still lower than the world average (http://apps.fas.usda.gov/psdonline/) and the increase is very slow, which seriously affects the cultivation of rapeseed Benefits and international competitiveness of the rapeseed industry. Therefore, my country's rapeseed production needs to be improved urgently (Yin Yan and Wang Hanzhong, 2012).
在同一种植密度条件下,油菜单产取决于单株产量,而单株产量直接决定于全株角果数、每角粒数和粒重三个构成因子。虽然油菜产量的三个构成因子之间呈现一定程度的负相关,但其系数往往不大,因此可以通过提高产量构成因子(如每角粒数)来增加产量。研究表明,每角粒数可以进一步分解为每子房胚珠数、可育胚珠比、可育胚珠受精率、受精胚珠发育率这四个子性状的乘积(杨玉花,2017)。因此,油菜每子房胚珠数是每角粒数的上限,决定其最大潜力。Yang等(2017)利用每角粒数极端的9份材料进行系统的遗传分析和细胞学观察,结果表明每子房胚珠数和受精胚珠发育率是影响每角粒数的主要因素。相对于油菜产量构成三因子,每子房胚珠数表型的鉴定无法肉眼进行,必须经过前期繁琐的细胞学处理然后在体式显微镜下观察计数。因此,为了对每子房胚珠数进行准确和高通量的筛选,必须开发可靠实用的分子标记进行辅助选择。Under the condition of the same planting density, the yield of rapeseed depends on the yield per plant, and the yield per plant directly depends on the three components of the number of siliques in the whole plant, the number of grains per horn and the weight of grains. Although there is a certain degree of negative correlation among the three constituent factors of rapeseed yield, the coefficients are often small, so the yield can be increased by increasing the yield constituent factors (such as the number of seeds per corner). Studies have shown that the number of grains per horn can be further decomposed into the product of four sub-traits: the number of ovules per ovary, the ratio of fertile ovules, the fertilization rate of fertile ovules, and the development rate of fertilized ovules (Yang Yuhua, 2017). Therefore, the number of ovules per ovary of rapeseed is the upper limit of the number of seeds per horn, which determines its maximum potential. Yang et al. (2017) conducted systematic genetic analysis and cytological observations using 9 materials with extreme number of grains per horn. The results showed that the number of ovules per ovary and the development rate of fertilized ovules were the main factors affecting the number of grains per horn. Compared with the three factors constituting the yield of rapeseed, the identification of the ovule number phenotype per ovary cannot be carried out with the naked eye, and must go through tedious early cytological processing and then observe and count under a stereomicroscope. Therefore, for accurate and high-throughput screening of ovule number per ovary, it is necessary to develop reliable and practical molecular markers for assisted selection.
随着分子标记技术的不断发展,其在作物中的应用越来越广泛。Grodzicker等(1974)创立了限制性片段长度多态性(restriction fragment length polymorphism,RFLP)标记技术。RFLP是第一代分子标记,具有数量丰富、稳定遗传、专一性、重复性好、共显性等特点。但是,该标记需要的DNA量比较大;且操作程序繁琐、耗时费力、周期长;还需要使用放射性同位素对探针进行标记,这些因素都局限了RFLP标记的广泛使用。AFLP标记结合了PCR和RFLP标记技术,在作物遗传多样性研究、细胞学研究、品种纯度鉴定和抗病等研究中得到广泛的应用(宋顺华等,2006;袁素霞,2009;王雪,2004)。但AFLP标记也存在一些缺点:成本较高,过程复杂,技术难度大;标记大多为显性标记;对DNA质量和限制性内切酶质量要求较高。SSR标记,也叫微卫星DNA标记(microsatellite DNA),已经被广泛应用于作物的基因定位、分子标记辅助选择、DNA指纹图谱、品种纯度鉴定、种质资源的保存及利用和遗传多样性分析等研究中(陈烨丽,2010;缪体云,2007;荆赞革,2010;王冬梅,2011)。SSR标记具有数量丰富、多态性高、操作简单、成本较低等优点,但难以实现高通量批量化的检测。近十年来,随着测序技术的持续进步,使得基于基因组序列信息的分子标记开发成为可能,如SNP标记和InDel标记(Hyten et al.,2010)。目前,全基因组选择育种芯片还只在水稻中开始尝试(Yu et al.,2014),油菜等其他作物仍以分子标记辅助选择为主。目前SNP检测方法主要分为两大类:一大类是以单链构象多态性(SS CP)、变性梯度凝胶电泳(DDGE)、酶切扩增多态性序列(CAPS)、等位基因特异性PCR(AS-PCR)等为代表的以凝胶电泳为基础的传统经典的检测方法,另一大类是以直接测序、DNA芯片、变性高效液相色谱(DHPLC)、质谱检测技术、高分辨率溶解曲线(HRM)等为代表的高通量、自动化程度较高的检测方法。这两类方法各有优缺点,目前已有将两者结合起来的检测方法如KASP(Lister et.,2013)和PARMS(Lu et al.,2020)等,不但操作简便、成本低廉而且能实现高通量的检测。With the continuous development of molecular marker technology, its application in crops is becoming more and more extensive. Grodzicker et al. (1974) created the restriction fragment length polymorphism (restriction fragment length polymorphism, RFLP) marker technology. RFLP is the first-generation molecular marker, which has the characteristics of abundant quantity, stable inheritance, specificity, good repeatability, and co-dominance. However, the amount of DNA required for this labeling is relatively large; the operation procedures are cumbersome, time-consuming and labor-intensive, and the cycle is long; radioactive isotopes are also required to label the probes. These factors limit the widespread use of RFLP labeling. AFLP marker combines PCR and RFLP marker technology, and has been widely used in crop genetic diversity research, cytology research, variety purity identification and disease resistance research (Song Shunhua et al., 2006; Yuan Suxia, 2009; Wang Xue, 2004). However, AFLP markers also have some disadvantages: high cost, complicated process, and technical difficulty; most of the markers are dominant markers; and they have high requirements for DNA quality and restriction endonuclease quality. SSR markers, also known as microsatellite DNA markers (microsatellite DNA), have been widely used in crop gene mapping, molecular marker-assisted selection, DNA fingerprinting, variety purity identification, preservation and utilization of germplasm resources, and genetic diversity analysis. In research (Chen Yeli, 2010; Miao Tiyun, 2007; Jing Zange, 2010; Wang Dongmei, 2011). SSR markers have the advantages of abundant quantity, high polymorphism, simple operation, and low cost, but it is difficult to achieve high-throughput batch detection. In the past decade, with the continuous advancement of sequencing technology, it has become possible to develop molecular markers based on genome sequence information, such as SNP markers and InDel markers (Hyten et al., 2010). At present, genome-wide selective breeding chips have only been tried in rice (Yu et al., 2014), and other crops such as rape are still mainly based on molecular marker-assisted selection. At present, SNP detection methods are mainly divided into two categories: one category is based on single-strand conformation polymorphism (SS CP), denaturing gradient gel electrophoresis (DDGE), enzyme-cut amplified polymorphic sequence (CAPS), allele The traditional classic detection methods based on gel electrophoresis represented by gene-specific PCR (AS-PCR), and the other major detection methods are direct sequencing, DNA chips, denaturing high-performance liquid chromatography (DHPLC), and mass spectrometry detection technologies. High-throughput and highly automated detection methods represented by , high-resolution melting curve (HRM), etc. These two types of methods have their own advantages and disadvantages. At present, there are detection methods that combine the two, such as KASP (Lister et., 2013) and PARMS (Lu et al., 2020), which are not only easy to operate, low in cost, but also can achieve High-throughput detection.
每子房胚珠数是一个典型的由多基因控制的复杂数量性状,表型连续分布而且容易受环境条件的影响。数量遗传学和分子标记技术的结合,可将复杂的数量性状分解为单个的数量性状基因位点(quantitative trait loci,QTL),然后像研究质量性状一样对控制数量性状的多个基因进行研究。QTL定位就是在遗传分离群体的基础上,借助分子标记和遗传图谱,利用QTL作图软件对分离群体的数量性状表型数据进行分析,从而确定数量性状基因在染色体上的位置和效应。目前,利用连锁作图(linkage mapping)和关联作图(association map ping)的方法,在油菜中已定位了数以千计的QTL,涉及许多重要性状如产量、株型、品质和抗性等(Hu et al.,2017)。然而关于油菜每子房胚珠数的QTL定位研究才刚开始起步,Khan等(2019)利用521份油菜关联群体和60K油菜SNP芯片进行全基因组关联分析,获得8个与每子房胚珠数显著关联的SNP标记。本发明通过对油菜每子房胚珠数性状进行四个环境下的全基因组关联分析,旨在找到对油菜每子房胚珠数具有改良效应的稳定位点,并据此开发实用的高通量低成本的分子标记,用于对油菜胚珠数进行选择。The number of ovules per ovary is a typical complex quantitative trait controlled by multiple genes, and the phenotype is continuously distributed and easily affected by environmental conditions. The combination of quantitative genetics and molecular marker technology can decompose complex quantitative traits into a single quantitative trait loci (quantitative trait loci, QTL), and then study multiple genes controlling quantitative traits like studying qualitative traits. QTL mapping is based on the genetic segregation population, with the help of molecular markers and genetic maps, using QTL mapping software to analyze the quantitative trait phenotype data of the segregation population, so as to determine the position and effect of the quantitative trait gene on the chromosome. At present, using linkage mapping and association mapping methods, thousands of QTLs have been located in rapeseed, involving many important traits such as yield, plant type, quality and resistance, etc. (Hu et al., 2017). However, the QTL mapping research on the number of ovules per ovary in rapeseed has just begun. Khan et al. (2019) used 521 rapeseed related populations and 60K rapeseed SNP chips to conduct genome-wide association analysis and obtained 8 QTLs that were significantly associated with the number of ovules per ovary. SNP markers. The present invention conducts genome-wide association analysis on the number of ovules per ovary of rapeseed under four environments, aiming to find stable loci that have an improvement effect on the number of ovules per ovary of rapeseed, and develop a practical high-throughput low-efficiency Molecular markers of cost for selection on ovule number in rapeseed.
发明内容Contents of the invention
本发明的目的是在于提供了油菜A03染色体上和每子房胚珠数显著关联的稳定位点qO NPO.A3-2。The purpose of the present invention is to provide a stable site qONPO.A3-2 on the rape A03 chromosome that is significantly associated with the number of ovules per ovary.
本发明另一个目的在于提供了针对每子房胚珠数关联位点qONPO.A3-2峰值SNP标记seq-new-rs32414设计的PARMS检测引物。Another object of the present invention is to provide PARMS detection primers designed for the ovule number per ovary associated locus qONPO.A3-2 peak SNP marker seq-new-rs32414.
本发明还有一个目的在于提供了上述检测引物在油菜每子房胚珠数选择育种中的应用。为了达到上述目的,本发明采取以下技术措施:Another object of the present invention is to provide the application of the above detection primers in the selective breeding of the number of ovules per ovary of rapeseed. In order to achieve the above object, the present invention takes the following technical measures:
油菜每子房胚珠数显著关联PARMS标记的获得The number of ovules per ovary in rapeseed is significantly associated with the acquisition of PARMS markers
(1)收集331份油菜总DNA,利用油菜60K SNP芯片对每个样本进行基因型分析。(1) Collect 331 rapeseed total DNA, and use the rapeseed 60K SNP chip to perform genotype analysis on each sample.
(2)利用Illumina BeadStudio基因分型软件(http://www.illumina.com/)计算群体材料在每个位点的标记杂合率(heterozygous rate)、缺失率(missing rate)、最小等位基因频率(minor allele frequency)。去掉无多态性、缺失比列高、无纯合基因型、等位基因频率低、杂合基因型频率高、位置不确定和多拷贝标记,最后获得327个株系的21242个高质量SNP标记用于后续分析。(2) Use the Illumina BeadStudio genotyping software (http://www.illumina.com/) to calculate the marker heterozygous rate (heterozygous rate), missing rate (missing rate) and minimum allele of the population material at each site Gene frequency (minor allele frequency). Removed no polymorphism, high deletion ratio, no homozygous genotype, low allele frequency, high heterozygous genotype frequency, uncertain position and multi-copy markers, and finally obtained 21242 high-quality SNPs from 327 lines markers for subsequent analysis.
(3)将关联群体的331份品系分别在2020青海、2020阳逻、2021武昌、2021阳逻四个环境种植(代号为20QH,20YL,21WC,21YL),开花期挑选关联群体的各个品系中长势均匀的5个单株,选取主花序下部5个蕾,剥离出子房。对子房进行整体透明后,在体视显微镜下拍照,并对胚珠进行计数(朱瑶瑶,2019)。(3) Plant 331 strains of the associated population in four environments (codenamed 20QH, 20YL, 21WC, 21YL) in 2020 Qinghai, 2020 Yangluo, 2021 Wuchang, and 2021 Yangluo, and select each strain of the associated population during the flowering period For 5 individual plants with uniform growth, 5 buds at the lower part of the main inflorescence were selected, and the ovary was stripped out. After the overall transparency of the ovary, photographs were taken under a stereomicroscope, and the ovules were counted (Zhu Yaoyao, 2019).
(4)结合关联群体的每子房胚珠数表型数据、基因型数据和群体结构,利用TASSEL5.0软件(Bradbury et al.,2007)进行关联分析。最终在油菜DarmorV4参考基因组A03染色体上获得与每子房胚珠数显著关联的位点qONPO.A3-2,其峰值SNP标记seq-new-rs32414位于第16,137,692位碱基处(碱基为T/A或C/G),可以在20YL和21YL两个环境和多个模型中被重复检测到。(4) Combining the ovule number per ovary phenotype data, genotype data and population structure of the associated population, use TASSEL5.0 software (Bradbury et al., 2007) for association analysis. Finally, the locus qONPO.A3-2 significantly associated with the number of ovules per ovary was obtained on the A03 chromosome of the rapeseed DarmorV4 reference genome, and its peak SNP marker seq-new-rs32414 was located at the 16th, 137,692th base (the base is T/A or C/G), which could be detected repeatedly in both 20YL and 21YL environments and multiple models.
(5)提取油菜A03染色体第14,943,779位碱基上下游各100bp的序列,按照引物设计原则获得PARMS检测引物序列:qONPO.A3-2Ft:GAAGGTGACCAAGTTCATGCTTTGGACTCTTGTTAAGGTAATAATATATAAGT;qONPO.A3-2Fc:GAAGGTCGGAGTCAACGGATTTGGACTCTTGTTAAGGTAATAATATATAAGC;qONPO.A3-2R:AATTTATGAAGTCAATTGTTGACTAAAAT。(5) Extract the sequences of 100 bp upstream and downstream of the 14th, 943, and 779th base of rape A03 chromosome, and obtain the PARMS detection primer sequence according to the primer design principle: qONPO.A3-2Ft: GAAGGTGACCAAGTTCATGCT TTGGACTCTTGTTAAGGTAATAATATATAAGT; qONPO.A3-2Fc: GAAGGTCGGAGTCAACGGATT TGGACTCTTGT TAAGGTAATAATATATAAGC;qONPO.A3 -2R: AATTTATGAAGTCAATTGTTGACTAAAAT.
本发明的保护内容包括:检测上述分子标记的试剂在甘蓝型油菜每子房胚珠数选择育种中的应用。The protection content of the present invention includes: the application of the reagent for detecting the above-mentioned molecular marker in selective breeding for the number of ovules per ovary of Brassica napus.
与现有技术相比,本发明具有以下优点:Compared with the prior art, the present invention has the following advantages:
(1)本发明获得了与油菜每子房胚珠数显著关联的位点qONPO.A3-2,且可被重复检测,平均可解释8.0%的表型方差,平均加性效应为1.01,可有效应用于油菜每子房胚珠数的遗传改良。(1) The present invention obtains the locus qONPO.A3-2 that is significantly associated with the number of ovules per ovary of rapeseed, and can be repeatedly detected. On average, it can explain 8.0% of the phenotypic variance, and the average additive effect is 1.01, which can be effective Genetic improvement of the number of ovules per ovary applied to rapeseed.
(2)本发明获得油菜每子房胚珠数显著关联的PARMS标记,其检测方法简便而且成本低廉,可对每子房胚珠数关联的基因组单倍型区域进行高通量筛选,提高选择效率和准确性。(2) The present invention obtains PARMS markers significantly associated with the number of ovules per ovary in rapeseed. The detection method is simple and low-cost, and high-throughput screening can be performed on the genomic haplotype region associated with the number of ovules per ovary to improve selection efficiency and accuracy.
附图说明Description of drawings
图1为油菜关联群体327份材料在四个环境中的每子房胚珠数表型分布。Figure 1 shows the phenotypic distribution of the number of ovules per ovary in 327 materials of rape related populations in four environments.
具体实施方式Detailed ways
本发明所述技术方案,如未特别说明,均为本领域的常规技术;所述试剂或材料,如未特别说明,均来源于商业渠道。本发明中使用的甘蓝型油菜基因组的版本号是Darmor-bzh(Version 4.1)https://www.genoscope.cns.fr/brassicanapus/。The technical solutions of the present invention, if not specified, are conventional techniques in the art; the reagents or materials, if not specified, are all derived from commercial channels. The version number of the Brassica napus genome used in the present invention is Darmor-bzh (Version 4.1) https://www.genoscope.cns.fr/brassicanapus/ .
实施例1:油菜每子房胚珠数显著关联SNP标记的获得Example 1: Acquisition of SNP markers significantly associated with the number of ovules per ovary in rapeseed
(1)收集331份来自世界各个国家的甘蓝型油菜自交系作为油菜核心关联群体,采集关联群体各株系的单株叶片,用CTAB法提取总DNA,利用油菜60K SNP芯片对每个样本进行基因型分析(Li et al.,2020)。(1) Collect 331 Brassica napus inbred lines from various countries in the world as the core related population of rapeseed, collect individual leaves of each strain of the related group, extract the total DNA by CTAB method, and use the rape 60K SNP chip to analyze each sample Genotype analysis was performed (Li et al., 2020).
(2)利用Illumina BeadStudio基因分型软件(http://www.illumina.com/)计算群体材料在每个位点的标记杂合率(heterozygous rate)、缺失率(missing rate)、最小等位基因频率(minor allele frequency)。利用SPAGeDi软件计算331份甘蓝型油菜种质资源间亲缘关系(Hardy and Vekemans,2002)。以缺失率≤0.2、杂合率≤0.2、最小等位基因频率>0.05以及SNP标记在甘蓝型油菜基因组中唯一匹配为筛选标准进行SNP标记的过滤,最终获得327份材料21242个高质量SNP标记用于全基因组关联分析。(2) Use the Illumina BeadStudio genotyping software (http://www.illumina.com/) to calculate the marker heterozygous rate (heterozygous rate), missing rate (missing rate) and minimum allele of the population material at each site Gene frequency (minor allele frequency). The genetic relationship among 331 Brassica napus germplasm resources was calculated by SPAGeDi software (Hardy and Vekemans, 2002). The deletion rate ≤ 0.2, the heterozygosity rate ≤ 0.2, the minimum allele frequency > 0.05, and the unique match of the SNP marker in the Brassica napus genome were used as the screening criteria to filter the SNP markers, and finally obtained 21,242 high-quality SNP markers from 327 materials for genome-wide association analysis.
(3)将关联群体的331个株系分别于2020青海、2020阳逻、2021武昌、2021阳逻四个环境种植(代号为20QH,20YL,21WC,21YL)。花期挑选关联群体各个株系长势均匀的5个单株,每株选取5个蕾。剥离出子房进行整体透明后,在体视显微镜下拍照并对胚珠进行计数,计算各株系平均值。通过对关联群体各个环境中的每子房胚珠数数据进行分析,结果表明所有四个环境中的每子房胚珠数均表现出很大的变异幅度而且呈正态分布(图1),可以直接用于后续的全基因组关联分析。(3) The 331 strains of the related population were planted in four environments of 2020 Qinghai, 2020 Yangluo, 2021 Wuchang, and 2021 Yangluo (code-named 20QH, 20YL, 21WC, 21YL). At the flowering stage, 5 individual plants with uniform growth of each line of the associated population were selected, and 5 buds were selected for each plant. After the ovary was stripped out for overall transparency, photographs were taken under a stereomicroscope and the ovules were counted to calculate the average value of each strain. By analyzing the data of the number of ovules per ovary in each environment of the associated population, the results show that the number of ovules per ovary in all four environments shows a large range of variation and is normally distributed (Figure 1), which can be directly for subsequent genome-wide association analysis.
(4)结合关联群体的基因型数据和每子房胚珠数表型数据,利用TASSEL 5.0软件(B radbury et al.2007)的四个模型(GLM-PCA,GLM-Q,MIM-PCA+K,MLM-Q+K)进行全基因组关联分析。通过对不同环境和模型中检测到的显著性关联SNP标记进行整合,在A03染色体获得与每子房胚珠数显著关联且重复性好的位点qONPO.A3-2,可以在20YL和21YL两个环境和四个模型中被重复检测到,其峰值SNP标记seq-new-rs32414位于Darmor V4.1参考基因组第16,137,692位碱基(T/A或C/G),最高显著性水平P=7.55E-06,平均可解释8.0%的表型方差,平均加性效应为1.01,因此理论上两种纯合基因型之间的每子房胚珠数差异有2.02。(4) Combining the genotype data of the associated population and the phenotype data of the number of ovules per ovary, using the four models (GLM-PCA, GLM-Q, MIM-PCA+K) of TASSEL 5.0 software (Bradbury et al.2007) , MLM-Q+K) for genome-wide association analysis. By integrating the significantly associated SNP markers detected in different environments and models, the locus qONPO.A3-2, which is significantly associated with the number of ovules per ovary and has good repeatability, is obtained on chromosome A03, which can be used in both 20YL and 21YL It was repeatedly detected in the environment and four models, and its peak SNP marker seq-new-rs32414 was located at base 16,137,692 (T/A or C/G) of the Darmor V4.1 reference genome, with the highest significance level P=7.55E -06, 8.0% of the phenotypic variance can be explained on average, and the average additive effect is 1.01, so the theoretical difference in the number of ovules per ovary between the two homozygous genotypes is 2.02.
表一、每子房胚珠数关联QTL-qONPO.A3-2的信息Table 1. The information of QTL-qONPO.A3-2 associated with the number of ovules per ovary
实施例2:一种与每子房胚珠数显著关联PARMS标记的开发Example 2: Development of a PARMS marker significantly associated with the number of ovules per ovary
实施例1中所获得的关联标记为来源于SNP芯片,只有用于分子杂交的探针序列信息。油菜SNP芯片一次可以检测数以万计的位点,但其操作比较繁琐而且需要用到特殊设备。另外,若用油菜SNP芯片对大量的育种中间材料进行检测则成本昂贵,因此需要将其转化为操作简单而且成本低廉的基于PCR扩增的检测方法,如PARMS(Penta-primerAmplifica tion Refractory Mutation System,五引物扩增受阻突变系统)标记。该标记系统包括一对荧光通用引物(FAM和HEX作为报告荧光)、一对SNP等位基因特异引物及一条反向共用引物,可快速简单地进行SNP等位基因型检测。The associated markers obtained in Example 1 are derived from the SNP chip, and only have the sequence information of the probes used for molecular hybridization. Rapeseed SNP chip can detect tens of thousands of sites at a time, but its operation is cumbersome and requires special equipment. In addition, it is expensive to use rapeseed SNP chips to detect a large number of breeding intermediate materials, so it needs to be converted into a detection method based on PCR amplification that is simple to operate and low in cost, such as PARMS (Penta-primer Amplification Refractory Mutation System, Five-primer amplification hindered mutation system) marker. The labeling system includes a pair of fluorescent universal primers (FAM and HEX as reporter fluorescence), a pair of SNP allele-specific primers and a reverse common primer, which can quickly and easily detect SNP alleles.
(1)针对与qONPN.A3-2关联的峰值SNP标记seq-new-rs32414,提取油菜DarmorV4.1参考基因组A03染色体第16,137,692位碱基上下游各100bp的序列。按照引物设计原则获得PARMS标记检测引物序列为:(1) For the peak SNP marker seq-new-rs32414 associated with qONPN.A3-2, extract the sequences of 100 bp upstream and downstream of bases 16, 137, and 692 on chromosome A03 of the rapeseed DarmorV4.1 reference genome. According to the primer design principle, the sequence of the PARMS marker detection primer is obtained as follows:
qONPO.A3-2Ft:gaaggtgaccaagttcatgctttggactcttgttaaggtaataatatataagt;qONPO.A3-2Ft: gaaggtgaccaagttcatgct ttggactcttgttaaggtaataatatataagt;
qONPO.A3-2Fc:gaaggtcggagtcaacggatttggactcttgttaaggtaataatatataagc;qONPO.A3-2Fc: gaaggtcggagtcaacggatt tggactcttgttaaggtaataatatataagc;
qONPO.A3-2R:aatttatgaagtcaattgttgactaaaat。qONPO.A3-2R: aatttatgaagtcaattgttgactaaaat.
(2)以油菜关联群体的基因组DNA为模板,利用上述引物进行荧光定量PCR扩增,采用Tecan F200扫描FAM、HEX和ROX信号并输出结果,最后转换成基因型。(2) Using the genomic DNA of rape related populations as a template, the above primers were used for fluorescence quantitative PCR amplification, and Tecan F200 was used to scan the FAM, HEX and ROX signals and output the results, and finally converted into genotypes.
实施例3:PARMS标记在油菜每子房胚珠数选择中的应用Example 3: Application of PARMS markers in the selection of the number of ovules per ovary in rapeseed
在关联群体331份材料中,以实施例2提供的PARMS标记qONPO.A3-2检测出基因型为CC的材料有76份,基因型为TT的为237份(表二)。两种基因型的每子房胚珠数在三个环境中达到极显著水平,CC和TT两种基因型的每子房胚珠数均值差异从1.53到2.16,均值为1.83。Among the 331 materials in the related population, 76 materials were genotyped as CC and 237 materials as genotype TT were detected by the PARMS marker qONPO.A3-2 provided in Example 2 (Table 2). The number of ovules per ovary of the two genotypes reached a very significant level in the three environments, the mean difference of the number of ovules per ovary between CC and TT genotypes ranged from 1.53 to 2.16, and the average value was 1.83.
表二、PARMS标记qONPO.A3-2两种基因型在关联群体四环境每子房胚珠数的比较Table 2. Comparison of the number of ovules per ovary of the PARMS marker qONPO.A3-2 two genotypes in the associated population four environments
*,**和***分别代表显著性水平P=0.05,0.01和0.001。*, ** and *** represent significance levels P=0.05, 0.01 and 0.001, respectively.
以上结果足以说明我们制备的PARMS分子标记qONPO.A3-2与油菜的每子房胚珠数是高度关联的,具有很好的筛选效果。The above results are enough to show that the PARMS molecular marker qONPO.A3-2 prepared by us is highly correlated with the number of ovules per ovary of rapeseed, and has a good screening effect.
序列表sequence listing
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