CN109929945B - Molecular marker BrSF2604 primer of main effect QTL sites in flowering phase and mature phase of brassica napus and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biology and genetic breeding, and discloses a molecular marker BrSF2604 primer of a main QTL site in flowering and mature periods of brassica napus and application thereof, wherein an applicant obtains phenotypic data of flowering characters by performing field experiments and character investigation on isolated populations of F2 and F2:3 families of Zhongshuang No. 11 and 73290; combining the genotype and the genetic map of the F2 segregation population to carry out QTL detection, and obtaining the major gene locus which controls the flowering phase and the mature phase of the rape together on the C2 linkage population, wherein the contribution rate of the major gene locus to the flowering phase of the rape is 21.1 percent, the additive effect is-2.8 percent, and the dominant effect is 0.3 percent; the contribution rate to the rape maturity period is 10.4%, the additive effect is-0.3, and the dominant effect is 0.4. The length of the flowering period and the mature period can be predicted by detecting the molecular markers related to the flowering period and the mature period, and then early-maturing varieties can be accurately and quickly screened.

Description

Molecular marker BrSF2604 primer of main effect QTL sites in flowering phase and mature phase of brassica napus and application thereof

Technical Field

The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker BrSF2604 primer of a main QTL site in flowering and mature periods of brassica napus and application thereof.

Background

Rape is the first major oil crop in our country, accounting for about 20% of world rape yield (Hu et al, 2016). Rapeseed oil was the first major source of domestic edible vegetable oil, accounting for 57.2% of the total amount of domestic edible vegetable oil (fangchining et al, 2018). The external dependence of domestic vegetable oil in China exceeds 60%, and the vegetable oil has a tendency of increasing continuously (Wang Han Zhong, etc., 2014). In addition, the rapeseed oil and the diesel oil have similar fatty acid composition, and are green renewable energy sources.

Although China is the biggest rapeseed oil producing country in the world, the supply of vegetable oil is still seriously insufficient, and a large amount of vegetable oil is still imported every year. The main reason for this phenomenon is that the economic benefit of rape is not high, although the total rape planting area is the first in China in the world, the planting area is small, and in recent years, the crop rotation contradiction between rape and grain and cotton crops is prominent, and the rape planting area is continuously reduced (Guangchunyun, etc. 2010). The season contradiction between double cropping rice and rape is the main reason for idle placement of a large number of fields in winter in double cropping rice planting areas in south China, and the fundamental way for solving the contradiction is the premature breeding of rape. The existing research shows that the precocious rape generally blossoms, and the precocious blossoms are positively correlated with the precocious maturity (Campbell and Kondra 1978; Gaoyongong 1979), so the research on the QTL related to the flowering period of the rape has important significance for improving the precocious maturity of the rape and accelerating the precocious breeding of the rape.

Although the traditional breeding method provides a plurality of excellent rape varieties for production once, the requirements of the current rape production cannot be completely met due to long breeding period and low selection efficiency. With the development of molecular biology and molecular genetics, selection of traits by breeders is gradually transitioning from phenotypic selection to genotypic selection. The molecular marker assisted breeding is a new breeding means which effectively combines molecular genetics and traditional phenotype selection, and the basic principle is that in the rape breeding process, molecular markers which are closely linked and coseparated with target character genes are directly utilized to carry out target region and whole genome screening on selected individuals, so that the purposes of improving the target character selection efficiency and shortening the breeding period are achieved. The key of the molecular marker assisted selective breeding technology is to identify DNA molecular markers closely linked with important agronomic traits. In recent years, research work in this area has been invested in enormous quantities in developed countries such as the united states. With the development of molecular markers for agronomic traits of important crops such as rice, corn, wheat and the like, the auxiliary selective breeding by using the screened molecular markers is gradually mature, and the target traits are also expanded from simple single-gene quality traits to complex multi-gene quantitative traits. With the rapid development of genomics and sequencing technologies, rape molecular marker research is receiving more and more attention, and the research field relates to aspects such as species genetic diversity analysis, genetic map construction, gene marking and positioning, species purity identification, combining ability prediction, marker-assisted selection and the like, and has made important progress. However, compared with developed countries, the research on the molecular breeding of rape in China has a large gap, which is mainly reflected in that: the method can not effectively discover and utilize beneficial genes in germplasm resources, and lacks genes and markers with independent intellectual property rights and breeding value.

With the continuous development of molecular marker technology, the application of the molecular marker technology in crops is more and more extensive. Grodzicker et al (1974) have created a Restriction Fragment Length Polymorphism (RFLP) tagging technique. The R FLP is a first-generation molecular marker and has the characteristics of abundant quantity, stable heredity, specificity, good repeatability, codominance and the like. However, the marker requires a relatively large amount of DNA; the operation procedure is complicated, time-consuming, labor-consuming and long in period; the need to label the probe with a radioisotope has also limited the widespread use of RFLP labeling. The AFLP marker combines P CR and RFLP marker technologies, and is widely applied to researches on crop genetic diversity, cytology, variety purity identification, disease resistance and the like (Song cishun, et al, 2006; Yuan Suxia, 2009; Wang Xue, 2004). However, AFLP markers also have some disadvantages: the cost is high, the process is complex, and the technical difficulty is high; the markers are mostly dominant markers; the requirements on the quality of DNA and the quality of restriction enzyme are high. SSR markers, also called microsatellite DNA markers, have been widely used in studies on crop gene mapping, molecular marker-assisted selection, DNA fingerprinting, variety purity identification, seed quality resource preservation and utilization, genetic diversity analysis, and the like (Chen Yeli, 2010; Miao clouds, 2007; Jing Zan leather, 2010; Wang Dongmei, 2011). SSR markers have the advantages of abundant quantity, high polymorphism, simple operation, low cost and the like, and are widely introduced to molecular marker-assisted selection for a long time. In recent decades, with the continuous progress of sequencing technologies, the development of molecular markers based on genomic sequence information has become possible, such as SNP markers and InDel markers (Hyten et al, 2010). At present, the whole genome selective breeding chip only starts to try in rice (Yu et al, 2014), and other crops such as rape are still mainly selected by the aid of molecular markers.

Most important agronomic characters (such as yield, quality, resistance and the like) show the genetic characteristics of quantitative characters, phenotypes are continuously distributed and are easily influenced by environmental conditions, so that the conventional breeding method based on phenotypic selection has poor selection effect on complex quantitative characters, the breeding efficiency is low, and the breeding period is prolonged. Due to the development and integration of molecular marker technology and quantitative genetics, one has decomposed complex quantitative traits into single Quantitative Trait Loci (QTL) and then studied multiple genes that control quantitative traits like quality traits. QTL positioning is that on the basis of genetic segregation population, quantitative trait phenotypic data of the segregation population are analyzed by using QTL mapping software by means of molecular markers and genetic maps, so that the position and the effect of quantitative trait genes on chromosomes are determined. The genetic research results of the flowering phase of the brassica napus all indicate that the flowering phase is controlled by multiple genes and has quantitative characters of major gene effects (Zaman et al, 1995; Lagercrantz et al, 1996; Osborne et al, 1997; Chuanchang et al, 2007). Raman et al (2013) utilize two spring rape Skipton and Ag-Spectrum as parent DH colony, research shows that the flowering phase is a complex character, QTL of the flowering phase is regulated and controlled to be distributed on 10 different chromosomes, at least 20 QTL sites participate in regulation and control, and the single phenotype variation rate is 2.4-28.6%; comparing the QTL position with the physical positions of flowering-related genes annotated by the sequenced cabbage genome, the results showed that the flowering-stage-related QTLs located on a02, a03, a07 and C06 probably represent homologous genes of flowering-stage-related genes known in arabidopsis thaliana. Wei et al (2014) perform flowering QTL positioning and detect 17 QTL related to flowering; meanwhile, two flowering-period-related QTLs are found on the A02 linkage group, and the contribution rate of phenotypic variation of a single QTL is 3.29-5.17%. The research aims to screen related QTL sites in flowering and mature periods of the rape through QTL positioning and is used for marker-assisted selection of early flowering and early maturing traits of the rape.

Disclosure of Invention

The invention aims to provide a molecular marker BrSF2604 primer of a main QTL site in flowering and mature periods of brassica napus, wherein the primer is as follows: CGAGAATCTCATCTGGCTCC, and TGCATGACTAATGCTTCGGA.

The invention also aims to provide application of the molecular marker BrSF2604 primer of the major QTL site in the flowering phase and the mature phase of the brassica napus, wherein the effect value and the contribution rate of the QTL site are high, the primer plays a key role in regulation and control of the flowering phase and the mature phase of the brassica napus, and the primer can be used for map cloning, rape premature breeding and molecular marker assisted selection. In order to achieve the purpose, the invention adopts the following technical measures:

the method for obtaining the QTL locus of the rape flowering phase character comprises the following steps:

(1) the F1 generation hybrid is selfed to generate an F2 population and an F2:3 family thereof by utilizing the double 11 and 73290 hybrids in rape varieties with very obvious differences in flowering period.

(2) The CTAB method (Doyle et al 1987) was used to extract total DNA from leaves of the parental double 11 and 73290 and F2 isolates.

(3) Rape public (http:// www.ukcrop.net/Brassica DB) and SSR and InDel primers which are independently developed are synthesized, the parental DNA is subjected to PCR amplification, products are electrophoresed in denaturing polyacrylamide gel, the size of a band is distinguished after dyeing and developing, and polymorphic primers are screened.

(4) And (3) carrying out molecular marker analysis on the F2 segregation population by using the polymorphic primer pair to obtain genotype data.

(5) Inputting genotype data of the F2 segregating population into the Joinmap4.0 software (commercially available) to construct a genetic linkage map;

(6) genotype data (limited to markers mapped on genetic maps) of the F2 population and flowering and maturity trait data of the F2 population and its F2:3 family are input into WinQTLctart 2.5 software for QTL mapping. Wherein, a QTL with one position point on the C2 linkage group can be repeatedly detected in three groups, and the effect value and the contribution rate are larger.

By adopting the technical measures, the applicant finally obtains a main QTL site for simultaneously controlling the pleiotropic main effect of the cabbage type rape in the flowering phase and the mature phase, the main QTL site is closely linked with an SSR marker BoSF2604 independently developed by the applicant, and the primer sequence of the main QTL site is 5'-CGAGAATCTCATCTGGCTCC-3' by aiming at the BoSF 2604F; BoSF2604R:5 '-TGCATGACTAA TGCTTCGGA'. The method is characterized in that the contribution rate of the rape to the flowering phase is 21.1 percent, the additive effect is-2.8 percent and the dominant effect is 0.3 percent, which are measured by utilizing WinQTLCart2.5 software analysis; the contribution rate to the rape maturity period is 10.4%, the additive effect is-0.3, and the dominant effect is 0.4.

The application of the molecular marker BrSF2604 primer of the major QTL sites in the flowering period and the mature period of the cabbage type rape comprises the application of the primer provided by the invention, and the primer can be used for breeding the cabbage type rape, including screening early-flowering early-maturing single plants, and being used for map-based cloning of the cabbage type rape or molecular marker-assisted selection of the cabbage type rape.

Compared with the prior art, this has following advantage:

the invention positions the important QTL site for controlling flowering phase of double 11 in rape variety, the genetic distance between the marker and the major gene site is very close (<2cM) and the marker is a codominant SSR marker based on genome sequence information, so that the marker is reliable and convenient to use, and great convenience is provided for breeding double 11 derived strains in the future. The phenotypic variances of 21.1% and 10.4% can be explained, respectively. In the conventional breeding method, the phenotype identification of the characters in the flowering phase and the mature phase respectively needs to wait until the flowering phase and the mature phase are investigated, which wastes time and labor and has low selection efficiency (the phenotype in the flowering phase and the mature phase is greatly influenced by the environment). By detecting the major gene loci of the characters in the flowering phase and the mature phase, the major gene loci can be eliminated in the seedling phase, so that the production cost is saved, and the selection efficiency is greatly improved. The position of the major gene locus is clear, and the detection method of the major gene locus is convenient and quick and is not influenced by the environment. The mark is used for detecting the offspring group derived from the amphiphilic sample, and the method proves that the effect is stable and reliable, the detection method is convenient and quick, and the method is not influenced by the environment. By detecting the molecular markers related to the characters of the early-maturing stage of the flowering phase, the length of the flowering phase and the mature phase can be predicted, and the early-maturing variety can be accurately and quickly screened.

Drawings

FIG. 1 is a frequency distribution diagram of flowering period of family F2:3 when planted in different environments;

the result shows that the phenotype in the flowering phase is normally distributed, the variation range is wide, and the flowering phase is proved to belong to quantitative traits.

FIG. 2 is a frequency distribution diagram of maturity of family F2:3 when planted in different environments;

the results show that the phenotype of the mature period is normally distributed, the variation range is wide, and the mature period is proved to belong to quantitative traits.

Detailed Description

The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

constructing a rape flowering phase segregation population and determining characters:

in the examples, the hybrid F1 generation was selfed to produce the F2 population and its F2:3 using the crossing of double 11 in the sequenced variety of rape and another variety of rape 73290 with large genetic differences. The comprehensive test base of the Yangtze logical unit of the oil institute of the Chinese academy of agricultural sciences plants F2 population in 2009 and F2:3 family population in 2010, 2011 and 2012. The character survey data shows that: the mean values of the flowering phase and the mature phase in 3 environments are normally distributed, and the quantitative genetic characteristics of the flowering phase and the mature phase characters are shown (figure 1 and figure 2)

Example 2:

development and synthesis of primers:

SSR primers utilized by applicants include two classes: one is the published primer sequences in the published articles and Brassica databases (http:// www.brassica.info/resource/markers/ssr-exchange. php); the other is developed by the applicant according to Chinese cabbage and cabbage scaffold sequences and named as BrSF and BoSF series respectively, and the specific development method is to search SSR in each scaffold by SSR Hunter software and then design SSR primers by Primer3.0 software. The autonomously developed InDel primers were derived by aligning 73290 the re-sequenced sequence to the midduplex 11 reference genomic sequence, first mapping 73290 the re-sequenced sequence to the midduplex 11 whole genomic reference sequence using BW a software, and then searching InDel using samtools software.

Example 3:

the process of screening primer polymorphism includes the following steps:

(1) 10 DNA strains randomly selected from each parent were mixed in equal amounts and used as templates for screening primers.

(2) Carrying out PCR amplification on the parent DNA by using the dissolved primer,

reaction system:

PCR reaction procedure:

(3) gel electrophoresis band pattern interpretation

Example 4:

f2 population genotype analysis, genetic linkage map construction and QTL positioning, which comprises the following steps:

(1) extracting DNA of 179 individuals of an F2 population by adopting a CTAB method;

(2) the DNA of 179 individuals of F2 population is amplified by PCR with selected polymorphic primers, and then the PCR products are subjected to polyacrylamide gel electrophoresis, development, staining and banding pattern interpretation. The molecular markers to which the invention relates are co-dominant markers, i.e. the differential bands show a variation in position (i.e. amplification product size), and the banding patterns of the segregating population are read as A, B and H, respectively, indicating the banding patterns from Mediterranean 11, 73290 and heterozygous, respectively.

(3) And (4) judging the band type of the molecular marker obtained after dyeing to obtain the genotype data of the molecular marker.

(4) The molecular marker genotype data of the F2 population are subjected to linkage analysis by using Joinmap4.0 software to construct a molecular marker genetic linkage map, so that 19 linkage populations (containing 805 molecular markers) are obtained, and the linkage populations exactly correspond to 19 chromosomes of the brassica napus.

(5) Based on the genetic map, genotype data of an F2 population, a F2 population and trait data of the F2: flowering phase and mature phase of a 3-family line, QTL detection is carried out by using QTLCart2.5 software, two main effect QTL loci with good repeatability are detected near a C2 chromosome SSR marker BoSF2604 (table 1), LOD values and contribution rates of the two main effect QTL loci are both large (table 2), wherein the contribution rate of one of the two main effect QTL loci to the flowering phase of rape is 21.1%, additive effect is-2.8, and dominant effect is 0.3; the contribution rate to the rape maturity period is 10.4%, the additive effect is-0.3, and the dominant effect is 0.4.

The primers designed aiming at the BoSF2604 molecular marker are shown in Table 1, and a band with the size of 287bp and marked as A in the invention is obtained by amplifying the primer in Zhongshuang No. 11; a band obtained by amplification in 73290, 292bp in size, marked as B in the invention; two bands, 287bp and 292bp, were amplified in the heterozygotes.

TABLE 1C 2 primer sequence of major QTL linkage marker BoSF2604 for linkage group per Kernel number

TABLE 2C 2 basic information of linkage group flowering phase major QTL

Example 5:

the validity verification of the major QTL linkage marker BoSF2604 in the flowering period and the mature period comprises the following steps:

(1) f3 generation seeds of F2 single plants are selected and planted in multiple points in multiple years.

(2) F3 single plant is sampled before final singling, total DNA of leaves is extracted, and the genotype of the main effect QTL in the flowering period is analyzed by utilizing a molecular marker BoSF 2604.

(3) F3 individuals were recorded for flowering and maturity. The results show that the mean of the flowering period (172.5 days) of the medium double 11 background single plants selected by the molecular marker assisted selection is shorter than that of the 73290 background single plants (177.8 days), and the population ratio of the single plants of the medium double 11 background single plants, the flowering period of which is shorter than that of the 73290 background population, is 87.5% (table 3); meanwhile, the average maturity period (233.0 days) of the Zhongshuang No. 11 background individuals was shorter than that of the 73290 background individuals (233.4 days), and the population ratio of the Zhongshuang No. 11 background individuals whose maturity period was shorter than that of the 73290 background population was 75.0% (Table 4). The early-maturing strains can be eliminated in the seedling stage, so that the production cost is saved, the selection efficiency is greatly improved, and the early-maturing strains can be quickly screened out for rape breeding.

TABLE 3 flowering time survey data of F3 individuals obtained by SSR marker-assisted selection with BoSF2604

Note: A. b represents molecular marker band patterns derived from double 11 and 73290 in parent

TABLE 4 maturation stage survey data of F3 individuals obtained by SSR marker-assisted selection with BoSF2604

Sequence listing

<110> institute of oil crop of academy of agricultural sciences of China

Molecular marker BrSF2604 primer of main effect QTL sites in flowering phase and mature phase of brassica napus and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cgagaatctc atctggctcc 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgcatgacta atgcttcgga 20

Claims (2)

1. The application of molecular marker primers of main effect QTL sites in flowering period and mature period of cabbage type rape in early flowering or early maturing screening breeding of cabbage type rape is as follows: CGAGAATCTCATCTGGCTCC, and TGCATGACTAATGCTTCGGA.

2. The application of molecular marker primers of main QTL sites in flowering and mature periods of cabbage type rape in molecular marker-assisted selection of early flowering or early-maturing cabbage type rape is as follows: CGAGAATCTCATCTGGCTCC, and TGCATGACTAATGCTTCGGA.

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