CN108913804B - Molecular marker closely linked with major QTL (quantitative trait locus) of wheat plant height character and application thereof - Google Patents

Abstract

The invention discloses a molecular marker closely linked with a wheat plant height trait major QTL and application thereof. The invention provides a primer pair for detecting the plant height of wheat to be detected, which is a primer pair for amplifying a target fragment; the target fragment is a fragment containing a fragment obtained by amplifying the primer 1 and the primer 2; the primer 1 is a single-stranded DNA molecule shown in a sequence 1; the primer 2 is a single-stranded DNA molecule shown in a sequence 2. Experiments prove that the primer pair SSR-2433 is used for auxiliary selection of the wheat segregation population. The result shows that the single plant with the genotype consistent with the high-stalk parent Jing 411 is obviously higher than the single plant with the genotype consistent with the short-stalk parent, which shows that the primer pair SSR-2433 is practical and effective for the molecular marker-assisted selection of wheat short-stalk breeding.

Description

Molecular marker closely linked with major QTL (quantitative trait locus) of wheat plant height character and application thereof

Technical Field

The invention belongs to the technical field of molecular biology-level genetic breeding, and particularly relates to a molecular marker closely linked with a major QTL (quantitative trait locus) of a wheat plant height character and application thereof.

Background

Wheat is the third major food crop in our country. With the increasing population and the reduction of the cultivated land area, the method further improves the per unit yield of wheat in China, and has important practical significance for guaranteeing the national food safety. The discovery and utilization of the key genes related to the yield play an important role in excavating the genetic potential of the wheat yield and improving the yield per unit of wheat. For example, the cultivation of semi-short stalk varieties in the 'green revolution' greatly improves the yield of crops such as wheat. The cloning and function research of the wheat dwarf gene Rht1 shows that one or a few genes can possibly cause great breakthrough of one-time yield breeding. Recent research shows that the Rht1 gene can reduce the plant height and simultaneously cause the grain weight to be obviously reduced. Therefore, in order to break the single yield level of the existing high-yield variety, a new dwarf gene needs to be utilized, and the positioning and cloning of related genes are also important bases for improving the wheat plant height property.

The discovery and utilization of dwarf genes began in the 19 th century japan's local varieties red wheat (Akagomugi) and dalm (Dar μ Ma). In 1916, the breeder Strampelli used the crossing of red wheat to breed dwarf wheat variety in Italy (Zhaoyu et al, 1991). In 1935, Japan bred famous agriculture and forestry No. 10 from the filial generation of Darman dwarf. Then, the high-yield varieties Gaines and Nugains are bred by introducing agriculture and forestry No. 10 in the 40 th of the 20 th century in the United states; in the 70's of the 20 th century, the earliest high yielding varieties Maris Fundin and Hobbit were developed by j.a.report in england using a derivative line of agriculture and forestry No. 10. In 1962, the world corn and wheat improvement center utilizes agriculture and forestry No. 10 to breed the first batch of short-stalked spring wheat with high yield, wide adaptability, plant height of 65cm-85cm, upright leaves of Mexican wheat Pitic62, Penjamo62 and the like, and the wheat is widely planted in China and America, China east, south Asia, south America, north Africa and the like. Derived offspring of Chilean wheat Walger system is used in 1964 to breed semi-short stalk winter wheat variety Fundin, and then semi-short stalk high-yield varieties such as Hobbit, Norman, Avelon and the like are bred, and the popularization area reaches 4000 kilohm²The above.

Scholars at home and abroad have conducted positioning analysis research on the QTL of plant height by utilizing different genetic backgrounds and different environmental conditions. Ellis et al (2005) located a number of genes that reduced plant height without affecting early growth. Keller et al (1999) utilized a population of recombinant inbred lines of common wheat Forno and spelt Oberkulmer to detect 11 QTLs distributed on 9 chromosomes in three environments, with a single QTL accounting for 7.9% -31.4% of the phenotypic variation and all QTLs accounting for 72.6% of the phenotypic variation. Liu Dong Cheng et al (2003) utilize F of ND3338 XF 390_2:3The family locates 7 QTLs related to the wheat plant height under 4 environmental conditions, wherein a single QTL can explain 5.2-37.2% of the phenotypic variation, and all QTLs can explain 64.8-75.0% of the phenotypic variation. Wang et al (2009) detected 6 plant height QTLs on 1D, 2D, 3D and 4D chromosomes, respectively, using the RIL population of heshangma × Yu8679, and a single QTL could account for 5.83% -25.24% of the phenotypic variation.

The dwarf gene of wheat is also utilized in the breeding of wheat in China, but the source of the dwarf gene is relatively single. The frequency analysis result of dwarf genes of 239 varieties of the Chinese main wheat district by the Yangrouje et al (2006) shows that Rht-B1B dwarf genes carried by the Chinese wheat variety or strain come from St2422/464 and agriculture and forestry No. 10, and Rht-D1B dwarf genes come from agriculture and forestry No. 10, water source 86, Huimihong and Scutigerella spp. The dwarf sources used in the production of China are mainly researched by Rht-B1B, Rht-D1B and Rht8, and the distribution frequency of 3 dwarf genes in different wheat regions is different (Zhang Xiaoke et al, 2007).

The development and utilization of molecular markers and the gradual maturity and perfection of molecular marker technology further provide a favorable tool for the positioning research of wheat quantitative trait genes. Cadalen et al (1998) used the DH population generated by the hybridization of Courtot and Chinese spring to locate 9 QTLs related to wheat plant height, wherein 2 RFLP markers Xfba1-4B, Xfba211-4D are linked with dwarf genes Rht1 and Rht2 respectively. Tao et al (2008) use the BSA method with F₂2 SSR markers wmc522 and wmc198 screened on the 2A chromosome of the segregating population are linked with dwarf genes.

Disclosure of Invention

The invention aims to provide a primer pair for detecting the plant height of wheat to be detected.

The primer pair provided by the invention is a primer pair for amplifying a target fragment;

the target fragment is a fragment containing a fragment obtained by amplifying the primer 1 and the primer 2;

the primer 1 is a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 1;

a2) a single-stranded DNA molecule which is obtained by deleting, inserting and/or changing one or more bases of the single-stranded DNA molecule defined by a1) and has the same function as the single-stranded DNA molecule defined by a 1);

the primer 2 is b1) or b2) as follows:

b1) a single-stranded DNA molecule shown in sequence 2;

b2) single-stranded DNA molecule obtained by deleting, inserting and/or changing one or more bases of the single-stranded DNA molecule defined by b1) and having the same function as the single-stranded DNA molecule defined by b 1).

The primer pair consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA shown in a sequence 2.

PCR reagents containing the primer pairs are also within the scope of the present invention.

In the PCR reagent, the final concentration of each primer in the PCR reagent is 2-4 mu M.

A kit comprising the above primer set or the above PCR reagent is also within the scope of the present invention.

The application of the primer pair or the PCR reagent or the kit in detecting the height of the wheat plant to be detected is also within the protection scope of the invention.

The application of the primer pair or the PCR reagent or the kit in predicting, screening or cultivating the short-stalk wheat to be detected is also within the protection range of the invention;

or the primer pair or the PCR reagent or the kit is also applied to prediction or screening or cultivation of high-stem wheat to be detected.

The invention also aims to provide a method for detecting the plant height of wheat to be detected.

The method provided by the invention comprises the following steps: carrying out PCR amplification on wheat to be detected by using the primer pair or the PCR reagent or the kit, and detecting a PCR product; the plant height of the wheat to be detected with the PCR amplification product size of 139-145bp (specifically 141bp) is lower than that of the wheat to be detected with the PCR amplification product size of 130-134bp (specifically 132 bp).

The 3 rd purpose of the invention is to provide a method for detecting the plant height of wheat to be detected.

The method provided by the invention comprises the following steps: carrying out PCR amplification on wheat to be detected by using the primer pair or the PCR reagent or the kit, and detecting a PCR product; carrying out the same amplification by taking Yumai 8679 and Jing 411 as controls;

the plant height of the wheat to be detected with the same banding pattern as the PCR amplification product of Yumai 8679 is lower than that of the wheat to be detected with the same banding pattern as the PCR amplification product of Jing 411.

The 4 th purpose of the invention is to provide a method for cultivating wheat to be tested with short stalks.

The method provided by the invention is used for cultivating the wheat to be detected with the PCR amplification product of 141bp obtained by the method or cultivating the wheat to be detected with the banding pattern consistent with that of the PCR amplification product of 8679 Yumai obtained by the method;

the 5 th purpose of the invention is to provide a method for cultivating high-stem wheat to be tested.

The method provided by the invention is used for cultivating the wheat to be detected with the PCR amplification product of 132bp obtained by the method or cultivating the wheat to be detected with the banding pattern consistent with that of the PCR amplification product of Jing 411 obtained by the method.

The wheat to be detected is Yumai 8679, Jing 411 or RIL group derived after the Yumai 8679 and the Jing 411 are hybridized or the selfing progeny of the residual hybrid line, in particular to F obtained by selfing the hybrid in the RIL line yj-171 of the Yumai 8679 and the Jing 411₂And (4) generation groups.

The primer pair SSR-2433 is a marker closely linked with the plant height major gene locus Rht8 and is a codominant SSR marker based on a PCR technology, so that the primer pair SSR-2433 is reliable and convenient to use. Experiments prove that the primer pair SSR-2433 is used for auxiliary selection of the wheat segregation population. The result shows that the single plant with the genotype consistent with the high-stalk parent Jing 411 is obviously higher than the single plant with the genotype consistent with the short-stalk parent, which shows that the primer pair SSR-2433 is practical and effective for the molecular marker-assisted selection of wheat short-stalk breeding. According to the invention, through QTL positioning of wheat plant height traits, the positioned QPht. cau-2D.2 and Rht8 gene positions of wheat are the same (reference document, (Gaspernii et al.2012)), and a molecular marker SSR-2433 tightly linked with the same is obtained, and the main effective gene locus QPht. cau-2D.2/Rht8 of wheat can explain 12.07% of phenotypic variation, and can be used for map-based cloning and molecular marker assisted selection. In the conventional breeding method, the plant height character can be identified only after the mature period, and the method is easily influenced by the environment, large in identification field error and low in selection efficiency. The wheat plant height character is predicted by detecting the main gene locus of the plant height in the seedling stage, so that the production cost is saved, the selection efficiency is greatly improved, and the breeding process is accelerated. The position of the main gene locus of the plant height is clear, and the detection method of the main gene locus is convenient and quick and is not influenced by the environment. The dwarf material can be accurately and rapidly screened by detecting the molecular marker which is closely linked with the main gene locus of the plant height.

Drawings

FIG. 1 is a non-denaturing polyacrylamide gel electrophoresis image of PCR products obtained by PCR of genomic DNA of Yumai 8679 and Jing 411, and RIL groups derived therefrom, using SSR-2433.

FIG. 2 is a linkage map corresponding to a wheat 2D chromosome; the genetic location of the marker is on the left and the marker name and confidence interval schematic for Rht8 (italics) are on the right.

FIG. 3 is a diagram showing the results of the positioning of the plant height trait of wheat; in the figure, the abscissa is a linkage map, the ordinate is a LOD value, and the position pointed by an arrow is the position of a main effective locus QPht. cau-2D.2/Rht8 of the wheat plant height.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction and Property measurement of wheat plant height-oriented population and segregating population

The populations used in this example were the RIL population and F derived from Yumai 8679 and Jing 411₂Population (F derived from one of the remaining heterolines yj-171 in the RIL population₂And (4) a group.

The RIL population and the heterozygous line yj-171 are described in the following documents: ZHai HJ, Feng ZY, Li J, Liu XY, Xiao SH, Ni ZF, Sun QX (2016) QTL Analysis of spark Morphological Traits and Plant Height in Winter Wheat (Tritic μ M aestiv μ M L.) use a High-sensitivity SNP and SSR-Based Linkage map. frontiers in Plant Science 7.doi: 10.3389/fpls.2016.01617. Plant height phenotype data for RIL populations also can be obtained from this reference, yj-171 derived F₂The phenotype of the population is measured.

yj-171 derived F₂The population is obtained by self-breeding heterozygotes in the RIL line yj-171Cross to obtain F₂And (4) a group.

First, extraction of total DNA of seedling leaf

(1) Taking fresh leaves with the length of 1cm, putting the fresh leaves into a centrifugal tube with the length of 1.2mL, adding steel balls with the diameter of 4mm, quickly freezing by liquid nitrogen, and grinding.

(2) Adding 300 μ L CTAB, and soaking in 65 deg.C water bath for 60min, mixing for 2-3 times.

(3) Add 300 μ L of chloroform: isoamyl alcohol (24:1, V/V), mixing by gently inverting, centrifuging at 10000rpm for 10min, and pipetting 100. mu.L of the supernatant to transfer to a new PCR plate.

(4) Adding 100 mu L of isopropanol, and freezing at-20 ℃ for 30min to precipitate DNA; centrifuging at 10000rpm for 5min to precipitate DNA, pouring out supernatant, washing the precipitate with 75% ethanol for 2 times, and drying the precipitate in a fume hood;

(5) add 100. mu.L of ddH₂And O, dissolving the DNA, measuring the concentration of the DNA by using an ultraviolet spectrophotometer, and storing the DNA in a refrigerator at the temperature of 20 ℃ below zero for later use.

Secondly, development and synthesis of primers

The inventors have synthesized public and newly developed SSR primers by bio-companies.

Using DNA of two parents Yumai 8679 and Jing 411 as templates of screening primers, and using the dissolved primers to perform PCR amplification on the parental DNA, wherein a PCR reaction system (10 mu L system) is as follows in the following table 1:

TABLE 1

The PCR reaction procedure (10. mu.L system) is as follows:

TABLE 2

Detecting the PCR product by polyacrylamide gel electrophoresis.

The DNA of line 191 of the RIL population was PCR amplified with primers that were polymorphic in the parents, and the PCR products were subjected to polyacrylamide gel electrophoresis, visualization, staining and banding pattern interpretation (FIG. 1). The SSR marker is a co-dominant marker, namely, a differential band shows variation on position (namely, the size of an amplification product), and the band types of the separated groups are respectively read as A and B according to circumstances and respectively represent the band types derived from Yumai 8679 and Jing 411. And (4) judging the band type of the molecular marker obtained after dyeing to obtain the genotype data of the molecular marker. The genetic linkage map of the molecular markers was constructed by linkage analysis of the genotype data of the molecular markers of the RIL population using the Joinmap4.0 software (FIG. 2). QTL detection is carried out by utilizing QTLCart2.5 software based on the genetic map, genotype data of RIL groups and plant height phenotype data, a main effect QTL locus (figure 3) with good repeatability is detected near a 2D chromosome SSR marker SSR-2433 (table 3), and the LOD value and the contribution rate of the locus are both large (table 4).

TABLE 32D linkage group plant height major QTL linkage marker SSR-2433 primer sequence

As shown in the result, the SSR marker SSR-2433 is selected as a target molecular marker, and consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA molecule shown in a sequence 2.

Example 2 application of molecular marker SSR-2433 in dwarf breeding

1. F for planting Yumai 8679 and Jing 411 in field₂And (4) a group.

2. Sampling by hanging a plate in the seedling stage, extracting total DNA of leaves, and carrying out PCR amplification on the total DNA by using a molecular marker SSR-2433, wherein the PCR amplification system is shown in table 1, and the PCR amplification reaction is shown in table 2.

Detecting the band type of the PCR product by polyacrylamide gel electrophoresis, and detecting F according to different band types₂The individual plants are classified into A-band type, B-band type and H-band type.

The A band type is the band type which is consistent with the parent Yumai 8679, and the band size is 141 bp;

the B band type is the same as the parent Jing 411, and the size of the band is 132 bp;

the H-band type is the simultaneous presence of two bands.

3. Investigation of F at maturity₂Plant height of the individual plants, the distance from the ground to the top of the ear (excluding the miscanthus floridulus) was measured before harvest, as shown in Table 5.

As can be seen from Table 5, the genotype of the molecular marker SSR-2433 is that the mean plant height of the single A-band type plant is significantly lower than that of the single B-band type plant.

Therefore, the molecular marker SSR-2433 can be used for assisting in detecting the plant height of wheat to be detected, and the specific steps are as follows:

amplifying wheat to be detected by using a molecular marker SSR-2433, detecting a PCR product,

the plant height of the wheat to be detected with the PCR amplification product of 141bp is lower than that of the wheat to be detected with the PCR amplification product of 132 bp.

Or, carrying out the same amplification by taking Yumai 8679 and Jing 411 as controls; the plant height of the wheat to be detected which is consistent with the banding pattern (banding pattern A) of the PCR amplification product of Yumai 8679 is lower than that of the wheat to be detected which is consistent with the banding pattern (banding pattern B) of the PCR amplification product of Jing 411.

The plant height data of the a-band type and the B-band type in table 5 were averaged, and the results are shown in table 6, and it can be seen that the average plant height of the a-band type wheat population to be tested was lower than the average plant height of the B-band type wheat population to be tested.

Therefore, the molecular marker SSR-2433 can be used for assisting in detecting the plant height of the wheat population to be detected, and the specific steps are as follows: amplifying the wheat population to be detected by using a molecular marker SSR-2433, detecting the banding pattern of a PCR product,

the plant height of the wheat population to be detected with the PCR amplification product of 141bp is lower than that of the wheat population to be detected with the PCR amplification product of 132 bp.

Or, carrying out the same amplification by taking Yumai 8679 and Jing 411 as controls; the plant height of the wheat group to be detected which is consistent with the banding pattern (banding pattern A) of the PCR amplification product of Yumai 8679 is lower than that of the wheat group to be detected which is consistent with the banding pattern (banding pattern B) of the PCR amplification product of Jing 411.

The wheat with low plant height can be used for cultivating short-stalk wheat.

TABLE 5F selection assisted by SSR marker SSR-2433₂Genotype and plant height data for individual plants of the population

TABLE 6

Sequence listing

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Claims (3)

1. The application of a primer pair or a PCR reagent containing the primer pair or a kit containing the primer pair in detecting the height of the wheat plant to be detected;

the primer pair consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA shown in a sequence 2;

the wheat is Yumai 8679 or Jing 411 or a group carried by the wheat.

2. The application of a primer pair or a PCR reagent containing the primer pair or a kit containing the primer pair in predicting, screening or cultivating the dwarf wheat to be detected;

the primer pair consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA shown in a sequence 2;

the wheat is Yumai 8679 or Jing 411 or a group carried by the wheat.

3. The application of a primer pair or a PCR reagent containing the primer pair or a kit containing the primer pair in predicting, screening or cultivating the high-stem wheat to be detected;

the primer pair consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA shown in a sequence 2;

the wheat is Yumai 8679 or Jing 411 or a group carried by the wheat.

Images