Molecular marker related to rape main inflorescence pod density and application thereof

Abstract

The invention belongs to the technical field of rape breeding and molecular biology, and discloses a molecular marker related to main inflorescence pod density (SDMI) and application of the molecular marker, the sequence of a forward primer for amplifying the molecular marker is 5 '-CCTAACCACCTACGCTGAG TTATATATAGATT-3', and the sequence of a reverse primer for amplifying the molecular marker is 5 '-TAG TCTGTAAATTTATTATGAGCTTCCTTGTCTT-3'. The total DNA (deoxyribonucleic acid) of a single rape plant is used as a template, and PCR (polymerase chain reaction) amplification is carried out by using the primer sequence, so that two bands of 279-bp and 254-bp can be obtained; the rape single plant only having a 279-bp single band is a sparse fruit individual, and the rape single plant only having a 254-bp specific band is a dense fruit individual. The molecular marker developed by the invention can be used for identifying and predicting the pod density, the operation is simple and convenient, the method is easy to implement, the offspring selection is more targeted, and the selection efficiency is improved.

Description

Technical field:

[0001] The invention belongs to the field of rapeseed breeding and molecular biology, and more specifically relates to a molecular marker related to rapeseed main inflorescence silique density (SDMI), and also relates to the application of a molecular marker related to SDMI degree in breeding rapeseed fruit density material. Background technology:

[0002] Rapeseed is a typical cruciferous vegetable. Its main inflorescence is located at the top of the plant. Siliques are mainly distributed on the tip of the main inflorescence and the side branches, which is the main economic part of rapeseed. The main inflorescence is very important for rapeseed yield and cultivating an ideal plant type. Plants with longer main inflorescences have higher plant heights, more main inflorescence siliques, and higher yields. In other words, it is difficult to increase the length of the main inflorescence without increasing plant height. Considering the current requirements for mechanical sowing in high-density rapeseed production, it is very important to select germplasm resources with appropriate main inflorescence length and high silique density. SDMI is closely related to rapeseed yield. Rapeseed mainly produces seeds from siliques on the main stem, and the number of siliques directly determines the number and yield of seeds. At present, there are few studies on SDMI of rapeseed. Therefore, studying how SD affects rapeseed yield can provide an important reference for high-yield rapeseed.

[0003] Genetic analysis of silique density in the main inflorescence (SDMI) revealed that SDMI may be controlled by multiple minor genes or not controlled by major genes. Through genetic linkage mapping, overlapping QTLs for SDMI and SNMI were found in the 11.6-27.3Mb interval on chromosome C06. Two 0.15Mb intervals were identified in the overlapping QTL region. Finally, a possible candidate gene BnARGOS was identified from these two intervals.

[0004] Based on the CDS, protein sequence and promoter sequence of the rapeseed reference gene BnARGOS, the CDS sequence (without intron) and promoter sequence of about -2000bp of BnARGOS of the fruit-dense and fruit-sparse parents were cloned respectively. Sequencing revealed that the BnARGOS gene sequence of the fruit-dense material (M) was consistent with the reference sequence (Zhongshuang 11) and the length was consistent with 408bp. Although the length of the fruit-sparse material (X) was also 408bp, there were 11 SNPs compared with the reference sequence and the fruit-dense parent, of which 4 were non-synonymous mutations. For the promoter region, the fruit-sparse material and the reference sequence were completely consistent, while the fruit-dense material and the reference sequence had 36 SNPs and 4 InDels. In summary, there were differences in the BnARGOS gene sequence and promoter region of the fruit-sparse and fruit-dense materials. The SDMI of transgenic Arabidopsis thaliana of OE-M-ARGOS and OE-X-ARGOS was significantly increased compared with WT, which speculated that the difference in promoters led to the difference in silique density of the parents.

[0005] The SDMI trait of rapeseed was finely mapped, and the candidate gene controlling the trait was identified. Based on the nucleotide sequence differences of the candidate gene, dense and sparse fruits, InDel markers with SDMI were developed. Summary of the invention:

[0006] The invention aims to provide a molecular marker related to rapeseed main inflorescence silique density (SDMI), the forward primer sequence for amplifying the molecular marker is 5'-CCTAACCACTACTCGCTGAGTTATATATATAGATT-3', and the reverse primer sequence is 5'-TAGTCTGTAAAATTTTAGTTATTTATTATGAGCTTTCCTTTGTCTT-3'.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A molecular marker associated with SDMI is obtained by the following method:

[0009] Using 6M023 and 6M029 with SDMI differences as parents, two sets of six-generation genetic analysis populations, P1 and P2, and reciprocal cross F1, F2, B1, B2, were constructed, and major gene + multi-gene mixed genetic analysis of SDMI traits was performed.

[0010] A DH positioning population was constructed, and simplified genome sequencing and genetic map construction were performed on the two parental materials and the DH population. Combined with the phenotypic data of the DH population in three environments over two years, it was found that SDMI and the number of siliques in the main inflorescence of rapeseed had overlapping QTLs in all three environments, located at 63cM-67.8cM on chromosome C06.

[0011] Using the identified rapeseed SDMI segregated DH population, including 103 DH lines, 17 extreme lines were selected from each to construct the SDMI extreme phenotype BSA mixed pool for resequencing and QTL-seq analysis. The resequencing-based QTL-seq identified a 0.15Mb QTL interval, which overlapped with the identified C06-QTL interval.

[0012] All rapeseed genes in the above interval were searched from the public database (http: / / yanglab.hzau.edu.cn / BnIR), and a gene BnARGOS related to SDMI was screened according to the corresponding functional annotations, existing literature related to plant siliques, and QRT-PCR.

[0013] DNA was extracted from the two parents, P1 and P2, and PCR amplification was performed. InDels were found in the CDS and promoter regions of the gene BnARGOS of both parents. Experiments suggested that the differences in promoters led to the differences in silique density between the parents.

[0014] According to the promoter sequence of rapeseed BnARGOS, primers that can amplify the above-mentioned InDel site were designed, the forward primer sequence was 5'-CCTAACCACTACTCGCTGAGTTATATATATAGATT-3', and the reverse primer sequence was 5'-TAGTCTGTAAAATTTTAGTTATTTATTATGAGCTTTCCTTTGTCTT-3'.

[0015] An application of a molecular marker related to rapeseed SDMI in rapeseed breeding comprises the following steps:

[0016] 1) Using DNA of one strain of oilseed oil as template;

[0017] 2) PCR was performed using the following primers:

[0018] The forward sequence of the primer is: 5'-CCTAACCACTACTCGCTGAGTTATATATATAGATT-3'

[0019] The reverse sequence of the primer was: 5′-TAGTCTGTAAAATTTTAGTTATTTATTATGAGCTTTCCTTTGTCTT-3′;

[0020] 3) After the enzyme digestion products were separated by 2% agarose gel electrophoresis at a voltage of 100 V and a current of 80 A, two bands of 279-bp and 254-bp were obtained; the single oilseed rape plant with only a single 279-bp band was a sparse fruit individual, and the single glycerol plant with only a 254-bp specific band was a homozygous dense fruit individual, which could be used for further breeding selection and cultivated into dense fruit materials to increase rapeseed yield.

[0021] Compared with the prior art, the present invention has the following advantages: the present invention can determine the density of rapeseed siliques (expressed as dense fruit) very early, distinguish it from sparse fruit materials, and thus retain it for the next round of selection, cultivate it into dense fruit materials, and increase rapeseed yield. The present invention can confirm dense fruit individuals as early as possible through early identification and auxiliary selection of SDMI molecular markers, reduce the planting scale of field materials and the later identification work, and effectively improve the efficiency and accuracy of selection. Description of the drawings:

[0022] Figure 1 Fine mapping of SDMI in rapeseed and the location of candidate genes.

[0023] Figure 2It is the difference in sequence of markers for sparse and dense fruits.

[0024] Figure 3 The figure is a schematic diagram of the SDMI detection of functional markers in parents and DH lines. m represents marker, M represents parents with dense fruits, X represents parents with sparse fruits, M-DH represents DH lines with dense fruits, and X-DH represents DH lines with sparse fruits.

[0025] Figure 4 This is a schematic diagram of the SDMI detection of functional markers in F2 and F3 generations. F2 and 3 represent the hybrid F2 and F3 generations obtained after self-pollination of F1 obtained by hybridization of P1 and P2, M-F2 and 3 represent the F2 and F3 generations with dense fruits, and X-F2 and 3 represent the F2 and F3 generations with sparse fruits. Specific implementation method:

[0026] The following is a specific implementation of the method of the present invention, but it is not intended to limit the method of the present invention. Any changes that do not depart from the essential content of the present invention should still fall within the protection scope of the present invention.

[0027] Embodiment 1:

[0028] This example takes a DH separation population obtained through a microspore experiment of a dense fruit rare fruit as an example, and describes in detail the method of obtaining molecular markers related to SDMI, as follows:

[0029] Population construction and phenotypic identification:

[0030] The two materials 6M023 and 6M029 with different SDMI were used as parents to prepare F1 hybrids. The pollen of F1 was taken for microspore culture and chromosome doubling treatment, and finally a DH population containing 103 genotypes was obtained.

[0031] SDMIQTL fine positioning

[0032] QTL-seq based on resequencing identified a 0.15Mb QTL interval, which overlapped with the C06-QTL interval located by QTL mapping based on the genetic linkage map.

[0033] (III) Candidate gene analysis:

[0034] All rapeseed genes in the above interval were searched from the public database (http: / / yanglab.hzau.edu.cn / BnIR), and a gene BnARGOS related to SDMI was screened according to the corresponding functional annotations, existing literature related to plant siliques, and QRT-PCR.

[0035] (IV) Functional marker conversion and detection:

[0036] DNA was extracted from the two parents, P1 and P2, and PCR amplification was performed, and it was found that the BnARGOS genes of both had InDels in the CDS region and promoter region.

[0037] We overexpressed the fruit density and fruit sparseness CDS in Arabidopsis thaliana and found that both increased SDMI compared with WT. Through experiments, we speculated that the differences in promoters led to the differences in silique density between the parents.

[0038] An InDel primer that can amplify the above site was designed, and the forward sequence of the primer was: 5'-CCTAACCACTACTCGCTGAGTTATATATATAGATT-3'; the reverse sequence of the primer was: 5'-TAGTCTGTAAAATTTTAGTTATTTATTATGAGCTTTCCTTTGTCTT-3'.

[0039] PCR amplification was performed in P1, P2 parents, and DH line plant materials. After the products were separated by 2% agarose gel electrophoresis, two bands of 279-bp and 254-bp were obtained. The single oleaginous plant with only the 279-bp single band was a sparse fruit individual, and the single oleaginous plant with only the 254-bp specific band was a homozygous dense fruit individual ( Figure 3 ). The differences in marker sequences between sparse and dense fruits are as follows Figure 2 shown.

[0040] Embodiment 2:

[0041] An application of a molecular marker related to rapeseed SDMI in rapeseed comprises the following steps:

[0042] 50 F2 plants obtained by self-pollination after hybridization of P1 and P2 were used as research materials. Among these plants, 11 showed sparse fruits, 14 showed dense fruits, and the remaining 25 showed medium silique density. Identification was performed using the markers obtained in Example 1, and the steps were as follows:

[0043] 1) Using DNA of one strain of oilseed oil as template;

[0044] 2) PCR was performed using the following primers:

[0045] The forward sequence of the primer was: 5′-CCTAACCACTACTCGCTGAGTTATATATATAGATT-3′;

[0046] The reverse sequence of the primer is: 5'-TAGTCTGTAAAATTTTAGTTATTTATTATGAGCTTTCCTTTGTCTT-3'

[0047] 3) PCR reaction system: The total volume is 10 μl, and the specific components are as follows:

[0048]

[0049] 4) PCR amplification program: 94°C for 5 min, [94°C for 45 s, 52°C for 45 s, 72°C for 30 s] × 35 cycles, 72°C for 10 min. Store at 4°C after the run.

[0050] 5) After the PCR products were separated by 2% agarose gel electrophoresis, two bands of 279-bp and 254-bp were obtained; the oil single strain with only the 279-bp single band was a homozygous individual with few fruits, and the glycerol single strain with only the 254-bp specific band was a homozygous individual with dense fruits ( Figure 4 ).

[0051] The above identification results show that in breeding, through the identification and screening of functional molecular markers, the density of rapeseed siliques (expressed as dense fruit) can be determined very early, and it can be distinguished from sparse fruit materials, so as to retain them for the next round of selection. Cultivating dense fruit materials can accelerate the breeding process and increase rapeseed yield.

Claims

1. An InDel molecular marker primer pair related to the density of siliques in the main inflorescence of rapeseed, characterized in that: The sequence of the forward primer was 5′-CCTAACCACTACTCGCTGAGTTATATATATAGATT-3′, and the sequence of the reverse primer was 5′-TAGTCTGTAAAATTTTAGTTATTTATTATGAGCTTTCCT TTGTCTT-3′.

2. An InDel molecular marker related to the density of siliques in the main inflorescence of rapeseed, characterized in that: The InDel molecular marker is obtained by amplifying the rapeseed genomic DNA as a template using the primer pair described in claim 1.

3. A kit for identifying the density of siliques in the main inflorescence of rapeseed, comprising the primer pair according to claim 1.

4. The kit according to claim 3, characterized in that Also contains reagents for PCR amplification.

5. Use of the primer pair according to claim 1, the molecular marker according to claim 2, or the kit according to claim 3 or 4 in the identification of silique density of rapeseed main inflorescence.

6. The use according to claim 5, characterized in that The steps include: 1) Using DNA of one strain of oilseed oil as template; 2) performing PCR amplification using the primer pair; 3) After the PCR products were separated by agarose gel electrophoresis, the single oil plant with only a 279-bp single band was a sparse fruit individual, and the single plant with only a 254-bp specific band was a dense fruit individual.

7. A method for identifying the density of siliques in the main inflorescence of rapeseed, characterized in that: The steps include: 1) Using DNA of one strain of oilseed oil as template; 2) performing PCR amplification using the primer set according to claim 2; 3) After the PCR products were separated by agarose gel electrophoresis, the rapeseed plant with only a single 279-bp band was a sparsely fruited individual, and the rapeseed plant with only a 254-bp specific band was a densely fruited individual.

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