CN112126711B - Molecular marker of maize 4 th chromosome rough dwarf disease resistance major QTL and application thereof - Google Patents
Molecular marker of maize 4 th chromosome rough dwarf disease resistance major QTL and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker closely linked with a major QTL for controlling maize rough dwarf virus resistance, which comprises SSR molecular markers CHR4-59/CHR4-67, and forward and reverse primer sequences for amplifying CHR4-59/CHR4-67 molecular markers are CHR4-59F/R and CHR 4-67F/R. The invention provides a molecular marker closely linked with a major QTL for controlling maize rough dwarf resistance, which can be used for detecting the rough dwarf resistance in maize strains; the molecular marker widens the marker selection of the maize rough dwarf disease resistance, defines the genetic rule of the maize rough dwarf disease resistance, can be used for the molecular marker-assisted breeding of the maize rough dwarf disease resistance, directionally and accurately selects breeding materials and shortens the breeding period.
Description
Technical Field
The invention relates to the technical field of genetic breeding and molecular biology, in particular to a molecular marker closely linked with a maize rough dwarf disease resistance major QTL and application thereof.
Background
Corn (Zea mays L.) is one of the most important grain crops in the world and has a very important strategic position in the grain production in China. However, corn is a natural host for a variety of pathogenic viruses, and diseases caused by some of these viruses can severely affect the yield and quality of corn. Maize Rough Dwarf Disease (MRDD) was first discovered in singkiang and gansu in china in 1954 and subsequently spread to other provinces. In recent years, due to the increase of extreme climate frequency and the popularization and application of the interplanting technology, the occurrence frequency of maize rough dwarf disease in some maize main production areas in China is increased, the incidence rate of severe plots is over 30 percent, the maize production is seriously influenced, and huge economic loss is brought to farmers. The pathogeny causing maize rough dwarf disease is very complex, and the main pathogenic viruses of the maize rough dwarf disease have significant difference in different maize producing areas and are mainly derived from four fijivirus viruses: maize rough dwarf virus MRDV, rice black-streaked dwarf virus RBSDV, southern rice black-streaked dwarf virus SRBSDV and MRCV, wherein MRDV mainly affects Europe, MRCV mainly affects south America and RBSDV mainly affects Asian regions. The main onset symptoms of maize rough dwarf disease include internode shortening, tassel deformity and even inability to carry out reproductive growth. At present, the prevention and control of the disease mainly depend on chemical pesticides, and long-term use of the pesticides wastes resources and pollutes the environment. Therefore, MRDD resistance breeding is the most effective and environment-friendly strategy for controlling diseases, and analysis of the disease-resistant molecular mechanism of maize rough dwarf disease plays an important guiding role in disease-resistant breeding. No germplasm material completely immune to MRDD has been found so far, most maize germplasm appears susceptible to rough dwarf disease, but some highly rough dwarf resistant material has been found in china, most of which are mainly derived from american hybrid P78599. In recent years, some studies have been conducted on maize MRDD resistant QTLs, where loci associated with resistance to rough dwarf disease have been detected on all chromosomes of maize. The novel rough dwarf resistant locus is developed and applied to molecular marker-assisted selective breeding practice, can improve the disease-resistant breeding efficiency, and has important guiding significance for breeding new maize rough dwarf resistant varieties.
Disclosure of Invention
In view of the above, the present invention provides a molecular marker for controlling tight linkage of a major QTL for maize rough dwarf disease resistance, which can improve the efficiency of breeding for disease resistance.
The invention provides a molecular marker closely linked with a major QTL for controlling maize rough dwarf virus resistance, which comprises SSR molecular markers CHR4-59/CHR4-67, and forward and reverse primer sequences for amplifying CHR4-59/CHR4-67 molecular markers are CHR4-59F1/R1 and CHR4-67F 2/R2.
Preferably, the primer sequences of the molecular markers for controlling the linkage of the maize rough dwarf resistance major QTL are respectively:
CHR4-59F NO:F1 GACAGGCCCAAGGCAATAAG
CHR4-59R NO:R1 GCTGCTGCTCTTCACAGGTTC
CHR4-67F NO:F2 TTAGGGTTTTCCCAAAGA
CHR4-67R NO:R2 TGAGATCGCAGAGTCGTA。
preferably, the major QTL for maize rough dwarf resistanceqMRDD4At chromosome 4 bin 4.07; preferably, the resistance gene is located between 168.91-169.03 Mb.
Preferably, the molecular markers closely linked to the maize rough dwarf resistance gene are obtained by the following steps: taking corn genome DNA to be detected as an amplification template, carrying out PCR amplification by using primers CHR4-59/CHR4-67, and detecting a PCR product by using an ABI3500XL genetic analyzer to obtain a polymorphic molecular marker band: if the PCR product has the same target band as the parent D863F, the maize material to be detected can be judged to contain the rough dwarf resistance gene and be a rough dwarf resistance maize strain; if the amplification product does not have the same target band as the parent D863F, the maize material to be tested can be judged to be a non-rough dwarf resistant maize strain.
The invention also provides application of the molecular marker in the positioning of maize rough dwarf disease resistance genes.
Preferably, the molecular marker is applied to the positioning of the maize rough dwarf resistance major QTL.
The invention also provides application of the molecular marker in screening and identifying maize rough dwarf disease resistant germplasm resources.
Preferably, the molecular marker is used for assisting breeding.
The invention has the beneficial effects that:
the invention provides a molecular marker closely linked with a major QTL for controlling maize rough dwarf resistance, which can be used for detecting the rough dwarf resistance in maize strains; the molecular marker widens the marker selection of the maize rough dwarf disease resistance, defines the genetic rule of the maize rough dwarf disease resistance, can be used for the molecular marker-assisted breeding of the maize rough dwarf disease resistance, directionally and accurately selects breeding materials and shortens the breeding period.
The invention firstly detects a major QTL for controlling maize rough dwarf disease resistance on the maize 4 th chromosome through QTL positioningqMRDD4Molecular markers closely linked to the major QTL were found. The molecular marker can be used for detecting the rough dwarf resistance of the corn material, and lays a solid foundation for the rough dwarf resistance breeding of the corn.
Drawings
FIG. 1 shows the identification and comparison of the rough dwarf in the field of parents D863F, ZS301 and Zheng 58 from left to right;
QTL of FIG. 2qMRDD-4Fine positioning map of (2);
FIG. 3 banding pattern of SSR molecular marker CHR4-59 in segregating population;
FIG. 4 banding pattern detection of SSR molecular marker CHR4-67 in segregating populations.
Detailed Description
The invention is further described below with reference to the figures and examples.
Example 1
1. Design of experiments
1.1 through field phenotype identification, carrying out rough dwarf disease resistance detection on 80 parts of maize inbred lines, and screening D863F, ZS301 and Zheng 58 which have excellent comprehensive agronomic characters and remarkable rough dwarf disease resistance difference.
1.2 hybridizing D863F and ZS301 with Zheng 58 to obtain F 1 And continuously selfing for 6 generations by using a single-grain genetic method to construct a population so as to obtain a recombinant inbred line population (RIL).
1.3, by carrying out field rough dwarf resistance identification on RIL groups, and simultaneously carrying out QTL positioning and linkage marker identification on genes controlling rough dwarf resistance by utilizing an SSR marker technology.
1.4 using the obtained linkage marker to evaluate the application of allele in the offspring.
2. Materials and methods
2.1 test Material and field test
The invention carries out the field identification of the maize rough dwarf disease resistance on 80 parts of maize germplasm resources, wherein the maize germplasm resources comprise a maize common inbred line and a self-selection line. The field test is arranged in the modern agricultural research and development base of Henan, the original Yang county of New county, Henan, and the environment around the test point has a large number of Laodelphax striatellus insect sources, thereby ensuring the field disease of the corn. And (4) random block sowing is adopted, the planting row length of each identification material is 5 m, the row spacing is 0.6 m, the plant spacing is 0.2 m, and the field management is normal.
Creating a Recombinant Inbred Line (RIL) group: hybridizing the above identified D863F, ZS301 and Zheng 58 with differences in rough dwarf resistance to obtain F 1 And establishing a Recombinant Inbred Line (RIL) group by adopting a single-grain propagation method.
The disease index identification method comprises the following steps: and (4) performing field disease investigation about 55 days after the corn seedlings emerge, and investigating the field disease level of the plants according to the 0-4 grade grading standard. The disease index is calculated according to the disease level of the material, and the disease index DSI is sigma (the number of diseased plants at each level x the disease level value)/(the total number of investigated plants x the highest disease level) x 100%. Disease indexes are divided into 5 disease-resistant types of high resistance (DSI:0-10%), resistance (DSI:10.1% -30%), resistance (DSI:30.1% -50%), feeling (DSI:50.1% -70%) and high feeling (DSI: more than 70%).
2.3 genetic map construction and QTL analysis
2.3.1 genomic DNA extraction
In the 6-leaf stage of the corn, selecting young leaves, quickly freezing and grinding by liquid nitrogen, extracting DNA by adopting a CTAB method, and storing a DNA sample in a refrigerator at the temperature of-20 ℃ for later use.
2.3.2 PCR amplification and electrophoretic detection
Near 1900 pairs of SSR primers are uniformly selected from a publicly published corn molecular marker genetic map, the polymorphism among parents is detected, and 215/181 of the SSR primers with the co-dominant polymorphism with clear band type and good repeatability is selected for the genotype analysis of individuals of a recombinant inbred line and the construction of a linkage map.
PCR Total reaction 10.0 mL: 1.0 mL of DNA, 0.4 mL of primer (M13/F/R), 0.15 mL of 2.5U/mL of Taq enzyme, 1.6 mL of 10 XBuffer (Mg2+), 0.15 mL of 10 mmol/L dNTP and 6.7 mL of double distilled water. The PCR amplification procedure was: 3 min at 94 ℃; 94 ℃ for 1 min, 65 ℃ for 1 min (-1 ℃/cycle), 72 ℃ for 1.5 min, 7 cycles; 94 ℃ 1 min, 58 ℃ 1 min, 72 ℃ 1.5 min, 7 times; 30 cycles; and 5 min at 72 ℃.
After the PCR amplification product is denatured, capillary fluorescence electrophoresis is carried out by using an ABI3500 genetic analyzer, the homozygous banding pattern of the disease-resistant parent is represented by '0', the homozygous banding pattern of the disease-resistant parent is represented by '1', and the heterozygous banding pattern is represented by '2', according to the banding pattern data.
3. Analysis of results
3.1 identification of rough dwarf resistance in maize germplasm
80 parts of maize inbred lines are planted in the Henan original sun in 2016, and the phenotype results show that: the disease index range of rough dwarf disease of the inbred line is between 3.5 percent and 100 percent. Materials can be classified into 5 types of high resistance, medium resistance, susceptibility and high susceptibility according to the order of the DSI index from low to high. The early 6 months is the transition high-peak period of the laodelphax striatellus, the number of the single-plant corn laodelphax striatellus is 5-10, and the severity and the consistency of the whole identification field corn rough dwarf disease are good. From the inbred lines tested, 2 parts of high resistant inbred line, 8 parts of resistant inbred line and 10 parts of medium resistant inbred line were selected, and the remaining maize material showed either a disease or a high disease (table 1). Wherein the inbred line D863F shows high resistance, ZS301 shows medium resistance, Zheng 58 shows high infection, and rough dwarf resistance localization research is carried out on the combined recombinant inbred line group.
TABLE 1 identification of rough dwarf resistance of partial maize inbred lines
3.2 genetic map construction and analysis of resistance to rough dwarf disease
The opened phenotype identification of the RIL population parents in Henan in 2016-2017 showed: the parents D863F, ZS301 and Zheng 58 have significant differences in resistance to MRDD under natural infection for more than two years (parent D863F, ZS301, Zheng 58 field rough dwarf identification is shown in figure 1), among the three parents, D863F has the strongest resistance to MRDD (2016 DSI: 2.82%, 2017 DSI: 4.20%), ZS301 has the second highest resistance (2016 DSI: 23.55%, 2017 DSI: 18.75%), and Zheng 58 has the lowest resistance (2016 DSI: 90.25%, 2017 DSI: 93.96%). The DSI of 241 RIL-Zheng 58 XD 863F strains and 242 RIL-Zheng 58 XZS 301 strains is also greatly changed, wherein the DSI change range of RIL-Zheng 58 XD 863F is 3.85-100%, and the DSI change range of RIL-Zheng 58 XZS 301 is 15.28-100%.
Based on Zheng 58 × D863F, a recombinant inbred line population containing 241 families is constructed, and parent polymorphism markers of the recombinant inbred line population and the parents Zheng 58 and D863F thereof are screened to obtain 232 polymorphism SSR markers. Using the Jionmap4 software, 1 genetic linkage map containing 215 SSR marker loci was constructed. Constructing a recombined inbred line group containing 242 families based on Zheng 58 xZS 301, and carrying out parent polymorphism marker screening on the recombined inbred line group and parents Zheng 58 and D863F thereof to obtain 190 polymorphism SSR markers. By using the Jionmap4 software, 1 genetic linkage map containing 181 SSR marker loci is constructed, 10 linkage groups are contained, and the marker distribution is relatively uniform.
Under 2 years of different environmental conditions, 2 RIL populations jointly detected QTLs associated with rough dwarf resistance at the maize 4 th chromosome bin4.07 positionqMRDD4The resistance sites were from D863F and ZS301 (table 2), respectively.
TABLE 2 RIL population 2 years consensus localization results
| QTL | Chromosome | Marker Interval | LOD | Add-DZ | Add-ZZ | |
| 2016 |
|
4 | umc1620-bnlg2162 | 5.25 | -5.16 | -2.43 |
| 2017 |
|
4 | umc1620-bnlg2162 | 4.52 | -3.09 | -2.62 |
3.3 QTL associated with rough dwarf resistance derived from inbred line D863FqMRDD-4Fine positioning of
By utilizing (Zheng 58 multiplied by D863F) multiplied by Zheng 58 backcross population, on the basis of early initial positioning, through constructing a near isogenic line and further developing a marker in a positioning interval, the disease-resistant major QTL is subjected toqMRDD4And carrying out fine positioning. To eliminate the interference of genetic background on the main effect QTL effect, further definingqMRDD4Subject group selected RIL137 strain (DSI: 29.37%) as donor parent in RIL (Zheng 58X D863F) populationZheng 58 in the constitution of the Benzhang backcross population, of which RIL137 isqMRDD8The major effective site is from Zheng 58,qMRDD4site is from D863F. After four rounds of backcross and two generations of selfing, the resistance allele carried by RIL137 is introduced into Zheng 58 genetic background.
Construction by hybridization of NIL-3 with Zheng 58qMRDD4F of site 2 Segregating the population to obtain 27qMRDD4Individuals that have recombined within the interval. Referring to B73 (V4) genome sequence information on MaizeGDB, 40 pairs of SSR markers uniformly distributed near a target interval are developed, wherein 14 pairs of markers have polymorphism between parental Zheng 58 and D863F and can be used for group genotype detection.
27 recombinant individuals were assigned to 11 recombination events using 14 newly developed SSR markers within the target segment. 27 recombinant individuals were selfed and 12 representative recombinant individuals were selected for progeny testing. By comparing the DSI differences of homozygous recombinant pedigree and the sick parent zheng 58, the results show that when the homozygous recombinant pedigree comprises the CHR4-59/CHR4-67 genomic fragment from the disease resistant parent D863F (RL-6, RL-9, RL-10, RL-11 and RL-12), the DSI of the homozygous recombinant pedigree is significantly lower than zheng 58; when the M9-M11 genomic fragment is derived from Zheng 58, there was no significant difference (QTL) between DSI and Zheng 58 of homozygous recombinant pedigrees (RL-1, RL-2, RL-3, RL-4, RL-5, RL-7 and RL-8)qMRDD-4See fig. 2). Therefore, presume thatqMRDD4The candidate gene is located in the interval CHR4-59/CHR 4-67.
By using the recombinant single strain in the segregating population to combine with resistance phenotype analysis, the disease-resistant gene is between the markers CHR4-59 and CHR4-67, and the resistance gene localization interval is reduced to be in the range of 168.91-169.03 Mb by combining with B73 reference genome physical location information, (the banding pattern of the SSR molecular marker CHR4-59 in the segregating population is shown in figure 3, and the banding pattern of the SSR molecular marker CHR4-67 in the segregating population is shown in figure 4).
Prediction of disease resistance candidate genes: to further determineqMRDD4Aligning the genomic reference sequence information of B73 on the MaizeGDB for one or more candidate genes by aligning CHR4-59/CHR4-67 marker intervals, aligning the sequence information over the mapped intervals, and predicting the targetThere are 4 target candidate genes in the segment (GRMZM 2G125378, GRMZM2G125544, GRMZM2G125512, GRMZM2G 125268). The homologous genes of GRMZM2G125378 and GRMZM2G125544 on grape and Arabidopsis are found to have disease resistance function by NCBI BLAST comparison (Table 3), and can be used as candidate genes of rough dwarf disease resistance genes.
TABLE 3 candidate Gene prediction, chromosomal location and functional Annotation
3.3 application of closely-linked SSR markers CHR4-59/CHR4-67 to maize rough dwarf disease resistance breeding
Zheng 58 and D863F derived F 8 In the recombinant inbred line group, background and foreground selection identification is carried out on SSR molecular markers by 232, a strain RIL137 with similar genetic background to parental Zheng 58 is selected, and the strain is introduced with a major QTL for controlling the rough dwarf resistance on the 4 th chromosome from D863FqMRDD4Hybridizing RIL137 and Zheng 58, selfing to obtain F 2 Isolating the population.
Statistical analysis of genotype and rough dwarf resistance of each strain in the segregating population: the marker CHR4-59/CHR4-67 selects 36 genotypes from the segregation population single plants, the genotypes of the selected 36 genotypes are consistent with those of the resistant parent D863F, and the average DSI value of the selected genotypes reaches 39.8%; 45 individuals with the same genotype as Zheng 58 have the average value of the DSI reaching 82.7 percent. Therefore, the combination of the molecular markers CHR4-59/CHR4-67 can directionally and accurately screen rough dwarf virus resistant materials, save breeding cost and time, and is used for corn rough dwarf virus resistant breeding.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> institute of plant protection of academy of agricultural sciences of Henan province
Molecular marker of <120> maize 4 th chromosome rough dwarf disease resistance major QTL and application thereof
<130> /
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 1
gacaggccca aggcaataag 20
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 2
gctgctgctc ttcacaggtt c 21
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 3
ttagggtttt cccaaaga 18
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 4
tgagatcgca gagtcgta 18
Claims (6)
1. A molecular marker closely linked with a major QTL for controlling maize rough dwarf disease resistance is characterized by comprising SSR molecular markers CHR4-59/CHR4-67, and forward and reverse primer sequences for amplifying CHR4-59/CHR4-67 molecular markers are CHR4-59F1/R1 and CHR4-67F 2/R2;
the primer sequences are respectively as follows:
CHR4-59F NO:F1 GACAGGCCCAAGGCAATAAG
CHR4-59R NO:R1 GCTGCTGCTCTTCACAGGTTC
CHR4-67F NO:F2 TTAGGGTTTTCCCAAAGA
CHR4-67R NO:R2 TGAGATCGCAGAGTCGTA。
2. a method for identifying the resistance of corn rough dwarf disease is characterized in that the genomic DNA of the corn to be detected and the genomic DNA of the corn D863F are used as amplification templates, the primers in claim 1 are respectively used for PCR amplification, if the PCR products of the two are the same, the corn material to be detected can be judged to contain the rough dwarf disease resistance gene and be a rough dwarf disease resistance corn strain; if the PCR amplification products of the two are different, the corn material to be detected can be judged to be a non-rough dwarf disease resistant corn strain.
3. Use of the molecular marker of claim 1 for mapping maize rough dwarf resistance gene.
4. The use of claim 3, wherein the molecular marker is used for the mapping of maize rough dwarf resistance major QTL.
5. Use of the molecular marker of claim 1 in screening and identifying maize rough dwarf resistant germplasm resources.
6. The use of claim 5, wherein the molecular marker is used in assisted breeding.
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