A SSR molecular marker primer and its application in identifying true and false mango F1 hybrids and analyzing genetic characteristics

Abstract

The present invention provides a SSR molecular marker primer, including primer MG-023, primer MG-030, primer MG-039, primer MG-046, primer MG-187, primer MG-055, primer MG-063, primer MG-079, primer MG-114, primer MG-146, primer MG-150, primer MG-177 and / or primer MG-061. The present invention uses the above 13 pairs of mango core primers to perform true and false hybrid detection and genetic characteristic analysis on the offspring population of different mango varieties. The present invention can effectively reveal the genetic diversity of mango seedlings, and can better identify the existing main cultivated varieties, and can provide theoretical guidance for mango hybrid breeding parent selection and the like.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to an SSR molecular marker primer and an application thereof in identifying true and false hybrids of mango F1 generation and analyzing genetic characteristics. Background Art

[0002] mango( Mangifera indica Linn) is a plant of the genus mango in the Anacardiaceae family. It originated in Southeast Asia and is one of the famous tropical fruits. It is known as the "king of tropical fruits" and has certain nutritional and economic values. As a cross-pollinating fruit tree, mango has a high genome heterozygosity, and most breeding parents have unclear self-compatibility and hybridization compatibility. The artificial pollination fruiting rate is low, which brings certain difficulties to the selection and matching of hybrid breeding parents.

[0003] Hybridization is an important part of variety innovation and breeding. The selection of parents is the key to determine whether mango hybrid breeding is successful. In the natural pollination population, mango parents are difficult to identify. Current studies have shown that 80% of most mango varieties are from seed breeding, and only about 20% of varieties are bred through controlled pollination and mixed natural pollination. As a crop with high difficulty in directional breeding and long breeding cycle, most breeding parents of mango are unclear. The fruit setting rate of conventional artificial hybrid breeding is only 0.3% internationally. Therefore, it is necessary to carry out early identification of mango hybrid offspring to exclude false hybrids and improve mango breeding efficiency. Early molecular marker methods such as ISSR, RAPD, AFLP, and SRAP are not suitable for hybrid identification due to unstable typing, too many genotypes, poor repeatability, and non-codominance. Molecular markers are a new technology that has emerged after traditional morphological markers, cell markers, and biochemical markers. Compared with other molecular marker technologies such as RFLP, PAPD, STS, and AFLP, SSR markers (simple sequence repeats) have the advantages of rich sites, good repeatability, strong identification ability, high polymorphism, and co-dominance. They have been applied to the identification of true and false hybrids and genetic diversity analysis of hybrid offspring of crops such as peanuts, adzuki beans, pomegranates, tomatoes, rice, tea trees, and apples. At present, there are few studies on the application of SSR in the analysis of mango genetic characteristics, and there are no reports on the identification of true and false hybrids of mango F1 generation using SSR markers. Summary of the invention

[0004] Based on the deficiencies of the prior art, this application proposes an SSR molecular marker primer and its application in identifying true and false hybrids and genetic characteristics analysis of mango F1 generation. This application takes large fruit, red color, high solids, high and stable yield as breeding goals, selects 13 varieties as parent materials, selects 1001 offspring according to the size of the offspring population of different varieties, and uses SSR technology to detect true and false hybrids and analyze genetic characteristics.

[0005] The scheme of the present invention includes the following contents:

[0006] In one aspect, the present invention provides an SSR molecular marker primer, comprising primer MG-023, primer MG-030, primer MG-039, primer MG-046, primer MG-187, primer MG-055, primer MG-063, primer MG-079, primer MG-114, primer MG-146, primer MG-150, primer MG-177 and / or primer MG-061;

[0007] The nucleotide sequence of primer MG-023 is shown in SEQ ID NO.1 and SEQ ID NO.2;

[0008] The nucleotide sequence of primer MG-030 is shown in SEQ ID NO.3 and SEQ ID NO.4;

[0009] The nucleotide sequence of primer MG-039 is shown in SEQ ID NO.5 and SEQ ID NO.6;

[0010] The nucleotide sequence of primer MG-046 is shown in SEQ ID NO.7 and SEQ ID NO.8;

[0011] The nucleotide sequence of primer MG-187 is shown in SEQ ID NO.9 and SEQ ID NO.10;

[0012] The nucleotide sequence of primer MG-055 is shown in SEQ ID NO.11 and SEQ ID NO.12;

[0013] The nucleotide sequence of primer MG-063 is shown in SEQ ID NO.13 and SEQ ID NO.14;

[0014] The nucleotide sequence of primer MG-079 is shown in SEQ ID NO.15 and SEQ ID NO.16;

[0015] The nucleotide sequence of primer MG-114 is shown in SEQ ID NO.17 and SEQ ID NO.18;

[0016] The nucleotide sequence of primer MG-146 is shown in SEQ ID NO.19 and SEQ ID NO.20;

[0017] The nucleotide sequence of primer MG-150 is shown in SEQ ID NO.21 and SEQ ID NO.22;

[0018] The nucleotide sequence of primer MG-177 is shown in SEQ ID NO.23 and SEQ ID NO.24;

[0019] The nucleotide sequence of primer MG-061 is shown in SEQ ID NO.25 and SEQ ID NO.26.

[0020] Preferably, the primers include primer MG-023, primer MG-030, primer MG-039, primer MG-046, primer MG-187, primer MG-055, primer MG-063, primer MG-079, primer MG-114, primer MG-146, primer MG-150, primer MG-177 and primer MG-061.

[0021] On the other hand, the present invention also provides the application of the SSR molecular marker primers in the analysis of mango genetic characteristics.

[0022] Furthermore, the present invention provides the application of the SSR molecular marker primers in mango genetic diversity analysis.

[0023] Furthermore, the present invention provides the application of the SSR molecular marker primers in mango genetic relationship analysis.

[0024] Furthermore, the present invention provides the use of the SSR molecular marker primers in identifying true and false hybrids of mango F1 generation.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The invention provides 13 pairs of SSR marker primers that can be used for mango true and false hybrid detection and genetic characteristic analysis. The research results of the invention can provide theoretical guidance for mango hybrid breeding parent selection and the like.

[0027] Among the 13 SSR markers of the present invention, 10 are highly polymorphic and 3 are moderately polymorphic, and the polymorphism is relatively high, which can effectively reveal the genetic diversity of mango seedlings and better identify the existing main cultivated varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 :Genetic differentiation analysis results among varieties;

[0029] Figure 2 : Cluster analysis results among populations based on genetic distance;

[0030] Figure 3 : Genetic population differentiation map;

[0031] Figure 4 : Genetic structure diagram based on Structure analysis; DETAILED DESCRIPTION

[0032] In order to clearly and completely describe the technical solutions in the present invention, the following is illustrated in conjunction with embodiments, but the following embodiments describe only a part of the embodiments of the present invention, rather than all the embodiments.

[0033] 1 Materials and methods

[0034] 1.1 Test materials

[0035] The materials were planted in an independent breeding nursery of the fifth team of Danzhou Experimental Base, Institute of Variety Resources, Chinese Academy of Tropical Agricultural Sciences. 13 mango varieties were mixed and naturally pollinated. The seedlings of the female parent included Guifei, Tainong No. 1, Jinhuang, Julie, Nandoumai, Yexiang, Willard, R2E2, Tommy, Hongyu, A61, Kate, 12 varieties, and a total of 1001 materials. From the 13 varieties of the male parent Guifei, Jinhuang, Tainong No. 1, White Ivory, Hongyu, R2E2, Yexiang, Tommy, Kate, Willard, Nandoumai, Julie, and A61, the possible male parent of the female parent seedling was identified.

[0036] 1.2 Test methods

[0037] 1.2.1 DNA extraction

[0038] Use a hole puncher to punch holes in the leaves, take 100 mg of fresh tissue, and use a magnetic bead genomic DNA extraction kit to extract mango genomic DNA. Use an ultraviolet spectrophotometer and 1% agarose gel electrophoresis to detect DNA concentration and purity, and store at -20℃ for later use.

[0039] 1.2.2 Primer screening

[0040] Based on the transcriptome data of mango obtained earlier, 200 pairs of primers were selected. The primers were synthesized by Tianyi Huiyuan Biotechnology Co., Ltd. The 200 pairs of synthesized primers were screened using the DNA of each parent as a template. The screened primers were used to perform SSR analysis on the samples.

[0041] 1.2.3 SSR-PCR system

[0042] Table 1 PCR reaction system (15 μL)

[0043]

[0044] The amplification reaction program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C-62°C for 30 s, extension at 72°C for 30 s, 10 cycles, with a 1°C decrease in each cycle; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, 25 cycles; end extension at 72°C for 20 min, and storage at 4°C.

[0045] 1.2.4 PCR product detection

[0046] Agarose gel electrophoresis (1% concentration) and capillary fluorescence electrophoresis were used for detection.

[0047] 1.2.5 Data Statistics and Analysis

[0048] Excel was used to calculate the site information of the amplified alleles. Cervus software was used to calculate genetic parameters such as the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), fixation index (F), and Shannon's information index (I). GenALEx6.0 software was used to calculate the polymorphic information index (PIC) and obtain the genetic differentiation index (Fst). R language was used to draw hotspot maps. Structure was used to analyze the genetic structure of the population. The unweighted group average method (UPGMA) was used to perform cluster analysis among mango seedlings using the selected SSR core primers.

[0049] 2 Results and Analysis

[0050] 2.1 SSR primer screening

[0051] By performing multiple repetitive tests on each parent, 13 primer pairs with stable amplification and clear bands were screened (Table 2), and 13 sites with good polymorphism and stability were identified.

[0052] Table 2 Sequences of 13 pairs of SSR primers screened

[0053]

[0054] 2.2 Genetic diversity analysis

[0055] 2.2.1 Genetic diversity at each site

[0056] A total of 80 alleles were obtained in mango using 13 primer pairs, and the number of alleles amplified by each primer pair ranged from 3 to 9, of which 9 alleles were detected in MG-039, MG-046 and MG-187. The average number of effective alleles per locus was 2.557. The average Shannon's information index was 1.027, and the Shannon's information index of the MG-023 locus was the highest. The average expected heterozygosity was slightly higher than the average observed heterozygosity. It is generally believed that when 0.25>PIC<0.5, it is moderately polymorphic, and when PIC>0.5, it is highly polymorphic. The PIC values ​​of the 13 loci ranged from 0.314 to 0.741, indicating that these 13 loci were highly polymorphic (Table 3).

[0057] Table 3 Genetic parameters of mango SSR marker loci

[0058]

[0059] Note: Locus: the name of the selected primer pair; N: the number of effective individuals in the total population; Na: the number of alleles; Ne: the number of effective alleles; I: Shannon's information index; Ho: observed heterozygosity; He: expected heterozygosity; F: fixation index; PIC: polymorphic information index.

[0060] 2.2.2 Analysis of variance

[0061] Since the P value is less than 0.05, it indicates that significant differences exist between groups, between individuals, and within individuals, indicating that the heterozygous alleles within individuals lead to significant differences between groups, that is, individual genetic diversity is high.

[0062] Table 4 Analysis of variance

[0063]

[0064] 2.2.3 Population genetic diversity

[0065] In terms of the effective number of individuals (N) and the number of alleles (Na), GF was the highest and Julie was the lowest, indicating that in the total population, the actual observed allele types at the corresponding locus of GF were the most. When the effective allele value (Ne) was closer to the absolute value of the allele number (Na), it indicated that the alleles were more evenly distributed in the population. Compared with other varieties, the alleles of GF, YX, and Julie were more evenly distributed in the population. The average Shannon's information index (I) of GF was the highest and that of Julie was the lowest, indicating that the genetic diversity in the GF population was higher and the population differentiation was the highest. On the contrary, Julie was the lowest. The observed heterozygosity (Ho) and expected heterozygosity (He) of GF were both the highest, indicating that GF was highly polymorphic. The fixation index (F) can measure whether the actual frequency of genotypes in a population deviates from the Hardy-Weinberg theoretical ratio. When there are too many homozygotes in the population, F>0; on the contrary, when there are too many heterozygotes, F<0 (Table 5).

[0066] Table 5 Population genetic diversity

[0067]

[0068] Note: Mean represents mean, SE represents standard error; uHe: unbiased expected heterozygosity;

[0069] GF: Guifei; HY: Hongyu; JH: Jinhuang; KT: Kate; NDM: Nan Dou Mai; TM: Tommy; TN: Tainong No. 1; YX: Coconut Fragrance.

[0070] 2.2.4 Hardy-Weinberg equilibrium test

[0071] The Hardy-Weinberg law was used to study the genes of 12 different mango varieties. The results showed that 11 varieties were in equilibrium, and variety HY deviated significantly from the equilibrium state (P<0.05).

[0072] 2.3 Genetic differentiation analysis

[0073] 2.3.1 Genetic differentiation index Fst among varieties

[0074] Fst refers to the average inbreeding coefficient between related local populations. When Fst is between 0.05 and 0.15, it indicates that there is a moderate degree of genetic differentiation between populations. When Fst>0.05, there is a large genetic differentiation between populations. The Fst results between the two varieties showed that there was obvious genetic differentiation between HY and R2E2, HY and YX, HY and NDM, Julie and R2E2, Julie and NDM, GF and NDM, and GF and Julie, while the genetic differentiation between the remaining varieties was small ( Figure 1 ).

[0075] 2.3.2 Genetic distance and cluster analysis among varieties

[0076] Genetic distance is an indicator to measure the size of the comprehensive genetic differences of different traits between varieties, which can directly reflect the genetic differences between parent varieties. The genetic distances between varieties R2E2 and HY, and R2E2 and Julie are the largest, which are 0.7377 and 0.6220 respectively, indicating that the genetic differences between these varieties are large. The genetic distances of the other varieties are small and the genetic differences are not obvious.

[0077] According to the genetic distance between samples and populations, Phylip software and UPGMA method (unweighted group average method) were used to perform cluster analysis between populations. Among them, mango varieties KT and TM were clustered into one category, and varieties HY and Julie were clustered into one category, with a close relationship ( Figure 2 ).

[0078] 2.3.3 Structure Analysis

[0079] Structure analysis shows that when K = 3, △ K=35 maximum value( Figure 3 ), all individuals can be divided into three groups. According to the population genetic structure ( Figure 4) It can be seen that different colors represent different genetic compositions, and the varieties with the highest proportion of the same color can be clustered into the same subgroup, that is, R2E2 is one cluster, HY is one cluster, A61, GF, JH, Julie, KT, NDM, TM, TN, Willard and YX are clustered into one cluster. Therefore, the 13 varieties can be divided into three major subgroups, and the genetic background can be divided into three types.

[0080] (Note: Structure is a group structure analysis software.)

[0081] 2.4 Parentage Testing

[0082] Parentage analysis was performed on unknown parental combinations, and then the offspring whose maternal parents were not the first candidate parents were selected and eliminated. A total of 191 samples were eliminated, with an elimination rate of 19.1%. The paternity relationship of 809 offspring and candidate fathers (the mother parent was known) was identified, and the actual identification rate was 87%.

[0083] 2.5 Hybridization identification results

[0084] The amplification is stable, the peak shape is good in the screening sample, there is no continuous peak, there is no interference from other peaks in the target peak range, the fluorescence detection signal value is within the normal value range and it is the optimal primer. When all parents are amplified successfully, these primers can distinguish all parents, and then identify whether the parents and offspring conform to Mendel's laws of inheritance.

[0085] From the results of the identifiable male parent, it was found that among the 5 hybrid offspring with A61 as the female parent, the true hybrid rate was 80%; among the 117 offspring with GF as the female parent, the self-pollination rate was 38.46% and the true hybrid rate was 61.54%; among the 167 offspring with HY as the female parent, the self-pollination rate was 74.8% and the true hybrid rate was 25.2%; among the 69 offspring with JH as the female parent, the self-pollination rate was low and the true hybrid rate was 82.61%; among the hybrid offspring with Julie as the female parent, 50% of its male parent came from HY and 50% from NDM; among the offspring with NDM as the female parent, the hybridization rate was low, the true hybrid rate was 25%, and its self-pollination rate was 75%; among the 77 offspring with KT as the female parent, Among them, the true hybrid rate was 72.73% and the self-pollination rate was 27.27%; in the offspring group with R2E2 as the mother parent, the self-pollination rate was 82.69% and the true hybrid rate was 17.31%; in the offspring with TM as the mother parent, the self-pollination rate was 56.57% and the true hybrid rate was 43.43%; in the 59 offspring with TN as the mother parent, the self-pollination rate was 16.95% and the true hybrid rate was 83.05%; in the 51 offspring with Willard as the mother parent, the self-pollination rate was 66.67% and the true hybrid rate was 33.33%; in the 11 offspring with YX as the mother parent, the self-pollination rate was 63.64% and the true hybrid rate was 36.36% (Table 6-1~Table 6-2).

[0086] The results showed that the self-pollination rates of NDM, R2E2, HY, Willard and YX were high, indicating that they had strong self-pollination compatibility and were very easy to self-pollinate; the hybridization rates of the other varieties were all greater than 50%, indicating that their hybridization compatibility was strong.

[0087] Table 6-1 True hybrid rate among varieties

[0088]

[0089] Table 6-2 True hybrid rate among varieties (continued)

[0090]

[0091] Mango is a highly heterozygous plant. During the production process, the offspring are easily affected by self-pollination, resulting in gene mutations, and natural hybridization is very easy to occur between different varieties. The present invention uses SSR markers to perform early identification of mango F1 hybrids. During the identification process, SSR primers with polymorphisms in the parents are screened, and when the primers also show polymorphisms in the offspring, they can be used to identify hybrid offspring.

[0092] For populations with small genetic variation, more possible paternal parents were identified, resulting in multiple paternal parents being unable to be eliminated. This experiment used SSR molecular marker technology to identify paternity. At 13 SSR loci, 809 of 1001 offspring fully complied with Mendel's laws of inheritance with their parents' genotypes, indicating that these loci can accurately identify the possible paternal parents of mango. The offspring of unidentified parents produced site mismatch errors in paternity identification, which may be caused by the inability to detect some heterozygous single bands due to invalid alleles. Among the offspring whose maternal parents were GF, JH, KT, TN, and A61, the paternal parents were derived from 12 varieties, and the hybridization rates were all greater than 50%. These varieties had high heterozygosity and strong hybridization affinity. NDM, R2E2, HY, Willard, and YX had low hybridization rates and were very easy to self-pollinate. In terms of expected heterozygosity or observed heterozygosity, the corresponding varieties TN, KT, Julie, JH, HY, and GF also had relatively high heterozygosity, which was also reflected in the Shannon Information Index (Table 5), that is, GF, JH, KT, TN, and A61 had richer genetic diversity than other varieties.

[0093] The genetic diversity of species is essentially the variation of the genetic material of organisms. The higher the level of genetic diversity, the stronger the adaptability of the species or population to the new environment. DNA molecular markers can reflect the degree of genetic variation. This study used 13 SSR markers to analyze the genetic diversity of 809 mango samples and clustered them into three different subgroups based on the genetic structure of the population. The SSR marker site showed that there were many alleles among mango varieties. The number of alleles varied from 3 to 9, and the average number of alleles (Na) was 6.154, with good polymorphism. However, the average effective number of alleles (Ne) was 2.557, the Shannon's information index (I) was 1.078, the observed heterozygosity (H0) was 0.571, the expected heterozygosity (He) was 0.579, and the polymorphism information index (PIC) was 0.526. The polymorphism information index was between 0<PIC<0.25, indicating low polymorphism, 0.25<PIC<0.5 indicating moderate polymorphism, and PIC>0.5 indicating high polymorphism. Among the 13 markers in this experiment, 10 were highly polymorphic and 3 were moderately polymorphic, indicating that the primers had high polymorphism, which could effectively reveal the genetic diversity of mango seedlings and better identify the existing main cultivated varieties.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. SSR molecular marker primer, characterized in that: Including primer MG-023, primer MG-030, primer MG-039, primer MG-046, primer MG-187, primer MG-055, primer MG-063, primer MG-079, primer MG-114, primer MG-146, primer MG-150, primer MG-177 and primer MG-061; The nucleotide sequence of primer MG-023 is shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleotide sequence of primer MG-030 is shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleotide sequence of primer MG-039 is shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequence of primer MG-046 is shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequence of primer MG-187 is shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequence of primer MG-055 is shown in SEQ ID NO.11 and SEQ ID NO.12; The nucleotide sequence of primer MG-063 is shown in SEQ ID NO.13 and SEQ ID NO.14; The nucleotide sequence of primer MG-079 is shown in SEQ ID NO.15 and SEQ ID NO.16; The nucleotide sequence of primer MG-114 is shown in SEQ ID NO.17 and SEQ ID NO.18; The nucleotide sequence of primer MG-146 is shown in SEQ ID NO.19 and SEQ ID NO.20; The nucleotide sequence of primer MG-150 is shown in SEQ ID NO.21 and SEQ ID NO.22; The nucleotide sequence of primer MG-177 is shown in SEQ ID NO.23 and SEQ ID NO.24; The nucleotide sequence of primer MG-061 is shown in SEQ ID NO.25 and SEQ ID NO.

26.

2. Application of the SSR molecular marker primers described in claim 1 in the analysis of mango genetic characteristics.

3. Application of the SSR molecular marker primers described in claim 1 in mango genetic diversity analysis.

4. Application of the SSR molecular marker primers described in claim 1 in mango genetic relationship molecules.

5. Use of the SSR molecular marker primers described in claim 1 in identifying true and false hybrids of mango F1 generation.

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