Gene BoYgl-3 controlling yellow-green leaf color of cabbage seedlings and its InDel marker

Abstract

The present invention provides a gene BoYgl‑3 and an InDel marker for controlling the yellow-green leaf color trait of cabbage seedlings. The InDel molecular marker is an insertion or deletion of a 15bp nucleotide sequence, and the nucleotide sequence is shown in SEQ ID No.9. The present invention discloses for the first time the gene BoYgl‑3 for controlling the yellow-green leaf color trait of cabbage young leaves by analyzing the yellow-green mutant 522Y of cabbage young leaves, and provides an insertion / deletion fragment (Indel molecular marker) related to the leaf color trait of cabbage young leaves and a detection method thereof. The present invention can quickly and accurately identify the yellow-green mutation type of cabbage young leaves, and is applied to cabbage leaf color auxiliary breeding.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to a gene BoYgl-3 for controlling yellow-green leaf color traits of cabbage seedlings and an InDel marker thereof. Background Art

[0002] Cabbage (Brassica oleracea L. var. capitata) is an important Brassica vegetable of the cruciferous family (Fang Zhiyuan, 2008). Leaf color is a very important agronomic trait of cabbage, which is usually green. In the process of cabbage breeding, leaf color mutations such as yellow-green and albino occur from time to time. These mutants are often due to the obstruction of chlorophyll synthesis or accelerated degradation, which significantly reduces the chlorophyll content. The chlorophyll content of some mutants is even less than 1% of the wild type, leading to plant death (Jung et al., 2003). Leaf color mutants have very important utilization value. Since the leaf color of mutants is obvious in the seedling stage, they can be used as marker traits for rapid identification of variety purity; yellowing mutants are excellent receptors for transgenics; leaf color mutants are ideal materials for studying plant photosynthesis, chlorophyll anabolism, gene regulation and other physiological and metabolic processes (Zhao et al., 2000; Chen Yanli, 2011; Yang Chong, 2014; Hou Ailin, 2018; Cui Huilin et al., 2019).

[0003] At present, various methods have been used to locate or clone leaf color genes in Brassica crops. In cabbage, Hou Ailin (2018) used Mut Map technology to locate a leaf yellowing gene lcm3 to the BraA01001067 gene on chromosome A01. In mustard rapeseed, Zhao Zhi and Du Dezhi (2013) located a purple leaf gene between AFLP markers PL01 and PL08, with genetic distances of 0.7 cM and 1.3 cM, respectively. In Arabidopsis, Dai Nan et al. (2017) located a yellow leaf gene in a region of about 231 kb between markers CH5-6.0 and CH5-6.24 on chromosome C05. In cabbage rapeseed, Chen Yanli (2011) located a yellow leaf gene bnaC.ygl on linkage group N17, and obtained a total of 28 markers linked to the bnaC.ygl gene. In carrots, Nothnagel and Straka (2003) located a yellow leaf gene YEL between AFLP markers AGL15 and AGL2, with genetic distances of 0.1 cM and 0.2 cM, respectively. In kale, Zhu et al. (2016) located a pink leaf gene between markers Ni2C12 and Boac04 at the start of chromosome C03, with genetic distances of 0.6 cM and 2.4 cM, respectively; Liu et al. (2017) located a purple leaf gene BoPr to gene Bo9g058630 on chromosome C09. In cabbage, Zhang Bin (2020) located two young leaf yellow-green genes BoYgl-1 and BoYgl--2 to the 392 Kb interval of chromosome C01 and the 232 Kb interval of chromosome C03, respectively, and identified Bo3g001140 as the candidate gene of BoYgl-2.

[0004] In cabbage, although there are reports of yellow-green young leaf mutants, the functional studies of their candidate genes have not been reported, and the molecular mechanism of leaf color formation has not been revealed. Therefore, in cabbage genetic breeding, it is very necessary to develop a molecular marker that is closely linked to the yellow-green young leaf gene, so as to provide a basis for subsequent research on the molecular mechanism of leaf color formation, and at the same time provide a new way for the accurate identification of yellow-green young leaf mutations in cabbage and the early rapid screening of yellow-green materials, greatly improving the efficiency of leaf color breeding.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a gene BoYgl-3 and its InDel marker for controlling the yellow-green leaf color trait of cabbage seedlings. The present invention can quickly and accurately identify the yellow-green mutation type of cabbage young leaves, and is applied to cabbage leaf color auxiliary breeding, and at the same time has important significance for the study of the molecular mechanism of leaf color formation.

[0007] The technical solution provided by the present invention is as follows:

[0008] In one aspect, the present invention provides a gene BoYgl-3 for controlling the yellow-green leaf color trait of young leaves of cabbage, wherein the nucleotide sequence of the gene BoYgl-3 is shown as SEQ ID No.1 or SEQ ID No.2.

[0009] In the cabbage young leaf yellow-green mutant, the gene has a 15 bp deletion in the second exon of the coding region, and the nucleotide sequence after the mutation is shown in SEQ ID No. 2. The nucleotide sequence of the cabbage young leaf yellow-green gene BoYgl-3 without deletion is shown in SEQ ID No. 1.

[0010] The present invention studies the cabbage 522Y natural mutant. According to the phenotypes of the cabbage young leaf yellow-green mutant 522Y and the kale normal green leaf inbred line 11-192 as parents after self-crossing or backcrossing, it is inferred that the cabbage 522Y natural mutant yellow-green leaves is controlled by a pair of recessive genes, and the mutant is named BoYgl-3.

[0011] In another aspect, the present invention provides an InDel molecular marker for identifying the yellow-green color of young leaves of cabbage, wherein the InDel molecular marker is linked to the yellow-green gene of young leaves of cabbage, and is an insertion or deletion of a 15bp nucleotide sequence, wherein the InDel molecular marker is named IDygl, and the deleted 15bp nucleotide sequence is SEQ ID No.9 (CGCATCATCCGTGCT). Insertion / deletion polymorphism (InDel) is a length polymorphic variation caused by the insertion / deletion of nucleotide fragments in the DNA sequence at the allele locus between different individuals.

[0012] In one aspect, the present invention provides a primer pair for amplifying the InDel molecular marker, and the sequences of the primer pair are shown in SEQ ID No.3 and SEQ ID No.4.

[0013] Forward primer IDyglF: 5'-ATCCCATCACTTCTCTCATCGC-3' (SEQ ID NO. 3), reverse primer IDyglR: 5'-GCAAGAGTTACCACATAGGGCTG-3' (SEQ ID NO. 4).

[0014] In one aspect, the present invention provides a kit for identifying the yellow-green leaf color of young cabbage leaves, which contains the primer pair as described above. In one embodiment, the kit also contains other components contained in conventional PCR kits in the art, such as polymerase, buffer, etc.

[0015] In one aspect, the present invention provides the use of the InDel molecular marker or the primer pair in the identification of yellow-green mutation types of cabbage seedlings and / or leaf color assisted breeding. The InDel molecular marker or the primer pair of the present invention can also be used in genetic map construction, QTL positioning, molecular assisted breeding and genetic diversity analysis.

[0016] In one aspect, a method for identifying a yellow-green mutation in young leaves of cabbage comprises detecting the insertion / deletion of the InDel molecular marker on chromosome 7 of cabbage.

[0017] In one embodiment, the InDel molecular marker is designed with corresponding InDel primers for detection. If the InDel molecular marker is inserted, it is a normal green-leaf plant; if the InDel molecular marker is missing, it is a young yellow-green plant.

[0018] In one embodiment, the method comprises:

[0019] (1) extracting genomic DNA of the cabbage sample to be tested;

[0020] (2) using the genomic DNA as a template and performing PCR amplification using the primer pair described in claim 3;

[0021] (3) Identify the leaf color phenotype of cabbage based on the gel electrophoresis results.

[0022] In one embodiment, in step (3), if the gel electrophoresis result amplifies a 193 bp band, it is a normal green-leaf plant; if the gel electrophoresis result amplifies a 178 bp band, it is a young yellow-green plant.

[0023] In the present invention, the genotype of the insertion / deletion polymorphic site is identified according to the electrophoresis results. The IDygl marker is co-segregated with the BoYgl-3 gene; the IDygl marker can amplify a 193bp band in the wild type 522G (sequence as shown in SEQ ID NO.7), and can amplify a 178bp band in the mutant 522Y and 16 recessive individual plants (sequence as shown in SEQ ID NO.8).

[0024] The agarose gel electrophoresis uses 8%-10% agarose gel by mass, preferably, 8% polyacrylamide gel electrophoresis. After gel electrophoresis and silver staining of the PCR product, genotype determination and phenotype prediction are performed according to the electrophoresis results.

[0025] In one embodiment, the PCR reaction system is: 1 μL of DNA template, 0.5 μL of 10 μmol / L forward and reverse primers, 5 μL of Taq Mix, and 3 μL of ddH2O.

[0026] In one embodiment, the annealing temperature in the PCR reaction program is 54-57°C;

[0027] Preferably, the PCR reaction procedure is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 40 s, 35 cycles; and extension at 72°C for another 5 min.

[0028] According to the present invention, the present invention also provides a method for identifying the BoYgl-3 genotype of cabbage or a method for predicting the leaf color of young cabbage leaves. If only the 193bp band is detected, it indicates that the genotype of the sample is a homozygote with the gene fragment inserted, and the leaf color is normal green leaves; if only the 178bp band is detected, it indicates that the genotype of the sample is a homozygote with the gene fragment missing, and the leaf color is yellow-green young leaves; if the 193bp and 178bp bands are detected at the same time, it indicates that the genotype of the sample is a heterozygote containing both the inserted allele and the deleted allele of the gene fragment, and the leaf color is normal green leaves.

[0029] The InDel molecular marker or the primer pair or the kit of the present invention can also be used in applications such as cabbage genotyping and leaf color prediction.

[0030] Beneficial effects:

[0031] The present invention provides for the first time a cabbage young leaf yellow-green gene BoYgl-3 and an InDel molecular marker within the gene, which can quickly and accurately identify the yellow-green mutation type of cabbage young leaves. The detection method is simple and easy, the results are stable and reliable, and can be applied to cabbage leaf color assisted breeding.

[0032] The InDel molecular marker of the present invention is co-separated with the cabbage young leaf yellow-green gene BoYgl-3, and can be used to identify whether the cabbage plant contains the mutant gene. The detection method is simple and easy, and the result is stable and reliable. The present invention can simply, quickly, cost-effectively and accurately detect the insertion / deletion polymorphism of the site through steps such as PCR amplification and agarose gel electrophoresis.

[0033] The InDel molecular marker (insertion / deletion polymorphism) of the present invention is linked to the leaf color trait of young cabbage leaves and can be used as a molecular marker for auxiliary selection in cabbage breeding; the present invention designs and screens specific and highly sensitive InDel marker primer pairs, which have the characteristics of high specificity, good sensitivity, etc.

[0034] The InDel molecular marker of the present invention can be used for the accurate identification of yellow-green mutations in young cabbage leaves and the early rapid screening of yellow-green materials, greatly improving the efficiency of leaf color breeding, and is of great significance for the study of the molecular mechanism of leaf color formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The wild type 522G and mutant 522Y phenotypes provided in Example 1 of the present invention;

[0037] Figure 2 The chlorophyll content of the wild type 522G and mutant 522Y provided in Example 1 of the present invention;

[0038] Figure 3 The initial location of the BoYgl-3 gene provided in Example 2 of the present invention;

[0039] Figure 4 The fine positioning of the BoYgl-3 gene provided in Example 2 of the present invention;

[0040] Figure 5 A partial sequence alignment of the BoYgl-3 gene in 522G and 522Y provided in Example 3 of the present invention;

[0041] Figure 6 It is the amplified band pattern of the IDygl marker in the parents and 16 recessive plants in Example 4 of the present invention; wherein M represents Marker, and 1-16 represents recessive plants;

[0042] Figure 7 It is the amplified band pattern of the IDygl marker in the parents and 108 cabbage green leaf inbred lines in Example 4 of the present invention; wherein M represents Marker, P1 represents the parent 522G, and P2 represents the parent 522Y. 1-108 represent different cabbage green leaf inbred lines. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1: Analysis of leaf color inheritance of cabbage 522Y mutant

[0045] The cabbage 522Y natural mutant originated from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences. The leaves of the mutant were yellow-green in the seedling stage, and the chlorophyll content was significantly reduced. However, the leaves gradually turned to normal green at maturity, and the chlorophyll content also recovered to 94% of the wild type 522G level ( Figure 1 and Figure 2 ).

[0046] The yellow-green mutant 522Y of young leaves of cabbage and the inbred line 11-192 with normal green leaves of kale were used as parents for hybridization and self-pollination or backcrossing to construct F2 and BC1 segregation populations. The segregation of green leaves and yellow-green leaves in the F2 and BC1 segregation populations was investigated, and the segregation ratio was consistent with 3:1 or 1:1 by the chi-square test (Table 1). It was inferred that the yellow-green leaf trait of cabbage 522Y was controlled by a pair of recessive genes, which were named BoYgl-3.

[0047] Table 1. Leaf color segregation of F2 and BC1 populations constructed using 4036Y and 11-192

[0048]

[0049] Example 2: Fine localization of the yellow-green gene in young leaves of BoYgl-3

[0050] Using the BC1 segregation population, 30 green-leaf and yellow-green-leaf plants were selected to construct a normal green-leaf pool and a seedling yellow-green pool. The two pools and the parents were resequenced, and according to the Δ(SNP-index) value of each site in the two pools, a threshold of 99% confidence level was selected, and the BoYgl-3 young leaf yellow-green gene was preliminarily located at the 4.46Mb interval of chromosome C07 (C07: 42,280,000bp-46,740,000bp) ( Figure 3 ).

[0051] Using the parental resequencing data, we designed 30 pairs of InDel primers in and around the initial positioning interval of BoYgl-3, of which 19 pairs were polymorphic between the parents. The modified CTAB method (Murray and Thompson, 1980) was used to extract genomic DNA of 632 recessive individuals in the BC1 population, and 11 markers with clear and stable amplified bands were selected for screening.

[0052] The PCR reaction system was as follows: 1 μL DNA template, 0.5 μL each of 10 μmol / L forward and reverse primers, 2× PCRSuperMix 5μL, ddH2O 3μL.

[0053] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 40 s, 35 cycles; and extension at 72°C for another 5 min.

[0054] The exchanged plants screened by each marker were counted, and the yellow-green gene of young leaves of BoYgl-3 was finally located in the 25Kb interval between InDel markers 522-32 and 522-43 on chromosome C07 (TO1000 genome) ( Figure 4 ).

[0055] Example 3: Analysis of BoYgl-3 candidate genes

[0056] According to the TO1000 reference genome and Arabidopsis homologous gene annotation information, this localization interval contains a total of 7 genes, among which Bo7g111960 encodes EMB1923 protein, which is located in the chloroplast. In cabbage, EMB1923 promoter mutation can cause leaf yellowing (Li et al., 2019), so Bo7g111960 is tentatively identified as a candidate gene for BoYgl-3.

[0057] According to the TO1000 reference genome sequence, the specific marker CE1923 was designed using Primer5 software to amplify the BoYgl-3 gene sequence. The nucleotide sequence of the CE1923 marker forward primer CE1923F is: 5'-GAGCGAATAAAGTAGGATGATGC-3' (SEQ ID NO.5), and the nucleotide sequence of the reverse primer CE1923R is: 5'-ATTACTAACCAGTGGGTAGAGAGG-3' (SEQ ID NO.6).

[0058] The PCR reaction system was as follows: 2 μL DNA template, 1 μL each of 10 μmol / L forward and reverse primers, 2× PCRSuperMix 10μL, ddH2O 6μL.

[0059] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 60 s, 35 cycles; and extension at 72°C for another 5 min.

[0060] The genomic DNA of 522G and 522Y was extracted by modified CTAB method. Sequencing analysis revealed that there was a 15 bp deletion in the second exon of the coding region of BoYgl-3 mutant 522Y ( Figure 5 ), the sequencing sequence of BoYgl-3 in 522G is shown as SEQ ID NO.1, and the sequencing sequence of BoYgl-3 in 522Y is shown as SEQ ID NO.2.

[0061] Example 4. Development and verification of InDel molecular markers of Bo Ygl-3 gene

[0062] According to the TO1000 reference genome sequence, specific primers were designed on both sides of the BoYgl-3 gene mutation site using Primer5 software to obtain the polymorphic InDel molecular marker IDygl between the wild type 522G (normal green leaves) and the mutant 522Y (young leaves yellow-green), whose nucleotide sequence is SEQ ID No.9 (CGCATCATCCGTGCT).

[0063] The IDygl marker was used to amplify the parental 522G and 522Y and 16 BC1 recessive individuals. The PCR reaction system was: 1 μL DNA template, 0.5 μL of each 10 μmol / L forward and reverse primers, 2× PCR SuperMix 5μL, ddH2O 3μL. The PCR reaction program was: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 40s, 35 cycles; 72℃ extension for another 5min. Then, 8% polyacrylamide gel electrophoresis was used for detection.

[0064] The results showed that the IDygl marker could amplify a 193 bp band in the wild type 522G (SEQ ID NO.7), and a 178 bp band in the mutant 522Y and 16 recessive strains (SEQ ID NO.8) ( Figure 6 ), indicating that the IDygl marker was co-segregated with the BoYgl-3 gene.

[0065] The genomic DNA of 108 cabbage green leaf inbred lines was extracted by the modified CTAB method and amplified using the IDygl marker. The PCR reaction system was: 1 μL DNA template, 0.5 μL of each 10 μmol / L forward and reverse primers, 2× PCRSuperMix 5μL, ddH2O 3μL. The PCR reaction program was: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 40s, 35 cycles; 72℃ extension for another 5min. Then 8% polyacrylamide gel electrophoresis was used for detection. The results showed that the genotypes of the 108 cabbage green leaf inbred lines were consistent with the parent 522G, indicating that the marker can be used to predict cabbage leaf color ( Figure 7 ).

[0066] In summary, the present invention provides a cabbage young leaf yellow-green gene BoYgl-3 and an InDel molecular marker within the gene, which can quickly and accurately identify the yellow-green mutation type of cabbage young leaves. The detection method is simple and easy, the results are stable and reliable, and can be applied to cabbage leaf color assisted breeding.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene that controls the yellow-green leaf color of young cabbage leaves BoYgl-3 , characterized in that, The gene BoYgl-3 The nucleotide sequence is shown as SEQ ID No. 1 or SEQ ID No.

2.

2. An InDel molecular marker for identifying the yellow-green leaf color of young cabbage leaves, characterized in that: The InDel molecule marker is an insertion or deletion of a 15 bp nucleotide sequence, and the nucleotide sequence is shown in SEQ ID No.

9.

3. A primer pair for amplifying the InDel molecular marker according to claim 2, characterized in that: The sequences of the primer pairs are shown in SEQ ID No. 3 and SEQ ID No.

4.

4. A kit for identifying the yellow-green color of young cabbage leaves, characterized in that: It contains the primer pair as shown in claim 3.

5. Use of the InDel molecular marker according to claim 2 or the primer pair according to claim 3 in the identification of yellow-green mutation types in cabbage seedlings and / or leaf color assisted breeding.

6. A method for identifying a yellow-green mutation in young cabbage leaves, characterized in that: The method comprises detecting the insertion / deletion of the InDel molecular marker described in claim 2 on chromosome 7 of cabbage.

7. The method according to claim 6, characterized in that The method comprises: (1) Extracting genomic DNA from the cabbage sample to be tested; (2) using the genomic DNA as a template and performing PCR amplification using the primer pair described in claim 3; (3) Identify the leaf color phenotype of cabbage based on the gel electrophoresis results.

8. The method according to claim 7, characterized in that In step (3), if the gel electrophoresis result amplifies a 193 bp band, it is a normal green-leaf plant; if the gel electrophoresis result amplifies a 178 bp band, it is a young yellow-green leaf plant.

9. The method according to claim 7, characterized in that: In step (2), the PCR reaction system is: 1 μL DNA template, 0.5 μL each of 10 μmol / L forward and reverse primers, 5 μL Taq Mix, and 3 μL ddH2O.

10. The method according to claim 7, characterized in that In step (2), the annealing temperature in the PCR reaction program is 54-57°C.

11. The method according to claim 10, characterized in that In step (2), the PCR reaction program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 40 s, 35 cycles; and extension at 72°C for another 5 min.

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