Sesame fertility molecular marker, primer, kit, application and breeding method of new high-lignan sesame variety
阅读说明：本技术 芝麻育性分子标记、引物、试剂盒、应用、高木酚素芝麻新品种选育方法 (Sesame fertility molecular marker, primer, kit, application and breeding method of new high-lignan sesame variety ) 是由 梅鸿献 郑永战 刘艳阳 崔承齐 杜振伟 武轲 江晓琳 于 2021-08-05 设计创作，主要内容包括：本发明涉及分子生物学及遗传育种技术领域,具体涉及芝麻育性分子标记、分子标记引物、应用、高木酚素芝麻新品种选育方法。本发明提供的分子标记的不同基因型对应芝麻的确定育性,通过针对不同基因型设计特异性扩增引物,通过判断扩增片段的有无即可对芝麻的育性进行准确判断；本发明提供的选育方法,采用轮回选育的方式,在基础群体构建过程的中回交过程结合分子标记进行芝麻育性的辅助选择,与传统的在回交并自交的群体后代中再选择不育株进行回交或者直接连续回交的方法相比,能减少一半以上的时间或工作量,并且构建的基础群体使不育系亲本遗传成分在群体中的比例降至2%以下,提高了群体的遗传多样性。(The invention relates to the technical field of molecular biology and genetic breeding, in particular to a sesame fertility molecular marker, a molecular marker primer, application and a method for breeding a new high-lignan sesame variety. The different genotypes of the molecular marker provided by the invention correspond to the determined fertility of sesame, and the fertility of sesame can be accurately judged by designing specific amplification primers aiming at the different genotypes and judging whether amplified fragments exist or not; the breeding method provided by the invention adopts a recurrent breeding mode, the backcross process is combined with the molecular marker to carry out the auxiliary selection of sesame fertility in the construction process of the basic population, compared with the traditional method of selecting sterile plants again from the progeny of the backcrossed and selfed population to carry out the backcross or direct continuous backcross, the time or workload can be reduced by more than half, and the constructed basic population reduces the proportion of the genetic components of the sterile line parents in the population to below 2%, thereby improving the genetic diversity of the population.)
1. The sesame fertility molecular marker is characterized by consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and (b) the nucleotide sequence shown in the figure.
2. A primer for amplifying a sesame fertility molecular marker is characterized by consisting of a first primer pair and a second primer pair; wherein the upstream primer of the first primer pair is as follows: f1: 5'-AATGACGATGCCTAAGGCCC-3' as set forth in SEQ ID NO: 3 is shown in the specification; the upstream primer of the second primer pair comprises the sequence: f2: 5'-AATGACGATGCAGAGGCATA-3', as shown in SEQ ID NO: 4 is shown in the specification; the downstream primers of the first primer pair and the second primer pair are R: 5'-GCATTAGCCTCTCCACAAATGG-3', as shown in SEQ ID NO: 5, respectively.
3. The primer for amplifying the sesame fertility analysis marker according to claim 2, wherein the upstream primer sequence of the second primer pair is: f2: 5'-TAATTAATGACGATGCAGAGGCATA-3', as shown in SEQ ID NO: and 6.
4. A kit for detecting the molecular marker of claim 1, comprising the primer of claim 2.
5. The use of the molecular marker according to claim 1 for sesame fertility selection, wherein the first primer pair and the second primer pair according to claim 2 are used for amplifying sesame breeding progeny material DNA; if only the amplified segment of the first primer pair is obtained, the material can be predicted to be a homozygous fertile plant; if only the amplified fragment of the second primer pair is obtained, the material can be predicted to be a homozygous sterile plant; if the amplified fragments of the first primer pair and the second primer pair are obtained simultaneously, the material can be predicted to be a heterozygous fertile strain.
6. A method for breeding a new variety of high-lignan sesame is characterized by comprising the following steps:
1) constructing a basic population:
(1) determining male parent and female parent:
male parent: screening 30-50 parts of germplasm resources which meet the requirements of target shapes and have excellent comprehensive shapes to serve as male parents;
female parent: taking a recessive genic male sterile material sterile plant as a female parent;
(2) the female parent and the male parent are respectively configured with hybrid combination and backcrossed to obtain BC1；
(3) Using a first primer pair and a second primer pair BC1Carrying out PCR amplification on DNA of each individual plant material in the population; judging the material only obtaining the amplified segment of the first primer pair as a homozygous fertile plant; judging the material only obtaining the amplified fragment of the second primer pair as a homozygous sterile plant; obtaining the materials of the amplified fragments of the first primer pair and the second primer pair at the same time, and judging the materials as heterozygous fertile plants; wherein the upstream primer of the first primer pair is as follows: f1: 5'-AATGACGATGCCTAAGGCCC-3' as set forth in SEQ ID NO: 3 is shown in the specification; the upstream primer of the second primer pair comprises the sequence: f2: 5'-AATGACGATGCAGAGGCATA-3', as shown in SEQ ID NO: 4 is shown in the specification; the downstream primers of the first primer pair and the second primer pair are R: 5'-GCATTAGCCTCTCCACAAATGG-3', as shown in SEQ ID NO: 5 is shown in the specification;
(4) selecting BC according to the method of step (3)1The heterozygous fertile plants in the population are used as female parents and the corresponding male parents are used as recurrent parents, and are respectively backcrossed for 6 generations to BC6And selfing to obtain BC6F2Seeds;
(5) each parent BC obtained in the step (4)6F2After the seeds are mixed in equal quantity, the seeds are planted under the isolation condition, the plant pollen fertility is marked in the flowering period, and the seeds on the sterile single plants are obtained in a mixed mode to form a basic population C0;
2) and (3) recurrent selection of lignan content:
planting a basic population C0 under the isolation condition, and marking the pollen fertility of the plant at the flowering stage; during mature harvest, selecting fertile single plants with excellent agronomic characters according to visual inspection, and harvesting sesame seeds from the single plants;
determining the contents of sesamin and sesamolin in the sesame seeds harvested from each individual plant;
according to the arrangement of the total lignan content, screening plant seeds with high lignan content and excellent comprehensive shape, and equivalently mixing to form a first group C1;
c1 is planted under isolation conditions, the pollen fertility of plants is marked in the flowering phase, and seeds on sterile single plants are harvested in a mixed mode to complete population recombination;
repeating the stepsAnd the step ofSo as to complete a new round of recombination and selection and finally breed a new variety with high lignan content and excellent comprehensive shape.
7. The method for breeding a new variety of high lignan sesame as claimed in claim 6, wherein the female parent in step (1) is ms 86-1.
8. The method for breeding a new variety of sesame having high lignan content as set forth in claim 6, wherein the step (A) isIn the basic population C0, the planting scale is 10000-20000 strains, and the fertile individual strain with excellent agronomic shape is 1000-2000 strains.
9. The method for breeding a new variety of sesame having high lignan content as set forth in claim 8, wherein the step (A) isFirstly, arranging according to the content of the total lignans, screening 500 strains before screening, then screening 100 strains with excellent comprehensive shapes, and mixing in equal quantity to form a first group C1; step (ii) ofThe planting scale of the medium C1 is 5000-10000 plants; and (5) planting 5000-10000 plants.
10. The method for breeding a new variety of sesame with high lignan content as claimed in any one of claims 6 to 9, wherein the step of breeding comprisesThe sesamin and sesamolin contents of the medium sesame seeds are detected by an infrared spectrometer.
Technical Field
The invention relates to the technical field of molecular biology and genetic breeding, in particular to a sesame fertility molecular marker, an analysis marker primer, a kit, application and a method for breeding a new high-lignan sesame variety.
Background
Sesame is an important characteristic high-quality oil crop in China, compared with other oil crops, sesame seeds have the fat content of 55 percent, wherein unsaturated fatty acids such as oleic acid and linoleic acid are 85 percent, but can be stored for a long time without rancidity, and have higher stability, mainly because sesame contains a specific natural antioxidant substance, namely lignans (lignans), and the active ingredients have stronger antioxidation (Huhuali and the like, North China agricultural science and report 2014,29: 190-. Studies show that lignan not only can prevent the oxidative rancidity of grease, but also has high physiological activity in human bodies, can eliminate free radicals in the bodies, and has the effects of reducing blood fat, reducing blood pressure, resisting thrombosis, relieving atherosclerosis, increasing the detoxifying activity of livers, enhancing the immunity of the organisms, inhibiting breast cancer and the like (Namiki et al, Crit Rev Food Sci, 2007, 47: 651-. The sesame lignan serving as a natural antioxidant can be used as a food additive and can also be used for developing health care medicines, and the sesame variety with high lignan content has wide market prospect.
The lignan components in the sesame seeds mainly comprise sesamin and sesamolin, the total content is 0.3-0.5%, but the variation is large in different varieties of resources. Lignan biosynthesis is a relatively complex process, and sesame lignan content is also a typical complex quantitative trait, is controlled by multiple genes, and is easily influenced by the environment. The technical scheme for breeding the sesame varieties with high lignans in the prior art comprises the following steps: a few parents with high lignan content and local fine varieties are used for artificial preparation, hybridization or backcross combination, and then offspring selection is carried out by a pedigree method. The breeding of the variety with genetic base gradually narrows due to long term hybridization with a few parents, and the breeding of the variety with breakthrough variety is difficult. The recurrent selective breeding method utilizes a plurality of parents to construct a basic population, and can break the linkage of unfavorable genes and increase the accumulation of excellent genes through continuous multiple rounds of genetic recombination and progeny selection, thereby being an important means for widening the genetic basis of varieties and breeding breakthrough varieties. However, the recurrent selection recombination process requires free pollination within the population, and conventional methods involve extensive manual detasseling and free pollination, which limits the applicability of recurrent selection to self-pollinated crops like sesame. In the last 80 th century, the discovery of recessive male sterile genes provides possibility for applying a recurrent selection breeding method in sesame, and breeding experts at home and abroad carry out genetic improvement on sesame yield-related characters by using the recurrent selection method.
However, the existing method for sesame recurrent selection by taking recessive male nuclear sterility as a medium still has two main problems, which limits the application of the method in sesame lignan content improvement, and is mainly characterized in that the same recessive male nuclear sterility material is adopted as a female parent when a basic population is constructed, and is respectively prepared and combined with different excellent materials, the genetic component of a sterile gene donor parent accounts for 50% of the population total, so that the genetic diversity of the basic population is easily reduced, the inheritance type and the character of a progeny are single, and the variety with excellent comprehensive characters is difficult to breed, a new method needs to be found, the proportion of the genetic component of a sterile gene donor to the population total is reduced, and the method is used for recurrent selection of sesame lignan content, so that a new sesame variety integrating high lignan content, high yield, stress resistance and broad adaptability can be quickly and effectively cultivated.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a sesame fertility molecular marker which can accurately screen sesame homozygous sterile characters, homozygous fertile characters and heterozygous fertile characters.
The second purpose of the invention is to provide a primer for amplifying a sesame fertility molecular marker, which is used for specifically amplifying the molecular marker, judging the genotype of the molecular marker based on the state of an amplification product and further accurately screening the sesame fertility.
The invention also aims to provide a kit comprising the primer provided by the invention, which is used for detecting the molecular marker provided by the invention and judging the fertility of sesame.
The fourth purpose of the invention is to provide an application of the primer provided by the invention in the aspect of sesame fertility selection.
The invention also provides a breeding method of the new sesame variety with high lignans, which is characterized in that the molecular marker is used for breeding sesame fertility in the process of constructing a basic population, so that the proportion of genetic components of a sterile gene donor in the population is reduced, the genetic components of the basic population are enriched, and the new sesame variety with high lignans content, high yield, stress resistance and wide adaptability is quickly and effectively cultivated.
A sesame fertility molecular marker consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and (b) the nucleotide sequence shown in the figure.
Wherein SEQ ID NO: 1 is sesame male fertile genotype; SEQ ID NO: 2 is the male nuclear sterile genotype of sesame.
A primer for amplifying a sesame fertility molecular marker consists of a first primer pair and a second primer pair; wherein the upstream primer of the first primer pair is as follows: f1: 5'-AATGACGATGCCTAAGGCCC-3' as set forth in SEQ ID NO: 3 is shown in the specification; the upstream primer of the second primer pair is: f2: 5'-AATGACGATGCAGAGGCATA-3', as shown in SEQ ID NO: 4 is shown in the specification; the downstream primers of the first primer pair and the second primer pair are R: 5'-GCATTAGCCTCTCCACAAATGG-3', as shown in SEQ ID NO: 5, respectively.
A kit for detecting the molecular marker, comprising the primer.
The application of the molecular marker in sesame fertility selection is to amplify the material DNA of sesame breeding progeny by adopting the first primer pair and the second primer pair; if only the amplified segment of the first primer pair is obtained, the material can be predicted to be a homozygous fertile plant; if only the amplified fragment of the second primer pair is obtained, the material can be predicted to be a homozygous sterile plant; if the amplified fragments of the first primer pair and the second primer pair are obtained simultaneously, the material can be predicted to be a heterozygous fertile strain.
The first primer pair and the second primer pair can be used for amplifying the DNA of sesame breeding progeny material respectively, and if only the amplified segment of the first primer pair is obtained, the material can be predicted to be a homozygous fertile plant; if only the amplified fragment of the second primer pair is obtained, the material can be predicted to be a homozygous sterile plant; if the amplified fragments of the first primer pair and the second primer pair are obtained simultaneously, the material can be predicted to be a heterozygous fertile plant;
at least 5bp sequences can be randomly added at the 5' end of the upstream primer of the second primer pair, the first primer pair and the second primer pair are adopted to simultaneously amplify the sesame breeding progeny material DNA, the difference exists in the sizes of the fragments of the specific amplification products of different primers due to the difference in the lengths of the upstream primers of the first primer pair and the second primer pair, so that the amplified fragments of different primers can be distinguished through the sizes of the fragments of the amplified products, and if only the amplified fragment of the first primer pair is obtained, the material can be predicted to be a homozygous fertile plant; if only the amplified fragment of the second primer pair is obtained, the material can be predicted to be a homozygous sterile plant; if the amplified fragments of the first primer pair and the second primer pair are obtained simultaneously, the material can be predicted to be a heterozygous fertile strain. More preferably, the sequence of the upstream primer of the second primer pair is F2: 5'-TAATTAATGACGATGCAGAGGCATA-3', as shown in SEQ ID NO: and 6.
A method for breeding a new variety of high-lignan sesame comprises the following steps:
1) constructing a basic population:
(1) determining male parent and female parent:
male parent: screening 30-50 parts of germplasm resources which meet the requirements of target shapes and have excellent comprehensive shapes to serve as male parents;
female parent: taking a recessive genic male sterile material sterile plant as a female parent;
(2) the female parent and the male parent are respectively configured with hybrid combination and backcrossed to obtain BC1；
(3) The first primer pair and the second primer pair are used for respectively pairing BC1Carrying out PCR amplification on DNA of each individual plant material in the population; judging the material only obtaining the amplified segment of the first primer pair as a homozygous fertile plant; judging the material only obtaining the amplified fragment of the second primer pair as a homozygous sterile plant; obtaining the materials of the amplified fragments of the first primer pair and the second primer pair at the same time, and judging the materials as heterozygous fertile plants;
(4) selecting BC according to the method of step (3)1The heterozygous fertile plants in the population are used as female parents and the corresponding male parents are used as recurrent parents, and are respectively backcrossed for 6 generations to BC6And selfing to obtain BC6F2Seeds;
(5) each parent BC obtained in the step (4)6F2And after the seeds are mixed in equal amount, the seeds are planted under the isolation condition, the plant pollen fertility is marked in the flowering period, and the seeds on the sterile single plants are obtained in a mixed mode to form a basic population C0.
2) And (3) recurrent selection of lignan content:
planting a basic population C0 under the isolation condition, and marking the pollen fertility of the plant at the flowering stage; during mature harvest, selecting fertile single plants with excellent agronomic characters according to visual inspection, and harvesting sesame seeds from the single plants;
determining the contents of sesamin and sesamolin in the sesame seeds harvested from each individual plant;
according to the arrangement of the total lignan content, screening plant seeds with high lignan content and excellent comprehensive shape, and equivalently mixing to form a first group C1;
planting under isolated conditions CMarking plant pollen fertility at the flowering stage, and harvesting seeds on sterile single plants in a mixed manner to complete group recombination;
repeating the stepsAnd the step ofSo as to complete a new round of recombination and selection and finally breed a new variety with high lignan content and excellent comprehensive shape.
Optionally, the parent in step (1) is ms 86-1.
Optional, step(s)In the basic population C0, the planting scale is 10000-20000 strains, and the fertile individual strain with excellent agronomic shape is 1000-2000 strains.
Optional, step(s)In China, 500 strains are firstly screened according to the high and low arrangement of the total lignan content, then 100 strains with excellent comprehensive shapes are screened, and the strains are equally mixed to form a first round of population C1.
Optional, step(s)The planting scale of the medium C1 is 5000-10000 plants; and (5) planting 5000-10000 plants.
Optional, step(s)The sesamin and sesamolin contents of the medium sesame seeds are detected by an infrared spectrometer.
The invention has the beneficial effects that:
1. according to the sesame fertility molecular marker provided by the invention, different genotypes correspond to sesame fertility, specific amplification primers are designed aiming at different genotypes, and the sesame fertility can be accurately judged by judging whether amplification fragments exist or not;
2. the invention provides a breeding method of a new sesame variety with high lignan content, which adopts a recurrent breeding mode, and in the process of constructing a basic population, firstly, a recessive male nuclear sterile material sterile plant is determined as a female parent and is hybridized and backcrossed with a selected male parent to obtain BC1, the male sterility character of a BC1 population is screened by using a molecular marker screened by the method and a primer for amplifying the molecular marker provided by the invention, the heterozygous plant is used as the female parent and is backcrossed with the corresponding male parent, and after 6 generations of continuous backcrossing, a single plant of grains are obtained by selfing and are equivalently mixed to obtain the basic population. The proportion of the genetic components of the sterile line parents in the population can be reduced to below 2 percent, and the genetic diversity of the population is improved; the genetic improvement of the content of the lignan of the sesame is carried out through recurrent selection, a plurality of favorable alleles can be polymerized, a high lignan variety with excellent comprehensive properties can be bred, and excellent materials can be added into the population at any time to expand the genetic basis of the variety.
In addition, the auxiliary selection of the sterile character of the sesame is carried out in the backcross process by combining with the molecular marker, and compared with the traditional method of selecting sterile plants again from the progeny of a backcross and selfing group for backcross or direct continuous backcross, the method can reduce more than half of time or workload; under the condition of Hainan or greenhouse generation adding, the construction of continuous 6-generation backcross and basic groups can be completed within 2 years.
3. Preferably, the method for performing nondestructive determination on the lignan content of the group progeny by using the near infrared spectrum obviously reduces the detection cost and realizes the rapid and efficient determination on the quality of large-scale samples.
Drawings
FIG. 1 shows the result of BSA analysis of sesame male sterility genes;
FIG. 2 is a fine mapping of the ms gene;
FIG. 4 shows the amplified banding patterns of the molecular marker primers determined in example 1 in the progeny of the population of the validation experiment in example 2;
FIG. 3 shows the banding patterns amplified from the parents and the progeny of the population in the breeding process of the new sesame variety with lignan content in example 3 by using the molecular marker primers determined in example 1.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
Example 1
The embodiment provides development of primers of a sesame fertility molecular marker and a specific amplification molecular marker, and the specific steps comprise:
(1) using sesame recessive male sterile line ms86-1 sterile strain (msms) Yuzhi No. 11 (a) as female parentMsMs) For male parent, an F containing 398 individuals was constructed2A population;
(2) respectively selecting 50 sterile and fertile plant individuals, mixing the DNAs in equal amount, and constructing a BSA (bulked segregant analysis) mixed gene pool;
(3) the method comprises the steps of utilizing an SLAF-seq (specific-logic-amplified fragment sequencing) technology to carry out simplified genome sequencing, namely combining two enzymes HaeIII and Hpy166II to carry out enzyme digestion on sample DNA, adding A to the enzyme-digested fragments to process, connecting the enzyme-digested fragments by utilizing a Dual-index sequencing linker, carrying out PCR amplification and then carrying out gel cutting, selecting a fragment with the enzyme digestion length of 264-364 bp, and constructing a sequencing library. Paired-end (PE) sequencing was performed using the Illumina HiSeq 2500 platform. Comparing the read sequences (reads) to a reference genome No. 13 of the middle glossy ganoderma by using BWA software, and carrying out SNP and InDel detection by using GATK and SAMtools software;
(4) BSA analysis was performed by MutMap method, as shown in FIG. 1, i.e., counting the genotype frequencies of the SLAF markers in the fertile and sterile pools, calculating the SNP-index between the two pools, and analyzing the SNP-index by△SNP-index finds relevant sites which are possibly separated from characters,in order to eliminate false positive sites, the position of the marker on the genome is used to obtain the gene with the polymorphic SLAF label△SNP-index value, occurrence of correlation threshold after completing loess regression fitting, and mixed pool fitting of sterile and fertile plants△The correlation threshold of (SNP-index) was fitted to 0.62 by loess regression. Of the 16 linkage groups in the reference genome, only LG1△(SNP-index) value greater than the threshold, indicating that this region is associated with a male sterility gene;
(5) determination of molecular markers: in order to further reduce the target gene interval, the two parents are subjected to re-sequencing, namely, genomic DNAs of ms86-1 and Yuzhi No. 11 are extracted, the DNAs are randomly broken by ultrasonic waves, and sequencing joints are connected to construct a sequencing library; paired-end (PE) sequencing was performed using the Illumina Hiseq 2500 sequencing platform. High quality reads were aligned to the reference genome of Zhongzhi No. 13 using BWA software, and SNP and InDel mutation detection was performed using GATK and SAMtools software. In the target genome segment, 20 pairs of SNP and InDel markers are developed, and 8 pairs of primers with polymorphism in parents are screened, wherein 2 pairs of SNP and 6 pairs of InDel are respectively selected. F derived from 398 strain "ms 86-1 XYuzhi No. 11" by using 8 pairs of polymorphic marker pairs2The population is subjected to genotype analysis, and through linkage analysis, the population is subjected to genotype analysismsThe gene is positioned between markers 629090 and 509788 (shown in figure 2), and the direct correlation of sesame XM-011095623 gene (shown in SEQ ID NO: 1 and SEQ ID NO: 2) on sesame fertility is determined through target segment gene annotation;
(6) determination of amplification primers: the DNA and CDNA sequence of the amphipathic gene XM _011095623 gene is analyzed, the gene comprises two exons and one intron, the total length of the cDNA is 1581bp, and the DNA and the CDNA sequence encode 526 amino acids. Sequence alignment analysis shows that two non-synonymous mutations exist in the two parents, one occurs in the first intron, the base G is mutated into A, the 26 th amino acid Val at the N terminal is mutated into Ile, the other mutation occurs in the second intron, the base CTAAGGCCC is mutated into AGAGGCATA, and the 507 th and 510 th amino acids ProLysALAGlu at the C terminal is mutated into GlnArg HisLys. The sequences of two mutation sites of 50 progeny single strains of the F2 population are analyzed, and the N-terminal sequence mutation is related to sesame fertility.
In order to screen sesame fertility through one-step amplification reaction, two pairs of amplification primers with different lengths are designed according to the N-terminal mutation sequence, wherein the upstream primer F1: 5'-AATGACGATGCCTAAGGCCC-3' is specific to a fertile genotype, and is shown as SEQ ID NO: 3, and an upstream primer F2: 5'-TAATTAATGACGATGCAGAGGCATA-3' specific to the sterile genotype, which is shown as SEQ ID NO: 6 is shown in the specification; the downstream primer R: 5'-GCATTAGCCTCTCCACAAATGG-3' is the same for the fertile genotype and the sterile genotype, and is shown as SEQ ID NO: 5, respectively. Adding a fertile genotype specific primer pair and a sterile genotype specific primer pair in one-step amplification reaction simultaneously, and distinguishing through the size of product fragments, wherein if only an amplification fragment of the fertile genotype specific primer is obtained, the plant is homozygous and fertile; if only the amplified segment of the sterile genotype specific primer pair is obtained, the plant is homozygous sterile; if the fragments of the two pairs of primers are obtained simultaneously, the plant is heterozygous and fertile.
It is to be noted that the upstream primer F2 specific for the sterile genotype can be adjusted: 5'-AATGACGATGCAGAGGCATA-3', as shown in SEQ ID NO: and 4, respectively amplifying the material to be screened twice by respectively adopting a fertile genotype primer pair and a sterile genotype primer pair, and determining the fertility of the material to be screened by judging whether amplification products exist or not.
Example 2
In this example, the accuracy of the molecular marker and primer thereof determined in example 1 on sesame fertility screening is verified, and the specific verification process is as follows:
(1) respectively preparing hybridization combinations by taking a recessive male sterile material ms86-1 sterile plant as a female parent and 63 germplasm resources as male parents, and selfing to obtain 63F 2 populations;
(2) selecting 5 fertile plants and 5 sterile plants from each population, extracting leaf DNA, and performing genotype analysis by using a MarkerSims marker, as shown in FIG. 3, performing fertility marking on 10 materials screened from 5 of the populations by using the molecular marker determined in example 1 and the primer thereof, wherein 1-5 are fertile plants; 6-10 are sterile plants, which shows that the marker distinguishes fertile plants and sterile plants by 100 percent.
Example 3
The embodiment provides a breeding method of a new sesame variety with high lignan content, which comprises the following specific steps:
1. basic population construction
(1) Screening 50 parts of germplasm resources with excellent comprehensive characters from Huang-Huai river basin from a resource library of sesame research center of academy of agricultural sciences of Henan province for construction of recurrent selection basic groups (P1);
(2) recessive male nuclear sterile material ms86-1 sterile plant (msms) is used as a female parent, 50 germplasm resources are used as male parents to respectively prepare hybrid combinations, and the hybrid combinations are backcrossed to obtain BC1；
(3) In each backcross population, extracting individual DNA, respectively using the specific fertile genotype primer pair and sterile genotype primer pair developed in example 1 to perform PCR amplification on the individual DNA, and observing the amplified product fragments after polyacrylamide gel electrophoresis and silver staining of the amplified product, as shown in FIG. 4; if only amplified fragments of a pair of primers of a fertile genotype are obtained (materials 1,2,6, 9,12,15, 19), the plant is homozygous fertile; if only the amplified fragment of the sterile genotype primer pair is obtained, the plant is homozygous sterile (materials 4,11,16, 23); if the fragments of the two pairs of primers are obtained simultaneously, the plant is heterozygous and fertile (materials 3,5,7,8,10,13,14,17 and 18);
(4) using the heterozygous fertile plant in the backcross population as female parent and the corresponding male parent as recurrent parent, respectively and continuously backcrossing for 6 generations to BC6And selfing to obtain BC6F2Seeds;
（5）BC6F2after the seeds are mixed in equal amount, the seeds are planted under the isolation condition, the scale is 5000-10000 plants, the pollen fertility of the plants is marked at the flowering phase, and the seeds on sterile single plants are mixed and harvested to form a basic population P1C 0.
2. Recurrent selection of lignan content
(1) Planting P1C0 population under isolation condition, wherein the scale is 10000-; when the plants are harvested in a mature mode, firstly, 1000-2000 fertile single plants with excellent agronomic characters are selected according to visual inspection, and the single plants are harvested; after drying and threshing, the contents of sesamin and sesamolin in the seeds are measured.
(2) The sesamin and sesamolin determination adopts near infrared spectroscopy, specifically, a DA7200 diode array near infrared spectrometer of the Swedish wave communication (Perten) company is used for collecting spectrum data of test material seeds. Before spectrum scanning, all samples needing spectrum collection are placed in a laboratory where a near-infrared spectrometer is located for more than 24 hours, so that the environmental conditions of the samples are consistent with those of an instrument, and the influence of temperature on the samples is reduced. After the instrument is preheated for 30 min, setting working parameters of the instrument before an experiment, wherein the range of a scanning spectrum region is 950-1650 nm, and the sampling interval is 2 nm; analyzing the contents of sesamin and sesamolin by using an NIRS model of the contents of sesamin and sesamolin established by 800 parts of resources;
(3) according to the high-low arrangement of the total lignan content, selecting the first 500 plants, mixing the 500 plants in equal quantity to form a first group P1C1, screening 100 single plants with excellent comprehensive properties, and entering a system selection program;
(4) planting P1C1 under isolation condition, with scale of 5000-;
(5) and (5) repeating the step (3) and the step (4) to complete a new round of recombination and selection.
By 2020, 5 rounds of selection have been completed in example 2, and the average total lignan content of the P1C5 population single plant is 6.10 mg/g, which is 43.53% higher than that of the basic population single plant of 4.25 mg/g, and the average total lignan content per round is 8.71%.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Sequence listing
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aatgacgatg cagaggcata aacccttaat tgccattcct aggcccagac taccagccca 60
tttgtataat tagcggccca tttgtggaga ggctaatgc 99
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 3
aatgacgatg cctaaggccc 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
aatgacgatg cagaggcata 20
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 5
gcattagcct ctccacaaat gg 22
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 6
taattaatga cgatgcagag gcata 25