CN113229136B - Stevia rebaudiana leaf quality breeding parent matching method - Google Patents
Stevia rebaudiana leaf quality breeding parent matching method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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Abstract
The invention discloses a stevia rebaudiana quality breeding parent matching method, and belongs to the field of crop quality breeding. The method comprises the steps of detecting compounds contained in the leaves of the breeding material, extracting qualitative and quantitative information, and evaluating the utilization potential of the breeding material based on the content improvement direction of a target compound, so that a calculable and more accurate stevia rebaudiana leaf quality breeding parent matching method is established. According to the stevia rebaudiana quality breeding parent accurate matching strategy, the stevia rebaudiana quality breeding efficiency can be improved.
Description
Technical Field
The invention belongs to the field of crop quality breeding, and particularly relates to a quality breeding parent matching method for improving the content of related compounds in stevia rebaudiana leaves.
Background
Stevia rebaudiana (also called Stevia rebaudiana, Stevia rebaudiana Bertoni) is a perennial herb plant of the Stevia genus of the family of Compositae, which is originally produced in the arnan mountain of south America, and is widely planted and processed because the leaves thereof are rich in stevioside, which is a tetracyclic diterpenoid compound with the taste similar to sucrose to different degrees, the sweetness of 300 times that of sucrose 150 and the heat of 1/300. Since the introduction of the seventies of the last century, China has developed into the most important stevia rebaudiana planting and processing base in the world.
With the improvement of consumption level, diabetics and weight control crowds are more and more, the market demand for natural stevioside is more and more, and the breeding of new stevia varieties is directly related to the sustainable development of the whole stevia industry.
Stevia glycosides compounds contained in stevia leaves planted in the eighties of the last century are mainly STV (stevia) with green grass flavor. With the improvement of the requirements of people on the taste of stevioside, the main stevioside compounds contained in stevia leaves are gradually changed from STV with grass flavor into RA (Rebaudioside A) with the taste closer to that of cane sugar. Since 2010, with the development of the stevioside contained in the stevia rebaudiana leaves by researchers, people are concerned about more and more types of stevioside: RC (Rebaudioside C) with sweetening function and capable of being used as an aromatic; the taste is closer to that of cane sugar, and RD (Rebaudioside D) and RM (Rebaudioside M) with smaller aftertaste; rebaudioside B (RB) with shorter residence time on taste buds; rebaudioside E (RE) and the like with good water solubility, high stability and pure mouthfeel.
In stevioside application, high-purity RA, RD, RM, RB and the like which are purified to 95-97% are mostly compounded and applied in downstream application enterprises. Due to high capital and technical threshold and low purification efficiency, the water solubility of the purified stevioside is reduced, a large amount of low-grade stevioside mixture which is poor in taste and can be applied only after being modified by beta-cyclodextrin glucosyltransferase and other enzymes is left. The method increases processing links, improves the application cost of the stevioside, and restricts the substitution and market expansion of the natural stevioside on sucralose and other synthetic sweeteners.
Along with the expansion of the application market of stevioside, the application cost control requirements of application enterprises including coca cola and the like on stevioside are higher and higher, a scheme that the stevioside components are not purified and then are compounded for application, and the stevioside primary extract is directly used for adding and applying is provided, so that the processing cost for extracting the stevioside can be greatly reduced, the replacement of the stevioside on a chemically synthesized sweetener is promoted, and meanwhile, a low-grade stevioside mixture which can be accepted by downstream application enterprises after being modified by using enzymes such as beta-cyclodextrin glucosyltransferase is also avoided.
This requirement of the application segment poses a challenge to the stevia breeding efforts. The newly bred stevia rebaudiana variety is required to increase the content of high-grade stevioside such as RE, RD, RM, RA and RB on the basis of the existing variety, and simultaneously reduce the content of low-grade stevioside such as STV and RF.
Because the stevia rebaudiana is a self-incompatible plant, the previous stevia rebaudiana breeding is totally based on experience, the breeding strategy commonly used by breeders is a 'superior' hybrid combination, namely, aiming at a breeding target compound, a single plant line with high content of the target compound in leaves is selected to prepare the hybrid combination, and because the variety of compounds including stevioside in the stevia rebaudiana leaves is various, the compounds are mutually transformed, the information of the compounds depended by stevia rebaudiana breeding workers is limited, and the breeding efficiency is low because the hybrid combination preparation is only carried out by adopting the content information of single or limited compounds. In order to achieve the breeding goal, breeding workers can only prepare hybrid combinations as much as possible, and the cost is high and the success rate is low.
Similarly, because the stevia rebaudiana is a self-incompatible plant, the genetic background is complex, and the acquisition difficulty of the recombinant self-incompatible line is high, so that the variation range of the types and the contents of various compounds including stevioside in single plant leaves among filial generations of the stevia rebaudiana is large, and a breeding worker can only detect the prepared filial generation of the hybridized combination one by one in a large amount. However, since the types of compounds including steviol glycosides in stevia rebaudiana leaves are large, the conventional high performance liquid chromatography separation detection method has high detection cost, low separation efficiency, and little information on extracted compounds. On the other hand, since the compound is also large in number, the time required for the separation and detection of each sample is as long as 1.0 hour or more, which results in high time cost.
Generally, the existing high performance liquid chromatography detection method has low separation efficiency and less obtained compound information, so that stevia rebaudiana breeding workers cannot effectively prepare hybridization combinations on the basis of comprehensive evaluation of various breeding materials, and the breeding efficiency is low. Meanwhile, because the genetic background of stevia rebaudiana hybrid parents is complex, the variation degree of hybrid progeny is large, the detection time and the instrument cost of breeding materials and the hybrid progeny are high, and two major restriction factors in detection are combined with an empirical hybridization combination preparation method, the efficient development of the stevia rebaudiana breeding work is seriously restricted, and the sustainable development of the stevia rebaudiana industry is also restricted. The fundamental reason for this is that stevia rebaudiana breeding workers lack a method for effectively extracting the compound information contained in the breeding material and the leaf of the filial generation thereof.
Disclosure of Invention
The invention aims to: aiming at the current situation of low quality breeding efficiency caused by insufficient extraction of compound information contained in the leaves of breeding materials and randomness of matching of breeding parents in the current stevia rebaudiana quality breeding work, the invention evaluates the utilization potential of each breeding material in the aspect of improving the content of target compounds contained in the leaves of the stevia rebaudiana according to the breeding direction on the basis of fully extracting the compound information contained in the leaves of the stevia rebaudiana breeding materials, accurately matches each breeding parent, realizes the high-efficiency improvement of the content of compounds such as stevioside and the like in the leaves of the stevia rebaudiana and promotes the sustainable development of the stevia rebaudiana industry.
The purpose of the invention can be realized by the following technical scheme:
a stevia rebaudiana quality breeding parent matching method comprises the following steps:
1) and (3) detecting compounds contained in the leaves of stevia rebaudiana breeding materials.
Detecting compounds contained in the leaves of the stevia rebaudiana breeding material by adopting a liquid chromatography-mass spectrometry (LC-MS), wherein: the liquid phase separation system is as follows: using ACE Ultrocore 2.5Super C18 column (150mm × 4.6mm,2.5 μm) as stationary phase, and using 0.1% (v/v) formic acid (A) in purified water and acetonitrile (B) as mobile phase, and gradient elution conditions are as follows: 0min 22.0% B, 7.0min 26.5% B, 12.0min 26.5% B, 15.0min 38.0% B, 19.0min 100% B, 22.0min 100% B, 22.01min 22.0% B.
The mass spectrum conditions are as follows: the ion source is an electrospray ionization source, the negative ion scanning mode is adopted, the atomizing gas is nitrogen with the purity of 99.9 percent, the collision gas is nitrogen with the purity of 99.999 percent, the pressure of the atomizing gas is 50psi, the temperature of the drying gas is 350 ℃, the flow rate of the drying nitrogen is 10.0L/min, the voltage of a capillary tube is 3500V, the Mass range is 100-5000 m/z, and the collision fracture voltage is 175V.
2) Qualitative analysis of compounds detected in leaves of stevia rebaudiana breeding material.
The qualitative method of the compounds contained in the leaves of each breeding material comprises the following steps: each detected compound was characterized by co-separation features including, but not limited to, retention time, M-1 peak, M-1+36 peak, M +62 peak, 2M-1 peak, and 3M-1 peak. Wherein M-1 is the molecular ion peak of the compound, M-1+36 is the chloride ion peak, M +62 is [ M + NO ]3]-And 2M-1 is a bimolecular ion peak of the compound, 3M-1 is a trimolecular ion peak of the compound, and when 2 or more than other coseparation characteristic peaks simultaneously appear in the ion peaks except the molecular ion peak, the compound is judged to be actually present.
3) Relative quantification of compounds detected in leaves of stevia rebaudiana breeding material.
The relative quantification method for detecting each compound in the leaf of the breeding material comprises the following three steps:
the first step is to count the peak areas corresponding to a cracking peak with mass more than or equal to 1/2M, an M-1 peak, an M-1+36 peak, an M +62 peak, a bimolecular cracking peak with mass between M +62 and 2M-1, a 2M-1 peak, a trimolecular cracking peak with mass between 2M-1 and 3M-1, 3M-1 and the isotope peak of each peak from small to large according to the mass size.
The second step is to convert 4 kinds of characteristic ion with mass not less than 1/2M, including cracking peak, M-1+36 peak, M +62 peak, etc. into binary molecular cracking peak and 2M-1 peak with mass between M +62 and 2M-1 and isotopic peak area of these two kinds of peaks into double molecular weight of the compound (2M), and to convert three molecular cracking peak and 3M-1 peak with mass between 2M-1 and 3M-1 and isotopic peak area of these two kinds of peaks into triple molecular weight of the compound (3M).
And the third step is to sum the amount of the compound represented by the peak area of each kind of ion peak and its isotope peak converted in the second step to obtain the relative content of the compound in each breeding material leaf.
4) Screening the breeding material as a breeding parent.
The screening of breeding materials which can be used as breeding parents is divided into three steps:
the first step is to measure and calculate the theoretical content of the target compound in the harvesting organs of each breeding material.
Screening out compounds related to the breeding improvement target compounds according to the correlation coefficient from high to low, establishing a multiple linear regression relational expression, and calculating the theoretical content of the breeding improvement target compounds in the leaves of the breeding materials according to the multiple linear regression relational expression.
And secondly, screening the breeding materials serving as male parents.
Comparing the measured values of the improved target compounds contained in the leaves of the breeding materials with the measured values calculated according to the multiple linear regression relation, and determining the breeding materials as male parents according to the breeding direction: if the content of the target compound in the stevia rebaudiana leaves needs to be improved through breeding, breeding materials with high measured values, measured values close to the measured values, good disease resistance and other field properties are taken as male parents; on the contrary, if the content of the target compound in the stevia rebaudiana leaves needs to be reduced, the stevia rebaudiana breeding material with low measured value, approximate measured value to the measured value, good disease resistance and other field properties is taken as a male parent.
And thirdly, screening the breeding material serving as the female parent.
Comparing the measured value of the improved target compound contained in each breeding material leaf with the measured value calculated according to the multiple linear regression relation, and determining the breeding material which can be used as the female parent according to the breeding direction: if the content of the target compound needs to be improved, breeding materials with high measurement value, higher measurement value than the measured value and high yield are taken as female parents; on the contrary, if the content of the target compound in the stevia rebaudiana leaves needs to be reduced, the stevia rebaudiana breeding material with low calculated value, smaller calculated value than the measured value and high yield is taken as the female parent.
The technical scheme of the invention is as follows: the stevia rebaudiana breeding material is asexual cuttage propagation seedling of stevia rebaudiana hybrid seed seedling with wider genetic background, and has large variation amplitude in the aspects of field property, compound variety contained in leaves, relative content of each compound and the like, and meanwhile, the stevia rebaudiana breeding material also comprises historical popularization varieties which can be collected when breeding work is carried out so as to improve the universality of each breeding material and the breeding efficiency.
The technical scheme of the invention is as follows: the leaf sampling time of the stevia rebaudiana breeding material is equal to the actual harvest period of stevia rebaudiana leaves in a producing area, the determination factors of the harvest period comprise two aspects of yield traits of the stevia rebaudiana leaves, the content of target compounds in the stevia rebaudiana leaves and the like, if RB is taken as a main breeding target, the harvest detection evaluation is carried out 2-3 weeks before budding with the highest RB content, and if RA is taken as a main breeding target, the harvest detection evaluation is carried out in the bud period.
The technical scheme of the invention is as follows: the leaf sampling method of the stevia rebaudiana breeding material is a half-plant sampling method, namely, one leaf is taken from each stem node and branch position from the bottom to the top of a plant, the leaf is fully ground and uniformly mixed after being dried, and the whole leaf is sampled and detected by a 60-mesh sieve and a cone quartering method.
The technical scheme of the invention is as follows: the liquid phase separation system is required to separate each compound one by one, but the realization of the requirement is challenging due to the variety of compounds contained in stevia rebaudiana leaves, and related compounds can be separated one by one through fine identification of co-separation characteristic peaks in the later period.
The technical scheme of the invention is as follows: the setting requirement of the collision fracture voltage in the mass spectrum condition is that compounds as much as possible enter a mass detector in the form of molecular ion peaks and the addition ions of the molecular ion peaks and chloride ions, nitrate ions and the like, and the qualitative and relative quantitative accuracy of each compound is improved.
The technical scheme of the invention is as follows: the scanning range of the mass analyzer in the mass spectrum condition is set to ensure that both the 2M-1 peak and the 3M-1 peak of the currently known stevioside compounds can be scanned and detected, so that the relative quantitative accuracy of each compound is improved.
The technical scheme of the invention is as follows: the identification method of each detected compound comprises, but is not limited to, identifying the compound contained in each stevia rebaudiana breeding material leaf by using co-separation characteristic peaks such as an M-1 peak, an M-1+36 peak, an M +62 peak, a 2M-1 peak and a 3M-1 peak which appear in the same retention period, wherein M-1 is a molecular ion peak of the compound, M-1+36 is a chloride ion peak, and M +62 is [ M + NO ]3]-And the peak, 2M-1 is a bimolecular ion peak of the compound, 3M-1 is a trimolecular ion peak of the compound, and when more than 2 (including) other coseparation characteristic peaks exist in addition to the molecular ion peak in the ion peaks, the compound is judged to be actually present.
The technical scheme of the invention is as follows: the relative quantification method of each detected compound in the stevia rebaudiana breeding material leaves comprises the step of simultaneously counting peak areas corresponding to a cracking peak with the mass of more than or equal to 1/2M, an M-1 peak, an M-1+36 peak, an M +62 peak, a bimolecular cracking peak with the mass between M +62 and 2M-1, a 2M-1 peak, a trimolecular cracking peak with the mass between 2M-1 and 3M-1, 3M-1 and isotope peaks of the peaks.
The technical scheme of the invention is as follows: the relative quantitative method for detecting each compound from the stevia rebaudiana breeding material leaves is characterized in that the peak areas of 4 types of characteristic ions and isotope peaks thereof, such as a cracking peak, an M-1+36 peak, an M +62 peak and the like with the mass of the compound being more than or equal to 1/2M, are multiplied by the single molecular weight (M) of the compound to be converted, the peak areas of a bimolecular cracking peak and a 2M-1 peak with the mass of between M +62 and 2M-1 and the isotope peaks of the two types of peaks are multiplied by the double molecular weight (2M) of the compound to be converted, and the peak areas of a trimolecular cracking peak and a 3M-1 peak with the mass of between 2M-1 and 3M-1 and the isotope peaks of the two types of peaks are multiplied by the triple molecular weight (3M) of the compound to be converted.
The technical scheme of the invention is as follows: the relative quantitative method for detecting each compound in the stevia rebaudiana breeding material leaves is to sum the amounts of the compound represented by the peak areas of various ion peaks and isotope peaks thereof to obtain the relative content of the compound in each stevia rebaudiana breeding material leaf.
The technical scheme of the invention is as follows: the first step of screening the breeding material as a breeding parent is to measure and calculate the theoretical content of a breeding improvement target compound in each breeding material leaf.
The technical scheme of the invention is as follows: the method for measuring and calculating the theoretical content of the breeding improvement target compound in each breeding material leaf comprises the following steps: screening out compounds related to the breeding improvement target compounds according to the correlation coefficient from high to low, establishing a multiple linear regression relational expression, and calculating the theoretical content of the breeding improvement target compounds in the stevia rebaudiana breeding materials according to the multiple linear regression relational expression.
The technical scheme of the invention is as follows: the second step of the screening of the breeding material as a breeding parent is the screening of the breeding material as a male parent.
The technical scheme of the invention is as follows: the screening method as male parent breeding material comprises the following steps: comparing the measured values of the target improved compounds contained in the leaves of the breeding materials with the measured values calculated according to the multiple linear regression relation, and determining the breeding materials which can be used as male parents according to the breeding direction: if the content of the target compound in the stevia rebaudiana leaves needs to be improved through breeding, breeding materials with high measured values, measured values close to the measured values, good disease resistance and other field properties are taken as male parents; if the content of the target compound in the stevia rebaudiana leaves needs to be reduced, breeding materials with low measured value, approximate measured value and good field properties such as disease resistance are taken as male parents.
The technical scheme of the invention is as follows: the third step of screening the breeding material as a breeding parent is screening the breeding material as a female parent.
The technical scheme of the invention is as follows: the screening method of the breeding material as the female parent comprises the following steps: comparing the measured value of the target improved compound contained in each stevia rebaudiana breeding material leaf with the measured value calculated according to the multiple linear regression relational expression, and determining the breeding material which can be used as a female parent according to the breeding direction: if the content of the target compound needs to be improved, stevia rebaudiana breeding materials with high measured value, measured value larger than measured value, yield and other good field properties are taken as female parents; and on the contrary, if the content of the target compound in the stevia rebaudiana leaves needs to be reduced, the stevia rebaudiana breeding material with low calculated value, the calculated value smaller than the measured value, the yield and other good field properties is taken as the female parent.
According to the stevia rebaudiana leaf quality breeding parent matching method, the breeding materials can be widely collected, and a wide material basis is provided for the stevia rebaudiana leaf quality breeding parent matching based on the existing materials.
According to the stevia rebaudiana leaf quality breeding parent matching method, the effective evaluation on the current situation of the leaf quality of the breeding material in the harvest period can be realized, and effective compound data contained in the leaf harvest period of each breeding material is provided for the stevia rebaudiana leaf quality breeding.
According to the stevia rebaudiana leaf quality breeding parent matching method, the whole condition of a breeding material plant can be effectively evaluated, and effective data are provided for stevia rebaudiana leaf quality breeding parent matching based on the existing material.
According to the stevia rebaudiana leaf quality breeding parent matching method, the compounds contained in the leaves of the breeding material can be completely separated, and effective data are provided for the stevia rebaudiana leaf quality breeding parent matching based on the existing material.
According to the stevia rebaudiana leaf quality breeding parent matching method, comprehensive statistics can be carried out on various addition ions and isotope ions of compounds contained in breeding materials, and effective data are provided for stevia rebaudiana leaf quality breeding parent matching based on existing materials.
According to the stevia rebaudiana leaf quality breeding parent matching method, comprehensive statistics can be performed on all compounds contained in the leaves of breeding materials, particularly scanning detection of a 2M-1 peak and a 3M-1 peak, accurate calculation of the relative content of a certain compound with large concentration difference among the breeding materials and the relative content of all compounds with large concentration difference in the same material is facilitated, and effective data are provided for matching of stevia rebaudiana leaf quality breeding parents based on the existing materials.
According to the stevia rebaudiana leaf quality breeding parent matching method, the existence of each compound in the leaf of the breeding material can be effectively judged, and effective data are provided for the stevia rebaudiana leaf quality breeding parent matching based on the existing material.
According to the stevia rebaudiana leaf quality breeding parent matching method, the relative accurate quantification of each compound detected in the leaves of the breeding material can be realized, and effective data is provided for the stevia rebaudiana leaf quality breeding parent matching based on the existing material.
According to the stevia rebaudiana leaf quality breeding parent matching method, the application potential value of each breeding material serving as a breeding parent can be accurately evaluated, and a basis is provided for accurate matching of stevia rebaudiana leaf quality breeding parents based on the existing materials.
According to the stevia rebaudiana leaf quality breeding parent matching method, effective screening of breeding materials serving as male parents can be realized, and a basis is provided for accurate matching of stevia rebaudiana leaf quality breeding parents based on existing materials.
According to the stevia rebaudiana leaf quality breeding parent matching method, effective screening of the breeding material serving as a female parent can be realized, and a basis is provided for accurate matching of stevia rebaudiana leaf quality breeding parents based on the existing material.
According to the stevia rebaudiana leaf quality breeding parent matching method, the following problems in stevia rebaudiana leaf quality breeding parent matching are mainly solved:
1) the invention adopts the strategy of collecting and investigating the plant field properties, the types of the compounds contained in the leaves and the stevia rebaudiana breeding materials with large relative content difference of the compounds contained in the leaves, thereby providing a wide material basis for the stevia rebaudiana leaf quality breeding based on the existing materials.
2) According to the method, the strategy of evaluating each breeding material in the optimal harvest period of the target compound is adopted, so that the influence of the fluctuation of the content of each compound including the target compound in different growth periods on the later analysis is avoided, the evaluation efficiency of the breeding material is improved, and the effectiveness of evaluation data is guaranteed.
3) Because the invention adopts a half-plant sampling method comprising the leaf positions of all parts of the whole plant of the plant, the influence of different sampling parts on the detection data is avoided, the representativeness of the sample is improved, and the effectiveness of the evaluation data is ensured.
4) The invention adopts a gradient elution liquid chromatography-mass spectrometry detection method, realizes the comprehensive separation and detection of each compound contained in each breeding material leaf, simultaneously improves the information richness of each detected compound, and provides comprehensive and accurate data acquisition for the correlation analysis among each compound in the later period and the like.
5) The invention adopts the method of comprehensively judging the existence of each compound by utilizing the information of the co-separated molecular ion peak, the addition ion peak, the bimolecular ion peak and the like in the same time period, so that the judgment is more accurate, and more comprehensive and accurate data acquisition is provided for the later-stage correlation analysis among the compounds.
6) According to the invention, a strategy of adding and counting the amounts of target compounds represented by fragment ion peaks, addition ion peaks, molecular ion peaks, bimolecular ion peaks and trimolecular ion peaks of the compounds and isotope peaks of the ion peaks is adopted, so that the effectiveness of statistical data of the relative content of the compounds in the leaves of the breeding material is improved, and relatively comprehensive and accurate data acquisition is provided for correlation analysis among the compounds.
7) The invention adopts the method of counting the relative content of each detected compound in the leaf of the breeding material, thereby ensuring the reliability of the multiple linear regression relational expression for measuring and calculating the theoretical content of the breeding improvement target compound in each breeding material.
8) As the invention adopts the parent matching strategy of father and mother breeding materials determined by comparing the measured values of the target improved compounds contained in the leaves of the breeding materials with the difference between the measured values calculated according to the multiple linear regression relational expression and combining the breeding direction, the content of the target compounds in the leaves of the filial generation strains achieves the percentage of improvement expecters, the breeding efficiency is high, and the operability is strong.
In conclusion, the stevia rebaudiana leaf quality breeding scheme adopting accurate matching of breeding parents has the beneficial effects that:
the invention breaks through the traditional strategy that only the content of an improved target compound in a breeding material is investigated, and the stevia rebaudiana leaf quality breeding is carried out by virtue of empirical 'excellent' hybridization combination, but establishes a calculable and more accurate selection method of stevia rebaudiana leaf quality breeding parents based on wider breeding materials and more comprehensive qualitative and quantitative analysis of compound information contained in leaves of each breeding material, and provides a more reliable new method for the accurate selection of the breeding parents for harvesting organ quality of self-incompatible crops including stevia rebaudiana; the randomness of parent matching in the self-incompatible crop quality breeding work including stevia rebaudiana is avoided, and the improvement of the crop quality breeding efficiency is facilitated; the comprehensive evaluation of the utilization potential of each breeding material of self-incompatible crops including stevia rebaudiana is facilitated, and a basis is provided for the establishment of a core germplasm resource library for the quality breeding of each crop; can better play and utilize the functions of various crops including the stevia rebaudiana in improving the health welfare level of animals and human beings, and is also beneficial to the sustainable development of the links of seed production, planting, processing and the like of self-incompatible crops including the stevia rebaudiana.
The superiority of the stevia rebaudiana leaf quality breeding strategy accurately matched by utilizing breeding parents is mainly embodied in the following aspects:
1) through comprehensive evaluation of each detected compound contained in the breeding material, the method is beneficial to efficiently establishing a crop quality breeding core germplasm resource library, avoiding single evaluation of germplasm resources and better exerting the breeding potential of each breeding material;
2) through comprehensive collection of compound information detected from each breeding material, the evaluation efficiency of the utilization potential of each breeding material as a breeding parent is improved, and the matching accuracy of the breeding material of a parent for improving the target compound is improved;
3) through the accurate matching of the breeding parents and parents based on the target compound improvement direction, the content of the target compound in the harvested organs of the filial generation is efficiently increased or reduced, the effects of crop products in improving the animal and human health level are better exerted, and the sustainable development of industries such as seed industry, planting industry, extraction industry, breeding industry, food processing industry and the like is facilitated.
Detailed Description
Example 1
Stevioside has high sweetness which is about 150-300 times of cane sugar sweetness, and taste buds can generate more continuous stimulation after eating (drinking) due to the lack of stevioside degrading enzymes in human saliva. RE is a stevioside compound synthesized by stevia rebaudiana leaves, which has good water solubility, high stability and pure mouthfeel, has the same molecular weight as RA but more symmetrical configuration than RA, short retention time on taste buds and close mouthfeel to honey, and has been developed and applied as a novel stevioside compound.
Now, taking crossbreeding for improving the content of RE in stevia rebaudiana leaves as an example, the application of the breeding parent precise matching strategy based on comprehensive analysis of information of each compound contained in the leaves of breeding materials with wide genetic background and variation degree in stevia rebaudiana quality breeding is shown, and the method comprises the following steps:
(1) construction of a stevia rebaudiana germplasm resource library: 14 parts of field promoted varieties in all years, 123 parts of laboratory self-breeding evaluation screening preservation materials in 2010-2018, 70 parts of hybrid seed radiation mutagenesis single plant material of Nannong 1066 and Tiannong No. 1, and 207 parts in total, and the materials are used as breeding materials for improving the RE content in stevia rebaudiana leaves for analysis and matching.
(2) Collecting and preparing a stevia rebaudiana germplasm leaf sample: in the bud stage, selecting leaves of each strain by a half-plant sampling method, drying at 80 ℃ overnight, grinding, uniformly mixing, sieving with a 60-mesh sieve, taking 50mg, putting into 1.5mL of 50% ethanol, oscillating overnight, ultrasonically extracting for 40 minutes, centrifuging at 4 ℃ at 10000rpm for 10 minutes, taking supernatant, filtering with a 0.45-micron organic microporous filter membrane, and storing in a refrigerator at 4 ℃ to be detected.
(3) Detecting a compound contained in the stevia rebaudiana germplasm leaves by a liquid chromatography-mass spectrometry (LC-MS) method: the liquid phase separation system is as follows: using ACE Ultrocore 2.5Super C18 column (150mm × 4.6mm,2.5 μm) as stationary phase, and using 0.1% (v/v) formic acid (A) in purified water and acetonitrile (B) as mobile phase, and gradient elution conditions are as follows: 0min 22.0% B, 7.0min 26.5% B, 12.0min 26.5% B, 15.0min 38.0% B, 19.0min 100% B, 22.0min 100% B, 22.01min 22.0% B. The mass spectrum conditions are as follows: the ion source is an electrospray ionization source, the negative ion scanning mode is adopted, the atomizing gas is nitrogen with the purity of 99.9 percent, the collision gas is nitrogen with the purity of 99.999 percent, the pressure of the atomizing gas is 50psi, the temperature of the drying gas is 350 ℃, the flow rate of the drying nitrogen is 10.0L/min, the voltage of a capillary tube is 3500V, the Mass range is 100-5000 m/z, and the collision fracture voltage is 175V.
(4) Qualitative analysis of compounds detected in leaves of stevia rebaudiana breeding material. The compounds contained in the leaves of the stevia rebaudiana breeding material are characterized by using co-separation characteristic peaks such as retention time, an M-1 peak, an M-1+36 peak, an M +62 peak, a 2M-1 peak and a 3M-1 peak. Wherein M-1 is the molecular ion peak of the compound, M-1+36 is the chloride ion peak, M +62 is [ M + NO ]3]-The peak, 2M-1 is a bimolecular ion peak of the compound, 3M-1 is a trimolecular ion peak of the compound, when the ion peaks except the molecular ion M-1 peak also have 2 or more other coseparation characteristic peaks, the compound is judged to be actually present, and thus 1003 compounds are determined in 207 detected breeding material leaves.
(5) Relative quantification of compounds detected in leaves of stevia rebaudiana breeding material. Firstly, simultaneously counting the cracking peak with the mass more than or equal to 1/2M, the M-1 peak, the M-1+36 peak, the M +62 peak, the bimolecular cracking peak with the mass between M +62 and 2M-1, the 2M-1 peak, the trimolecular cracking peak with the mass between 2M-1 and 3M-1, the 3M-1 and the peak areas corresponding to the isotope peaks of the peaks in the order from small to large. Converting by multiplying the peak areas of 4 types of characteristic ions with the mass of more than or equal to 1/2M, such as a cracking peak, an M-1+36 peak, an M +62 peak and the like and the isotope peaks thereof by the single molecular weight (M) of the compound, converting by multiplying the peak areas of a bimolecular cracking peak and a 2M-1 peak with the mass of between M +62 and 2M-1 and the isotope peaks of the two types of peaks by the double molecular weight (2M) of the compound, and converting by multiplying the peak areas of a trimolecular cracking peak and a 3M-1 peak with the mass of between 2M-1 and 3M-1 and the isotope peaks of the two types of peaks by the triple molecular weight (3M) of the compound. And finally, summing the amounts of the compounds represented by the peak areas of various ion peaks and isotope peaks thereof to obtain the relative content of each compound in each breeding material leaf.
(6) The screening of breeding parents for improving the RE content in hybrid progeny leaves comprises the following three steps:
and step one, measuring and calculating the theoretical content of RE in each breeding material leaf.
Compounds related to RE are screened according to the correlation coefficient from high to low, and a multiple linear regression relation is firstly established, wherein [ RE ] ═ 376.272+0.429 [ Rubusoside ] +1.075 [787@16.766] -0.256 [741@16.559] +0.029 [ RA ] +1.560 [1097@12.704] +0.736 [919@16.094 ]. And calculating the theoretical content of RE in the leaves of the breeding materials according to the multiple linear regression relation.
And step two, screening male parent breeding materials for improving the RE content of hybrid progeny leaves.
Comparing the measured values of RE contained in the leaves of the breeding materials with the measured values obtained by operation according to the multiple linear regression relational expression, and selecting the material with high measured value, similar measured value to the measured value, good field properties such as disease resistance and the like and the serial number 913 as the male parent for improving the RE content of the leaves of the filial generations.
And thirdly, screening female parent breeding materials for improving the RE content of hybrid progeny leaves.
Comparing the RE measured value contained in each stevia rebaudiana breeding material leaf with the measured value obtained by operation according to the multiple linear regression relational expression, and selecting the material with high measured value, measured value greater than the measured value, good yield and character and number 6659 as the female parent for improving the RE content of the hybrid progeny leaf.
(7) Hybridization matching experiment
And (3) carrying out a matching experiment for improving the RE content of the leaf of the filial generation by using the material with the number 913 as a male parent and the material with the number 6659 as a female parent.
Comparative examples 1 to 1
Referring to the conventional "superior" hybridization combination for matching parent and female parents, the difference from example 1 is that the 4160 material with the highest content of leaf RE in the 207 materials is directly selected to be hybridized with the 6659 material, the 4160 material is taken as a male parent, and the 6659 material is taken as a female parent.
Control group 1
The material of number 6659 is used as female parent, and placed in field for natural pollination.
Table 1 shows the RE content in leaves of each combined parent single line and the filial generation thereof in the harvest stage (bud stage). The results show that:
1) because the genetic background of each parent is complex, the RE content separation degree in the bud stage leaves of each filial combination progeny is high;
2) in the three hybridization methods, the average value and the highest value of the RE content in the leaves of the filial generation population are high based on the accurate matching (example 1) by utilizing the full-component analysis result of the leaves of the breeding material;
3) the average value and the maximum value of the RE content in the leaves of the progeny group of the traditional 'excellent' hybrid combination (comparative example 1-1) and the control group by using two breeding materials with high RE content in the leaves are lower than those of the accurate matching hybrid (example 1) based on the whole-component analysis of the leaves of the breeding materials.
The result shows that compared with the traditional method, the method relies on germplasm resources with wider variation and more comprehensively adopts compound information closely related to the formation and accumulation of the leaf RE in a harvested organ, and the precise matching strategy of the stevia rebaudiana leaf quality breeding parents can obviously improve the breeding efficiency.
TABLE 1 RE content of each parent single line and each filial combination progeny in bud stage
Example 2
The STV is a series of stevia glycosides compounds with content only next to RA in leaves of stevia rebaudiana varieties such as Rundy, Julong, Bangxing and Haoyang which are widely popularized and planted in 2010, and because the stevia rebaudiana varieties have green grass flavor, the prior strategy is to remove or reduce the relative content of the stevia rebaudiana glycosides compounds in a stevioside mixture through a later refining step and then apply the stevia rebaudiana glycosides mixture to downstream food or beverage. If the relative content of the stevioside in stevia rebaudiana leaves can be reduced by a breeding method, the refining and processing links are reduced, the processing cost is reduced, and the market development capability of the stevioside is effectively improved.
Now, taking cross breeding for reducing the content of STV in stevia rebaudiana leaves as an example, the application of the precise parent matching strategy based on comprehensive analysis of compound information contained in the leaves of a breeding material in stevia rebaudiana quality breeding is shown, and the method comprises the following steps:
(1) - (5) same as example 1
(6) The screening of the breeding parent for reducing the STV content in the leaf of the filial generation is divided into three steps:
and step one, measuring and calculating the STV theoretical content in each breeding material.
Screening compounds related to the STV according to the correlation coefficient from high to low, and establishing a multiple linear regression relation:
[STV]＝-288528.498+3.182*[1031-2]+26.354*[537@19.723]+0.979*[836@14.407]+19.741*[571@5.013]+1.175*[353@1.25]+0.780*[RF]+2.456*[1051-10]-3.856*[357@19.773]
and calculating the theoretical content of STV in the leaves of the breeding materials according to the multiple linear regression relation.
And step two, screening male parent breeding materials for reducing the STV content of hybrid progeny leaves.
Comparing the STV measured value contained in each breeding material leaf with the measured value obtained by operation according to the multivariate linear regression relational expression, and selecting the material with the number 4147 as the male parent for reducing the STV content of the filial generation leaf, wherein the material has higher RA content, low STV measured value, close STV measured value to the measured value, good field properties such as disease resistance and the like.
And thirdly, screening the female parent breeding material for reducing the STV content of the hybrid progeny leaves.
Comparing the STV measured value contained in each breeding material leaf with the measured value obtained by operation according to the multiple linear regression relational expression, and selecting the material with high RA content, low STV measured value, STV measured value smaller than the measured value, good yield and character and number 6643 as the female parent for reducing the STV content of the filial generation leaf.
(7) Hybridization matching experiment
The mating experiment for reducing the STV content of the leaf of the filial generation is carried out by using the material with the number 4147 as a male parent and the material with the number 6643 as a female parent.
Comparative example 2-1
The difference from example 2 is that the material No. 810 with poor yield performance but the highest RA content in leaves is directly selected to be hybridized with the material No. 6643 in 207 parts of the above-mentioned materials, the material No. 810 is taken as a male parent, and the material No. 6643 is taken as a female parent.
Control group 2
The material of number 6643 is used as female parent, and placed in field for natural pollination.
Table 2 shows the STV content in leaves of each combined parent single line and its filial generation in the harvest stage (bud stage). The results show that:
1) because the genetic background of each parent is complex, the STV content separation degree in the bud stage leaves of the filial generation of each hybridization combination is high;
2) in the three hybridization methods, the average value and the minimum value of the STV content of the leaf of the filial generation group based on the precise matching (example 2) of the analysis result of the whole components of the leaf of the breeding material are all the lowest;
3) in comparison, the average value and the minimum value of the STV content in the leaves of the progeny population using the high RA content in the leaves and adopting the traditional "excellent" hybrid combination (comparative example 2-1) and the control group are higher than the STV content of the leaves of the progeny population of the precise matching hybrid based on the whole-component analysis result of the leaves of the breeding material (example 2).
The result shows that compared with the traditional method, the method relies on germplasm resources with wider variation and more comprehensively adopts compound information closely related to the formation and accumulation of the STV of the leaves in the harvested organs, and the precise matching strategy of the parents for stevia rebaudiana leaf quality breeding according to the invention can obviously improve the breeding efficiency.
TABLE 2 STV content of leaves in bud stage of each parent single line and each cross combination progeny
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (1)
1. A stevia rebaudiana leaf quality breeding parent matching method is characterized in that: the method comprises the following steps:
1) detection of Compound contained in leaf of stevia rebaudiana Breeding Material
Detecting compounds contained in leaves of various breeding materials by adopting a liquid chromatography-mass spectrometry method, wherein: the liquid phase separation system is as follows: taking an ACE Ultrocore 2.5Super C18 column as a stationary phase, taking pure water containing 0.1% (v/v) formic acid (A) and acetonitrile (B) as mobile phases, and carrying out gradient elution under the following conditions: 0min 22.0% B, 7.0min 26.5% B, 12.0min 26.5% B, 15.0min 38.0% B, 19.0min 100% B, 22.0min 100% B, 22.01min 22.0% B;
the mass spectrum conditions are as follows: the ion source is an electrospray ionization source, the negative ion scanning mode is adopted, the atomizing gas is nitrogen with the purity of 99.9 percent, the collision gas is nitrogen with the purity of 99.999 percent, the pressure of the atomizing gas is 50psi, the temperature of the drying gas is 350 ℃, the flow rate of the drying nitrogen is 10.0L/min, the voltage of a capillary tube is 3500V, the Mass range is 100-5000 m/z, and the collision fracture voltage is 175V;
2) qualitative analysis of compound detected in leaf of stevia breeding material
The qualitative method for detecting the compound in each breeding material leaf comprises the following steps: the characterization of each detected compound using co-separation features including but not limited to retention time, M-1 peak, M-1+36 peak, M +62 peak, 2M-1 peak and 3M-1 peak; wherein M-1 is the molecular ion peak of the compound, M-1+36 is the chloride ion peak, M +62 is [ M + NO ]3]A peak, 2M-1 is a bimolecular ion peak of the compound, 3M-1 is a trimolecular ion peak of the compound, and when 2 or more other coseparation characteristic peaks simultaneously appear in the ion peaks except the molecular ion peak, the compound is judged to be actually present;
3) relative quantitative analysis of compounds detected in leaves of stevia breeding material
The relative quantification method for detecting each compound in the leaf of the breeding material comprises the following three steps:
the first step is to count the peak areas corresponding to a cracking peak, an M-1+36 peak, an M +62 peak, a bimolecular cracking peak with the mass between M +62 and 2M-1, a 2M-1 peak, a trimolecular cracking peak with the mass between 2M-1 and 3M-1, a 3M-1 peak and an isotope peak of the peaks, wherein the cracking peak, the M-1 peak and the isotope peak are derived from the compound and have the mass more than or equal to 1/2M according to the mass sizes from small to large;
the second step is to convert the peak area of 4 kinds of characteristic ions and their isotope peaks with mass of the compound being greater than or equal to 1/2M by multiplying the single molecular weight (M) of the compound; converting a bimolecular cleavage peak with the mass between M +62 and 2M-1 and a 2M-1 peak, and multiplying the isotope peak area of the two types of peaks by the double molecular weight (2M) of the compound; converting three-molecule cracking peak with mass between 2M-1 and 3M-1 peak, and multiplying the isotope peak area of the two kinds of peaks by the triple molecular weight (3M) of the compound;
the third step is to sum the amount of the compound represented by the peak area of each kind of ion peak and its isotope peak converted in the second step, and the sum is used as the relative content of the compound in each breeding material leaf;
4) screening of breeding materials as breeding parents
The screening of the breeding material which can be used as a breeding parent is specifically divided into the following three steps:
first, calculating theoretical content of target compound in harvesting organs of various breeding materials
Screening out compounds related to the breeding improvement target compounds according to the correlation coefficient from high to low, establishing a multiple linear regression relational expression, and measuring and calculating the theoretical content of the breeding improvement target compounds in the leaves of each breeding material according to the multiple linear regression relational expression;
second step, screening of breeding material as male parent
Comparing the measured values of the improved target compounds contained in the leaves of the breeding materials with the measured values calculated according to the multiple linear regression relation, and determining the breeding materials as male parents according to the breeding direction: if the content of the target compound in the stevia rebaudiana leaves needs to be improved through breeding, breeding materials with high measured values, measured values close to the measured values, good disease resistance and other field properties are taken as male parents; on the contrary, if the content of the target compound in the stevia rebaudiana leaves needs to be reduced, the stevia rebaudiana breeding material with low measured value, approximate measured value to the measured value, good disease resistance and other field properties is taken as a male parent;
thirdly, screening the breeding material as female parent
Comparing the measured value of the improved target compound contained in each breeding material leaf with the measured value calculated according to the multiple linear regression relation, and determining the breeding material which can be used as the female parent according to the breeding direction: if the content of the target compound needs to be improved, breeding materials with high measurement value, higher measurement value than the measured value and high yield are taken as female parents; on the contrary, if the content of the target compound in the stevia rebaudiana leaves needs to be reduced, the stevia rebaudiana breeding material with low calculated value, smaller calculated value than the measured value and high yield is taken as the female parent.
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| CN106061987A (en) * | 2014-02-18 | 2016-10-26 | 麦克内尔营养有限公司 | Process for separation, isolation and characterization of steviol glycosides |
| CN108112474A (en) * | 2016-11-28 | 2018-06-05 | 山东农业大学 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
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| CN106061987A (en) * | 2014-02-18 | 2016-10-26 | 麦克内尔营养有限公司 | Process for separation, isolation and characterization of steviol glycosides |
| CN108112474A (en) * | 2016-11-28 | 2018-06-05 | 山东农业大学 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
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