A method for accurate identification of phenotypic traits and gene effects in molecular breeding fields

Abstract

The present invention discloses a method for accurately identifying phenotypic traits and gene effects in the field of molecular breeding, and belongs to the field of plant molecular marker-assisted breeding. The above method adopts a concentric ring planting method, plants the original parent in the center of the circle, and plants molecular breeding strains with different genes or gene combinations around the ring. According to the needs of special experimental purposes, a detachable moisturizing device and its accessories are covered on the ring planting material to ensure that the precise identification results of single gene effects or multi-gene aggregation effects are obtained through accurate investigation of agronomic traits and yield traits under completely consistent experimental conditions. This method is conducive to intuitively identifying the differences between materials containing different genes or combinations, and for the first time discloses the need that any molecular breeding process must eventually go through the field breeding and identification stage, and provides a method for accurately identifying phenotypic traits and gene effects in the field of molecular breeding based on ring planting.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular marker-assisted breeding, and in particular to a method for accurately identifying phenotypic traits and gene effects in molecular breeding fields. Background Art

[0002] The breeding of new crop varieties is a scientific innovation that has been carried out along with the development of human civilization. In the United States, a project entitled "Bringing Genomics to Wheat Breeding Fields" was launched in 2001, and successfully bred the first hard white spring wheat variety 'Patwin' that introduced the stripe rust resistance gene Yr17 and the leaf rust resistance gene Lr3. In Australia, the "Australian Winter Crop Molecular Marker Project" (AWCMMP) was established. Canada has launched an important project (40 million Canadian dollars) to "integrate molecular marker technology into traditional breeding procedures", which is mainly used to transfer excellent genes to cultivate new varieties. Molecular marker-assisted breeding in my country started relatively late, but has made rapid progress in the past decade. National key research programs such as 973 and 863 have supported molecular breeding research. For example, the applicant of the present invention participated in the National Key Basic Research and Development Program project "Applied Basic Research on Design and Breeding of High-Yield and High-Quality Wheat Varieties" (Project No. 2009CB118301), and identified 33 QTL loci and their molecular markers related to wheat yield, plant type, disease resistance and other traits, and bred more than a dozen new wheat varieties, including Shannong 20, a high-yield variety resistant to powdery mildew and stripe rust (which won the second prize of the National Science and Technology Progress in 2017).

[0003] A lot of progress has been made in the field of wheat molecular breeding both at home and abroad. However, it is undeniable that there are still many technical limitations in the work of breeding new varieties by aggregating excellent genes for multiple traits. Among them, "the inability to accurately identify the field phenotypic trait differences and gene aggregation effects between strains containing different trait genes / QTLs (or different numbers of genes for the same trait)" is the biggest problem, which even affects the breeding effect more than the molecular markers themselves. As we all know, no matter any high-tech breeding such as genetic modification, molecular markers and molecular design, good varieties can only be selected through field planting and selection in the end. However, molecular breeding experts often attach great importance to gene aggregation and indoor molecular identification, and relatively ignore the precise identification of field phenotypic traits and gene effects, and the identification methods all use traditional small plots (generally 6m 2 ) comparison method planting, due to the long distance between the materials and the difference in growth conditions, many agronomical comparisons between different treatments are difficult to compare with each other, and the results of gene-phenotype relationship identification are not accurate. Therefore, providing a method for accurate identification of field phenotypic traits and gene effects is very critical to solving the current technical problems. Summary of the invention

[0004] The purpose of the present invention is to provide a method for accurately identifying phenotypic traits and gene effects in the field of molecular breeding, so as to solve the problems existing in the above-mentioned prior art. The circular planting is adopted to ensure that all materials containing a gene or a combination of genes are under completely consistent growth conditions, thus laying the foundation for the accurate identification of phenotypic traits and gene effects in the field of molecular breeding.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for accurately identifying phenotypic traits and gene effects in the field of molecular breeding. The method adopts a concentric ring planting method, wherein the original parent is planted in the center of the circle, and molecular breeding strains with different genes or gene combinations are planted around the circle. Under completely consistent experimental conditions, accurate identification results of single gene effects or multi-gene aggregation effects are obtained through accurate investigation of agronomic traits and yield traits.

[0007] Preferably, the method specifically comprises the following steps:

[0008] (1) Selection of planting materials

[0009] Select planting materials with a defined target genetic composition obtained through conventional breeding or molecular breeding;

[0010] (2) Making a concentric ring planting device

[0011] The device for the concentric ring planting is made of galvanized iron material, and scales for fixed-point planting of the planting material are marked on the outer and inner rings of the concentric circles;

[0012] (3) Concentric ring planting

[0013] Carry out circular planting according to the device manufactured in step (2), wherein the parent material or the control material is planted along the inner ring wall according to the scale, and the planting material is planted along the outer ring wall according to the scale, and the spacing between plants is adjusted according to the amount of the planting material;

[0014] (4) Identification and evaluation of agronomic traits

[0015] According to the type of planting material planted in the ring in step (3) and the function of the target gene, the identification period and standard of agronomic traits and their gene effects are established;

[0016] (5) Identification and evaluation of gene / QTL locus effect values

[0017] The genetic data obtained by the gene / QTL site effect value identification are collated with the phenotypic trait data of each experimental strain obtained in step (4), and correlation analysis and path analysis are performed to determine the effect values ​​of different genes / QTLs of the same trait (but not limited to the same trait) on the phenotypic trait;

[0018] (6) Identification and creation of molecular breeding elements for wheat

[0019] According to the results of step (4) and step (5), a strain containing a known gene / QTL locus composition and having a significant contribution rate to the phenotypic traits is selected as a wheat molecular breeding element; the wheat molecular breeding element is a parental material that is known to contain the target trait gene / QTL and its marker and has a large contribution rate to the phenotypic traits and is used to prepare a hybrid combination.

[0020] Preferably, in step (2), the concentric ring planting device includes: an outer ring, an inner ring concentrically arranged with the outer ring, and a first connecting piece for fixing the outer ring and the inner ring; the outer ring and the inner ring are both made of galvanized iron material, the diameter of the outer ring is 0.6-1.5m, and the diameter of the inner ring is 0.1-0.3m.

[0021] Preferably, the device further comprises a detachable moisturizing frame covering the circular planting material, the moisturizing frame is configured as an equilateral triangular pyramid, the equilateral triangle at the bottom of the equilateral triangular pyramid is circumscribed to the outer ring, the tops of the three prisms of the equilateral triangular pyramid are configured as small circular rings, and the top circular ring parts of the three prisms are connected in series by an additional circular ring for easy installation and storage. The three sides of the equilateral triangular pyramid composed of three prisms can be attached with transparent plastic films to meet the moisturizing requirements of special planting (e.g., resistance to ergot) identification.

[0022] Preferably, the height of the equilateral triangle pyramid is set to 1.5, and the lengths of the three prisms are respectively set to 1.7-1.9 m.

[0023] Preferably, the additional ring passes through the small rings at the top of the three prisms and is sleeved on the top of the regular triangle pyramid. The diameter of the additional ring is 10 cm. A self-controlled humidity device can be installed on it to provide completely consistent growth conditions for breeding materials (especially disease-resistant materials).

[0024] Preferably, the bottom of each of the prisms is connected by a second connecting member to form the equilateral triangle, so as to fix the prism and make each side of the equilateral triangle circumscribe the outer ring.

[0025] Preferably, in step (3), the plant spacing is 20-30 cm; the circular planting is performed by sowing at equal distances, with 2-8 plant material seeds sown in each hole, and one plant is planted in time according to the growth conditions after germination.

[0026] The present invention discloses the following technical effects:

[0027] (1) The present invention discloses for the first time the requirement that any molecular breeding process must ultimately go through a field selection and identification stage, and provides a method for accurately identifying phenotypic traits and gene effects in the molecular breeding field based on ring planting.

[0028] (2) The identification method disclosed in the present invention ensures that all identification materials are within the same soil fertility, the same climate factors and the same distance from the control, that is, all materials containing a gene or gene combination are under completely consistent growth conditions, which is not only conducive to the observation, comparison and recording of field agronomic traits, but also more conducive to intuitively identifying the differences between materials containing different genes or combinations.

[0029] (3) For tests such as identification of resistance to fusarium head blight, which require special conditions (especially in northern wheat fields) to induce diseases, this can be achieved by modifying the circular planting of this patent. The present invention is the first to use a regular triangle cone field microclimate control device that promotes the induction of wheat fusarium head blight. It can be covered on the circular planting material during the heading and flowering period of wheat to increase humidity locally, providing technical support for field selection of fusarium head blight resistance and identification of fusarium head blight gene effects in the dry and rainless climate of northern wheat flowering period.

[0030] (4) The phenotypic traits of new varieties (lines) bred by high-tech technologies such as transgenic, molecular design, and gene editing accurately identified by the present invention can be analyzed by correlation analysis and gene analysis with the target genes contained therein, which can clarify the relationship between "phenotype---gene (or gene combination)", obtain the contribution rate of different genes to related phenotypic traits, and further determine the main effect gene / QTL according to the size of its effect value. Selecting strains (lines) containing one or more main effect genes / QTLs and having a significant effect on phenotypic traits as hybrid parents is a very necessary molecular breeding element. Therefore, the present invention provides technical support for improving molecular breeding technology, accelerating the molecular breeding process, and successfully breeding breakthrough new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 A schematic diagram of the concentric ring planting device;

[0033] Figure 2 A schematic diagram of a circumscribed triangle of an outer ring of a concentric ring implantation device;

[0034] Figure 3is a schematic diagram of the moisturizing framework;

[0035] 1-outer ring; 2-inner ring; 3-first connecting piece; 4-second connecting piece; 5-prism; 6-additional circular ring. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The present invention provides a method for accurately identifying phenotypic traits and gene effects in the field of molecular breeding. The method adopts a concentric ring planting method, wherein the original parent is planted in the center of the circle, and molecular breeding strains with different genes or gene combinations are planted around the circle. Under completely consistent experimental conditions, accurate identification results of single gene effects or multi-gene aggregation effects are obtained through accurate investigation of agronomic traits and yield traits.

[0042] In a preferred embodiment, the method specifically comprises the following steps:

[0043] (1) Selection of planting materials

[0044] Select planting materials with a target gene composition obtained through conventional breeding or molecular breeding; more specifically, select stable wheat lines (kinds) with known target gene composition obtained through transgenic, molecular marker polymer hybridization, molecular design breeding and other technologies, such as materials known to contain one or more disease resistance genes among Fhb1 to Fhb7. Of course, this patented method can also be used to accurately identify important traits of different strains or different new lines (kinds) of the same combination offspring in the conventional breeding process.

[0045] (2) Making a concentric ring planting device

[0046] The device for the concentric ring planting is made of galvanized iron material, and scales for fixed-point planting of the planting material are marked on the outer and inner rings of the concentric circles;

[0047] (3) Concentric ring planting

[0048] The device manufactured in step (2) is used for circular planting, wherein 1-2 plants of the parent or control material are planted along the inner ring wall according to a scale, and when there are many control materials, they can be planted at equal distances along the outer wall of the inner ring (for example, multiple parents of a three-way combination); the planting materials are planted along the outer ring wall according to a scale, and the spacing between plants is adjusted according to the amount of the planting materials;

[0049] (4) Identification and evaluation of agronomic traits

[0050] According to the type of planting material planted in the ring in step (3) and the function of the target gene, the identification period and standard of agronomic traits and their gene effects are formulated; for example, the precise identification of the effect of 5 dwarfing genes or gene combinations on plant height can be carried out after heading; the disease resistance identification of 7 ergot resistance genes or gene combinations can be carried out half a month after the flowering period. The identification method disclosed in the present invention can be used for the investigation of agronomic traits such as spikelet density, sub-spiking length, flag leaf length and width, spike length, number of spikelets, heading period, flowering period, maturity period, frost resistance, drought resistance, grain weight, and single plant yield (according to the "Wheat Variety Regional Trial Recording Items and Standards", NY / T1301-2007) and the precise identification of gene effects.

[0051] (5) Identification and evaluation of gene / QTL locus effect values

[0052] The phenotypic trait data and genotypic data of each experimental strain obtained in step (4) are sorted accordingly, and correlation analysis and statistical analysis are performed to determine the effect values ​​of different genes / QTLs of the same trait on the phenotypic trait. For example, by analyzing the plant height data and corresponding genes / QTLs of 5 dwarf strains, the precise effect values ​​of each gene / QTL on plant height can be obtained; the interactive effect values ​​of multiple gene / QTL combinations on the phenotypic trait can also be determined. For example, the test of the combination of Fusarium head blight resistance genes Fhb, Fhb2, Fhb5 and Fhb1+Fhb2, Fhb1+Fhb5 can identify the effect of a single gene, and can also determine the effect of each gene combination and the interactive effect between genes.

[0053] (6) Identification and creation of molecular breeding elements for wheat

[0054] According to the results of step (4) and step (5), the identification and creation of wheat molecular breeding elements are carried out; the wheat molecular breeding elements are parental materials for preparing hybrid combinations that are known to contain target trait genes / QTLs and their markers, and specifically have three standards: (1) containing target trait QTLs / genes with clear functions; (2) the target trait QTLs / genes have stable and effective molecular markers, and the gene aggregation or transmission can be tracked through molecular markers; (3) having a practical effect on the improvement of yield or target traits.

[0055] In a preferred embodiment, in step (2), the concentric ring planting device includes: an outer ring 1, an inner ring 2 concentrically arranged with the outer ring 1, and a first connecting member 3 for fixing the outer ring 1 and the inner ring 2; the outer ring 1 and the inner ring 2 are both made of galvanized iron material, the diameter of the outer ring 1 is 0.6-1.5m, and the diameter of the inner ring 2 is 0.1-0.3m.

[0056] For example: the diameter of the outer ring 1 is 0.8m (the diameter of the ring can also be adjusted appropriately according to the amount of planting materials), and a small ring with a diameter of 0.2m (the diameter can be increased or decreased when the diameter of the outer ring 1 is large) is designed at the center of the ring. The outer ring 1 and the inner ring 2 are connected by wire welding at the three-divided position of the circumference (the shape is as follows Figure 1 As shown in the figure, a scale (0.10 m) is made on the periphery of the outer circle in equal parts to serve as a reference for the spacing between plants when planting plants containing one or more genetic materials at fixed points.

[0057] In a preferred embodiment, the device also includes a detachable moisturizing frame, which is configured as an equilateral triangular pyramid. The equilateral triangle at the bottom of the equilateral triangular pyramid is circumscribed to the outer ring. The tops of the three prisms 5 of the equilateral triangular pyramid are configured as small circular rings, and the tops of the three prisms are fixed in series by adding a circular ring 6.

[0058] In a preferred embodiment, the height of the equilateral triangle pyramid is set to 1.5, and the lengths of the three prisms are respectively set to 1.7-1.9 m.

[0059] In a preferred embodiment, the additional ring 6 passes through the small rings at the top of the three prisms and is sleeved on the top of the regular triangle pyramid. The diameter of the additional ring 6 is 10 cm.

[0060] In a preferred embodiment, the bottom of each of the prisms 5 is connected by a second connecting member 4 to form the equilateral triangle, so as to fix the prism 5 and make each side of the equilateral triangle circumscribe the outer ring 1.

[0061] The above-mentioned moisturizing frame is set up for experiments such as identification of resistance to ergot disease, which require high air humidity (especially in northern wheat fields) to induce the occurrence of diseases. It is necessary to control the environmental humidity. Therefore, it is realized by modifying the above-mentioned concentric circle planting device. Specifically, on the outside of the outer ring, a transparent plastic shed built with an equilateral triangle pyramid frame is used to maintain the humidity in the microenvironment and promote the occurrence of wheat ergot disease. Specific approach: The three prisms of the equilateral triangle pyramid are made of galvanized iron material, and the equilateral triangle at the bottom is circumscribed to the outer ring (when the diameter of the outer ring is 0.8m, the side length of the equilateral triangle is about 1.4m, such as Figure 2 , Figure 3 As shown), the vertebral body height is 1.5m, and the prism length is 1.7m. For the convenience of disassembly, fixing, and hanging other subsequent devices, a ring with a diameter of 10cm is set at the top of the vertebral body. The tops of the three prisms are small ring-shaped, and the top ring parts of the three prisms are connected to the ring by ring-joining sleeves. The bottom of the prism is connected with a rope (the length of the rope is 1.4m of the side length of the bottom regular triangle), and the outside of the vertebral body is covered with a transparent plastic sheet. If necessary, a humidity sensing control and automatic spraying device is installed at the top position in the plastic shed, or a humidifier with an intelligent mode commonly used in the market is directly selected (when installing and using the humidifier, the humidifier position should be appropriately raised to make it higher than the wheat ear layer, and pay attention to adding water in time), and the humidity in the shed is controlled at about 75% during the wheat heading-flowering period. This additional device for identifying special requirements for ergot disease identification, while maintaining the advantages of the annular planting of the present invention, also meets the special needs of accurate identification of special traits. Precise identification of field agronomic traits and genetic effects of other specific traits may also allow for further improvements in ring planting structures.

[0062] In a preferred embodiment, in step (3), the plant spacing is 20-30 cm; the circular planting is performed by sowing at equal distances, with 2-8 plant material seeds sown in each hole, and one plant is planted in time according to the growth conditions after germination.

[0063] In order to more specifically explain how to implement the technical solution of the present invention, a specific embodiment is used to further illustrate it below.

[0064] Example 1

[0065] 1. Experimental Materials

[0066] Seven new lines containing three major QTL / gene loci for plant height and their combinations (provided by Shandong Agricultural University), namely, lines containing Qph5D.1, Qph4D.1, Qph5D.2, Qph5D.2+Qph5D.1, Qph5D.2+Qph4D.1, Qph5D.1+ Qph4D.1 and Qph5D.2+Qph5D.1+Qph4D.1 (Table 1), were named PHS1, PHS2, PHS3, PHS4, PHS5, PHS6, PHS7, PHS8, and PHS0 (CK, Yumai 57 without three major QTL / genes, provided by Shandong Agricultural University), a total of 8 materials, containing 1 to 3 major QTL / gene loci controlling plant height. Among them, the three main effect loci affecting plant height, Qph5D.2, Qph5D.1 and Qph4D.1, were obtained by Shandong Agricultural University through genetic analysis of plant height in the DH population (Huapei No. 3 X Yumai 57). Their maximum contribution rates and effect values ​​to wheat plant height were 21.97% and 2.413, 12.28% and 5.829, and 20.3% and 4.202, respectively.

[0067] Table 1 Materials containing different gene combinations for plant height

[0068]

[0069] 2. Planting and field management

[0070] This experiment used a circular planting method to further verify the effects of three main loci of plant height with known functions and markers under completely consistent planting conditions, and to identify the interactive effects of different combinations.

[0071] Seven materials (repeated once) were sown at 7 equal points along the planting circle with an outer ring diameter of 1.0m, with 6 seeds sown for each material; Yumai 57 was used as CK and 6 plants were planted at equal points along the outer side of the small circle. Water and fertilizer management was carried out according to conventional methods, and field characteristics of seedlings were investigated and recorded.

[0072] 3. Identification and evaluation of plant height in the field

[0073] The plant height of wheat was investigated after the wheat had fully headed, and the investigation and records were made with reference to the Technical Specifications for Regional Trials of Crop Varieties Wheat (NY / T 1301-2007). Six plants were investigated for each material, and the average value was taken as the plant height of the material (Table 2).

[0074] Table 2 Precise field identification of plant heights of different combinations of materials

[0075]

[0076] It can be seen from Table 2 that as the number of plant height controlling genes / QTLs increases, the plant height decreases. By actually measuring the plant height of each material, it is found that the relative errors with the theoretical plant height values ​​are small. It can be seen that the identification method of the present invention can accurately identify the field phenotypic traits and gene effects of strains containing different plant height genes.

[0077] The three genes / QTLs controlling plant height, PHS1, PHS2 and PHS3, have different effects on plant height, and their combination has positive effect. PHS5, PHS6 and PHS7 lines can be used as molecular breeding elements for creating dwarf stems.

[0078] Example 2

[0079] 1. Experimental Materials

[0080] Materials containing different ergot resistance genes and their different combinations were obtained by transferring Fhb1, Fhb2, Fhb4 and Fhb5 to PH691 (2009 national approved name: Shannong Youmai No. 2, bred by Shandong Agricultural University) provided by Nanjing Agricultural University. With variety PH691 (2009 national approved name: Shannong Youmai No. 2) as the control, a total of 16 materials (Table 3) were used for accurate identification of ergot resistance traits and their gene effects.

[0081] Table 3 Test materials containing different scab resistance genes and their combinations based on PH691

[0082] Test materials Contains different ergot resistance genes NMAS08 PH691-Fhb0 NMAS09 WSB / PH691-Fhb1 NMAS10 WSB / PH691-Fhb2 NMAS11 WSB / PH691-Fhb4 NMAS12 WSB / PH691-Fhb5 NMAS13 PH691-Fhb1+Fhb2 NMAS14 PH691-Fhb4+Fhb5 NMAS15 PH691-Fhb1+Fhb5 NMAS16 PH691-Fhb4+Fhb2 NMAS17 PH691-Fhb1+Fhb4 NMAS18 PH691-Fhb5+Fhb2 NMAS19 PH691-Fhb1+Fhb4+Fhb2 NMAS20 PH691-Fhb1+Fhb5+Fhb2 NMAS21 PH691-Fhb1+Fhb4+Fhb5 NMAS22 PH691-Fhb2+Fhb4+Fhb5 NMAS23 PH691-Fhb1+Fhb4+Fhb5+Fhb2

[0083] 2. Planting and field management

[0084] The planting method of the present invention was used to accurately identify the fusarium resistance traits and gene effects of 16 materials, including wheat materials containing 4 fusarium resistance genes and different combinations thereof from common wheat, plus control varieties, as follows:

[0085] Fifteen materials were sown along 15 equally divided points of a planting circle with an outer diameter of 1.5 m. Six seeds were planted for each material. PH961 was planted in a small circle as CK. Water and fertilizer management was carried out according to conventional methods, and field characteristics of the seedlings were investigated and recorded.

[0086] 3. Soil surface inoculation and resistance identification

[0087] In order to enhance the induction of disease, one month before wheat heading (late March), the diseased wheat grains were evenly spread on the soil surface in the planting ring, with an inoculation rate of 4 kg / 667 m 2 , pay attention to maintaining soil moisture, and refer to the "Technical Regulations for Identification of Resistance to Fusarium Head Blight in Wheat Regional Trial Varieties" (NY / T2954-2016) for the preparation of diseased wheat kernel inoculum. In late April (heading period), a plastic shed supported by a regular triangular cone specially made according to the present invention is used for covering, and a humidifier with intelligent mode (GREESCK-4011) is added in the shed to control the humidity in the shed to about 75% for a total of 21 days. From mid-May, disease investigation and resistance identification will be carried out, and agronomic traits will be investigated in a timely manner, as well as seed testing after wheat matures.

[0088] 4. Identification and evaluation of resistance to fusarium scurf

[0089] When wheat was flowering, 10 different ears were randomly marked. The number of diseased spikelets was investigated 20 days after flowering. The number of diseased spikelets and the location of the diseased spikelets were accurately observed and recorded. The identification and evaluation grading standards were referred to the "Technical Procedures for Identification of Wheat Regional Trial Varieties Resistance to Fusarium Head Blight" (NY / T2954-2016). The wheat fusarium head blight resistance traits of the planting materials were recorded, and the disease resistance traits were analyzed with the disease resistance genes and their gene combinations contained in the materials to determine the fusarium head blight resistance effects of the disease resistance genes and their gene combinations.

[0090] It can be seen from Table 4 that the materials containing different resistance genes have different resistance to ergot. When containing a single gene, the resistance gene effect of the Fhb1 gene was the highest, followed by the Fhb2 gene. Through field resistance identification, their resistance levels were moderate resistance and moderate sensitivity, respectively; when containing two genes, the resistance gene effect of the Fhb1+Fhb5 combination was the highest, followed by the Fhb1+ Fhb2 combination, and the resistance effects increased by 0.57464 and 0.48154, respectively. The field resistance was identified as moderate resistance, and the other combinations were moderately sensitive; when containing three resistance gene combinations, the resistance gene effects of the Fhb1+Fhb2+Fhb4 combination and the Fhb1+Fhb5+Fhb4 combination were the best, and the resistance effects increased by 0.57785 and 0.57464, respectively. The field resistance was identified as moderate resistance, and the other combinations were moderately sensitive; when the material contained four resistance genes, its resistance effect was the highest, 0.82183, and the field resistance was identified as high resistance. It can be seen that the patented method can be used to accurately identify the field phenotypic traits and gene effects containing different resistance gene materials.

[0091] Table 2 Results of the survey on the incidence of planting materials and the identification of resistance

[0092]

[0093]

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for accurately identifying phenotypic traits and gene effects in molecular breeding fields, characterized in that: The concentric ring planting method is adopted, with the original parent planted in the center of the circle and molecular breeding lines with different genes or gene combinations planted around the ring. Under completely consistent experimental conditions, precise investigation of agronomic traits and yield traits is conducted to obtain precise identification results of single gene effects or multi-gene aggregation effects. The method specifically comprises the following steps: (1) Selection of planting materials Select planting materials with clear target gene composition obtained through conventional breeding or molecular breeding; (2) Making a concentric ring planting device The device for the concentric ring planting is made of galvanized iron material, and scales for fixed-point planting of the planting material are marked on the outer and inner rings of the concentric circles; (3) Concentric ring planting Carry out circular planting according to the device manufactured in step (2), wherein the parent material or the control material is planted along the inner ring wall according to the scale, and the planting material is planted along the outer ring wall according to the scale, and the spacing between plants is adjusted according to the amount of the planting material; (4) Identification and evaluation of agronomic traits According to the type of planting material planted in the ring in step (3) and the function of the target gene, the identification period and standard of agronomic traits and their gene effects are established; (5) Identification and evaluation of gene / QTL locus effect values The genetic data obtained by gene / QTL site effect value identification are collated with the phenotypic trait data of each experimental strain obtained in step (4), and correlation analysis and path analysis are performed to determine the effect values ​​of different genes / QTLs of the same trait on the phenotypic trait; (6) Identification and creation of molecular breeding elements for wheat According to the results of step (4) and step (5), a strain containing a known gene / QTL locus composition and having a significant contribution rate to the phenotypic trait is selected as a wheat molecular breeding element; the wheat molecular breeding element is a parent material for preparing a hybrid combination that is known to contain a target trait gene / QTL and its marker and has a large contribution rate to the phenotypic trait; In step (2), the concentric ring planting device comprises: an outer ring, an inner ring arranged concentrically with the outer ring, and a first connecting member fixing the outer ring and the inner ring; the outer ring and the inner ring are both made of galvanized iron material, the diameter of the outer ring is 0.6-1.5m, and the diameter of the inner ring is 0.1-0.3m; The device also includes a detachable moisturizing frame covering the circular planting material, the moisturizing frame is configured as a regular triangular pyramid, the equilateral triangle at the bottom of the regular triangular pyramid is circumscribed to the outer ring, the top ends of the three prisms of the regular triangular pyramid are configured as small circular rings, and the top circular ring parts of the three prisms are connected in series by an additional circular ring for easy installation and storage.

2. The method according to claim 1, characterized in that The height of the equilateral triangle pyramid is set to 1.5, and the lengths of the three prisms are set to 1.7-1.9m respectively.

3. The method according to claim 1, characterized in that The additional circular ring passes through the small circular rings at the top of the three prisms and is sleeved on the top of the regular triangle pyramid. The diameter of the additional circular ring is 10 cm.

4. The method according to claim 1, characterized in that The bottom of each of the prisms is connected by a second connecting member to form the equilateral triangle, so as to fix the prism and make each side of the equilateral triangle circumscribe the outer ring.

5. The method according to claim 1, characterized in that In step (3), the plant spacing is 20-30 cm; the circular planting is performed by sowing at equal distances, with 2-8 plant material seeds being sown in each hole, and one plant being planted in time according to the growth conditions after germination.

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