阅读说明：本技术 利用标记辅助选择将小麦抗条锈性qtl与优良农艺性状结合的方法 (Method for combining wheat stripe rust resistance QT L with excellent agronomic traits by marker-assisted selection ) 是由 周新力 胡天 钟晓 杨随庄 于 2020-04-24 设计创作，主要内容包括：本发明公开了利用标记辅助选择将小麦抗条锈性QTL与优良农艺性状结合的方法,具体包括以下步骤：S1：确定杂交组合；S2：常规育种；S3：基因组DNA提取；S4：分子标记辅助选择；S5：抗病性鉴定；S6：产量相关农艺性状的调查；S7：成功筛选出具有高抗性和优良农艺性状的小麦品系,对分子标记辅助育种利用QYr.nafu-2BL和QYr.nafu-3BS抗病基因资源创制新型抗病材料防控小麦条锈病具有重要意义；另外,本实验获得的未检测出含有抗性QTL但对条锈病表现高抗且农艺性状良好的小麦品系也具有进一步研究运用的价值。(The invention discloses a method for combining wheat stripe rust resistance QT L with excellent agronomic characters by marker-assisted selection, which specifically comprises the following steps of S1, determining hybridization combination, S2, conventional breeding, S3, extracting genomic DNA, S4, performing molecular marker-assisted selection, S5, identifying disease resistance, S6, investigating yield-related agronomic characters, S7, successfully screening out wheat strains with high resistance and excellent agronomic characters, and developing a novel disease-resistant material for preventing and controlling wheat stripe rust by using QYr.nafu-2B L and QYr.nafu-3BS disease-resistant gene resources in molecular marker-assisted breeding, wherein the wheat strains which do not detect the existence of QT L, have high resistance to stripe rust and have good agronomic characters and further research and application values.)

1. A method for combining wheat stripe rust resistance QT L with excellent agronomic traits by using marker-assisted selection is characterized by comprising the following steps:

s1: determining the hybridization combination: hybridizing P9897 with Sichuan wheat 42, Ximai 25 and Zhengmai 9023 respectively, using P9897 as a male parent, Sichuan wheat 42, Ximai 25 and Zhengmai 9023 as female parents to obtain F1, and sowing in a field;

s2: conventional breeding: selection was performed by mixing before the F4 generation, retaining all genotypes, reducing the drift loss of good genes that are still highly heterozygous to F₄Selecting by pedigree method for manual screening of single plants with moderate plant height, more tillering and spikelet number and full seeds to obtain F₅Plant generation;

s3: extracting genome DNA: take 114F₅Of ancestor line and of each individual plant of four parentsExtracting genome DNA from leaves;

s4 molecular marker assisted selection F of three hybridization combinations using molecular markers linked to the QYr.nafu-2B L and QYr.nafu-3BS QT L sites in P9897₅Screening generation and offspring families, and carrying out QT L detection;

s5: and (3) identifying disease resistance: 114F of three hybridization combinations₅Family group is planted in the oil experimental field, reaction type and maximum severity are selected as disease resistance analysis indexes for F5 family group, and disease resistance identification is carried out when the disease severity of M169 reaches 50% and 90%, so as to obtain the identification result of the experiment;

s6: investigation of yield-related agronomic traits: for 114F₅Manually investigating and recording the plant height, tillering number and spikelet number of the family and the parent, and weighing thousand-grain weight after harvesting;

s7, successfully screening the wheat strain with high resistance and excellent agronomic traits by utilizing comprehensive evaluation of the field agronomic traits and molecular detection results of the resistance QT L.

2. The method for combining wheat stripe rust resistance QT L with excellent agronomic traits through marker assisted selection according to claim 1, characterized in that in step S4, QT L is detected by using the molecular markers Xbarc160, Xcfd73, Xgwm120, Xbarc87 and Xbarc133 linked to the QYR. nafu-2B L and QYR. nafu-3BSQT L sites for the 114F hybrid combinations of five markers including Xbarc160, Xcfd73, Xgwm120, Xbarc87 and Xbarc133₅PCR amplification and electrophoresis are carried out on the ancestral line and the four parents.

3. The method for combining the wheat stripe rust resistance QT L with the excellent agronomic traits through marker-assisted selection according to claim 1, characterized in that in step S7, the comprehensive evaluation of the field agronomic traits is that offspring families with plant height of 80-100 cm, tillering number not less than 4, spikelet number not less than 17 and thousand kernel weight not less than 42g are screened out by combining the plant height, tillering number, spikelet number and thousand kernel weight data of each parent.

Technical Field

The invention relates to the technical field of crop breeding, in particular to a method for combining wheat stripe rust resistance QT L with excellent agronomic traits by using marker-assisted selection.

Background

The development of resistant varieties, the control of stripe rust by using disease-resistant genes is the most economical and effective, and the environment-friendly mode for preventing and controlling stripe rust (Roebbelen and Sharp 1978; L origin Chen1995) with the continuous increase of abundance of molecular markers in wheat genomes, molecular Marker-assisted selection (Marker-assisted selection) can greatly improve the efficiency in breeding and reduce the consumption of manpower and material resources by using the molecular markers, the research work of wheat MAS has gained abundant results in recent years and has revolutionary influence on molecular breeding, MAS is one of the most ideal breeding modes (Yoon et al 2015), SSR, RGA, SNP, etc. due to short time consumption, high efficiency and high accuracy of target trait screening at present, MAS is widely used for positioning important trait genes (Yoon et al 2015), the molecular markers linked with the target genes are helpful for accurately screening individuals with the target genes, the accelerated success of the target gene breeding process, the selection of multiple varieties with the target genes is successful and the genetic selection of Sishen, particularly the genetic resistance of Siherr in traditional breeding methods (Cheynia).

The resistance source of China is single, and Yr26 genes are used in various places, so that the new small species CYR34 with toxicity is gradually increased in recent years, and the threat to wheat production is formed. Therefore, the method can make up the current situation that the wheat stripe rust resistance gene in China is single and seriously deficient at present by digging and developing excellent stripe rust resistance genes (such as P9897) and applying the genes to wheat breeding for disease resistance, and has very important theoretical and practical significance for sustainable control of the wheat stripe rust.

Based on the above, the invention designs a method for combining the stripe rust resistance QT L of the wheat with excellent agronomic traits by marker-assisted selection, and combines the traditional breeding with the MAS, thereby saving the consumption of time, manpower and material resources, improving the breeding efficiency, and reducing the cost for carrying out molecular experiments.

Disclosure of Invention

The invention aims to provide a method for combining wheat stripe rust resistance QT L with excellent agronomic traits by using marker-assisted selection so as to solve the problems in the background technology.

In order to achieve the aim, the invention provides the following technical scheme that the method for combining the stripe rust resistance QT L of the wheat with the excellent agronomic traits by using marker-assisted selection specifically comprises the following steps:

s1: determining the hybridization combination: hybridizing P9897 with Sichuan wheat 42, Ximai 25 and Zhengmai 9023 respectively, using P9897 as a male parent, Sichuan wheat 42, Ximai 25 and Zhengmai 9023 as female parents to obtain F1, and sowing in a field;

s2: conventional breeding: selection was performed by mixing before the F4 generation, retaining all genotypes, reducing the drift loss of good genes that are still highly heterozygous to F₄Selecting by pedigree method for manual screening of single plants with moderate plant height, more tillering and spikelet number and full seeds to obtain F₅Plant generation;

s3: extracting genome DNA: take 114F₅Extracting genome DNA from leaves of each individual plant of the generation family and the four parents by adopting a 2 × CTAB method;

s4 molecular marker assisted selection F of three hybrid combinations using molecular markers linked to the QYr. nafu-2B L and QYr. nafu-3BSQT L sites in P9897₅Screening generation and offspring families, and carrying out QT L detection;

s5: and (3) identifying disease resistance: 114F of three hybridization combinations₅Family group is planted in the oil experimental field, reaction type and maximum severity are selected as disease resistance analysis indexes for F5 family group, and disease resistance identification is carried out when the disease severity of M169 reaches 50% and 90%, so as to obtain the identification result of the experiment;

s6: investigation of yield-related agronomic traits: for 114F₅The plant height, tillering number and spikelet number of the family and the parent are manually investigated and recorded, and the thousand seed weight is weighed after harvesting.

S7, successfully screening the wheat strain with high resistance and excellent agronomic traits by utilizing comprehensive evaluation of the field agronomic traits and molecular detection results of the resistance QT L.

Preferably, in the step S4, the QT L detection is specifically performed by using 114F of three hybridization combinations of five markers including Xbarc160, Xcfd73, Xgwm120, Xbarc87 and Xbarc133, which are molecular markers linked to the QT L sites of QYr. nafu-2B L and QYr.nafu-3BS QT L₅PCR amplification and electrophoresis are carried out on the ancestral line and the four parents.

Preferably, in step S7, the comprehensive assessment of the field agronomic traits is: and (3) screening out offspring families with plant height of 80-100 cm, tillering number of more than or equal to 4, spikelet number of more than or equal to 17 and thousand kernel weight of more than or equal to 42g by combining the plant height, tillering number, spikelet number and thousand kernel weight data of each parent.

Compared with the prior art, the wheat strain with high resistance to stripe rust and excellent agronomic characters has important significance for molecular marker assisted breeding and creation of novel disease-resistant materials for preventing and controlling the stripe rust by using QYr.nafu-2B L and QYr.nafu-3BS disease-resistant gene resources, and in addition, the wheat strain which is not detected to contain the resistance QT L, but shows high resistance to stripe rust and excellent agronomic characters and is obtained in the experiment also has value of further research and application.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of the present Infection Type (IT) distribution;

FIG. 2 is a distribution of Disease Severity (DS) according to the present invention;

FIG. 3 is a histogram of plant height according to the present invention;

FIG. 4 is a histogram of spikelets according to the present invention;

FIG. 5 is a histogram of tillering number of the present invention;

FIG. 6 is a graph showing a frequency distribution of thousand kernel weight of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme of a method for combining wheat stripe rust resistance QT L with excellent agronomic traits by using marker-assisted selection, which specifically comprises the following steps:

s1: determining the hybridization combination: hybridizing P9897 with Sichuan wheat 42, Ximai 25 and Zhengmai 9023 respectively, using P9897 as a male parent, Sichuan wheat 42, Ximai 25 and Zhengmai 9023 as female parents to obtain F1, and sowing in a field;

s2: conventional breeding: selection was performed by mixing before the F4 generation, retaining all genotypes, reducing the drift loss of good genes that are still highly heterozygous to F₄Selecting by pedigree method for manual screening of single plants with moderate plant height, more tillering and spikelet number and full seeds to obtain F₅Plant generation; by adopting the method to carry out manual screening and elimination, the obtained F₅The characters of the generation family tend to be stable;

s3: extracting genome DNA: take 114F₅Extracting genome DNA from leaves of each individual plant of the generation family and the four parents, and extracting the genome DNA by adopting a 2 × CTAB method;

s4 molecular marker assisted selection F of three hybrid combinations using molecular markers linked to the QYr. nafu-2B L and QYr. nafu-3BSQT L sites in P9897₅Selecting generation offspring families to obtain the families with single QT L and two QT L, and carrying out QT L detection;

the QT L detection method specifically comprises the steps of using the QYr.nafu-2B L and QYr.nafu-Molecular markers linked to the 3BSQT L site Xbarc160, Xcfd73, Xgwm120, Xbarc87 and Xbarc133, five markers for 114F of three hybrid combinations₅PCR amplification and electrophoresis are carried out on the ancestral line and the four parents.

The progeny of the three crosses were classified according to the results of the QT L test into four categories, i.e., those containing QYr.nafu-2B L, those containing QYr.nafu-3BS, those containing the above two QTs L0, and those not containing QT L1. among the F5 families of Chuanmai 42/P9897, 4 families containing QYr.nafu-2B L, 3 families containing QYr.nafu-3BS, 7 families containing the two QTs L, 5 families containing no QT L, among the F5 families of Chuanmai 25/P9897, 25 families containing QYr.nafu-2B L, 9 families containing QYr.nafu-3BS, 15 families containing the two QTs L, 13 families containing no QT L, among the F5 families of Zheng 23/P9897, those containing QYr.nafu-3BS, 15 families containing the two QT L, 13 families containing no QT 3B, and 3, 7 families containing at least QT 469, 3, 7 families containing the QT 3, 7, 3, 7 containing the same QT 3, and7 containing the same QT 3, and7 containing the same QT 2, and the same.

TABLE 1 summary of F5 generation pedigrees in the absence of three crossing combinations in QT L, 2BQT L, 3BQT L, 2B +3BQT L

Hybrid combinations	QT-free L	2BQTLa	3BQTLb	2B+3BQTL	QT L Total
						Chuanmai 42/P9897	6	6	3	4	13
Xiangmai 25/P9897	24	22	6	10	38
						Zheng wheat 9023/P9897	14	9	9	1	19

Therein, 2BQT L^aRepresents QYr. nafu-2B L, 3BQT L^bRepresents QYr. nafu-3 BS.

S5: and (3) identifying disease resistance: planting 114F 5 family groups of the three hybridization combinations in a JiangYOU experimental field, selecting a reaction type and the maximum severity as disease resistance analysis indexes for the F5 family group, and identifying the disease resistance when the severity of M169 reaches 50% and 90%, so as to obtain the identification result of the experiment;

according to the identification result of the field test, the rust streak fungus fully attacks in all environments. All 114F 5 lines showed different degrees of disease resistance, while P9897 showed high resistance (IT 2, DS 5%), and chuan wheat 42, xiang wheat 25, zheng wheat 9023 showed moderate resistance (IT 6, 5, DS 40%, 30%). The offspring families of Sichuan wheat 42/P9897, Xiangmai 25/P9897 and Zhengmai 9023/P9897 mainly show high resistance, and respectively account for 89.5%, 88.7% and 93.9%; 5.3%, 6.5%, 3% of the families showed resistance and 5.3%, 4.8%, 3% showed immunity, respectively, and none of the three cross combinations showed highly susceptible families, as shown in FIG. 1-2 for the distribution plots of Infection Type (IT) and Disease Severity (DS), data for 114F 5 lines of 3 hybrids at different stages of adult worm growth, and Table 2.

Table 2: summary of infection types of F5 lines with three cross combinations of immunity, resistance, moderate infection, and susceptibility to infection

S6: investigation of yield-related agronomic traits: the plant heights, tillering numbers and spikelet numbers of 114F 5 families and parents were manually investigated and recorded, and the thousand-grain weights were weighed after harvesting.

S7, successfully screening out a wheat line with high resistance and excellent agronomic traits by utilizing comprehensive evaluation of the agronomic traits in the field and molecular detection results of the resistance QT L, wherein the comprehensive evaluation of the agronomic traits in the field is to screen out descendant families with the plant height of 80-100 cm, the tillering number of more than or equal to 4, the spikelet number of more than or equal to 17 and the thousand kernel weight of more than or equal to 42g by combining with the plant height, tillering number, spike number and thousand kernel weight of each parent, and to screen out 30F 5 families in total by combining with the result of QT L detection (namely at least containing one resistance L in P9897), wherein 6 families are screened out by combining Sichuan wheat 42/P9897, 20 families are screened out by combining Sichuan wheat 25/P9897, 4 families are screened out by combining Zheng wheat 9023/P9897, and as shown in FIGS. 3-6, 114F 5 strains of 3-3 hybrid combinations are distributed in the data of mature period, 3 strains (A, B, C and 5 varieties of wheat varieties with thousand kernel weight and 5.

Table 3: a summary of the detailed information of the male parent row and the selected row,

in 12 families of Sichuan wheat 42/P9897-3, Sichuan wheat 25/P9897-25, Sichuan wheat 25/P9897-26, Sichuan wheat 25/P9897-27, Sichuan wheat 25/P9897-39, Sichuan wheat 25/P9897-40, Sichuan wheat 25/P9897-56, Sichuan wheat 25/P9897-59, Sichuan wheat 25/P9897-60, Sichuan wheat 25/P9897-78, Sichuan wheat 25/P9897-92 and Sichuan wheat 9023/9897-105, QYr. nafu-2B L and QYr. nafu-3BS L resistant sites are provided at the same time under the condition of meeting the above agronomic characters and disease resistance standards, and have good application prospects.

At present, the Sichuan wheat 42 is known to contain Yr26 disease-resistant gene, and molecular markers on both sides of the Sichuan wheat 42/P9897F5 generation family are detected, so that 63.2% of the family contains Yr 26. The results are combined, and the five families of Sichuan wheat 42/P9897-3, Sichuan wheat 42/P9897-11, Sichuan wheat 42/P9897-12, Sichuan wheat 42/P9897-15 and Sichuan wheat 42/P9897-19 are found to contain Yr26 disease-resistant genes under the condition of meeting the standards, so the gene has good application prospect.

The specific working principle is as follows:

the research combines conventional breeding with MAS, performs mixed method selection on three hybrid combinations before F4 generation, then starts pedigree method selection for manual screening in F4 generation, and then detects resistance QT L in F5 family by using molecular markers, and successfully screens 30 wheat lines with high resistance and excellent agronomic characters to wheat stripe rust by using comprehensive evaluation of the field agronomic characters and molecular detection results of the resistance QT L.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.