Method for constructing female parent group and male parent group suitable for Huang-Huai-Hai region and application of female parent group and male parent group

Abstract

The invention belongs to the field of corn genetic breeding, and discloses a method for constructing a female parent group and a male parent group suitable for the Huang-Huai-Hai region and application of the female parent group and the male parent group. By collecting 201 parts of germplasm resources at home and abroad and combining genetic typing and field phenotype screening, germplasm is divided into 8 class groups, female parent materials are selected from a PA group and an SS group, and male parent materials are selected from a four-flat-head group and an NSS group. A single-cross hybrid and a four-cross hybrid are formed through pairwise hybridization, and a population with rich genetic diversity is obtained through two rounds of powder mixing balance treatment. Group structure analysis shows that the plant height, the ear height, the ear length and the ear thickness all accord with normal distribution (Pgt; 0.05), which shows that the group structure is reasonable. According to the invention, the genetic basis of Huang-Huai-Hai corn breeding can be effectively broadened, and germplasm support is provided for breeding new varieties which are lodging-resistant, close-planting-resistant and suitable for mechanical harvesting.

Description

Technical Field

[0001] The invention belongs to the field of corn genetic breeding and discloses a method for constructing a maternal group and a paternal group adapted to the Huanghuaihai region and an application thereof. Background Art

[0002] The Huanghuaihai summer corn region is a key corn production area in my country, with an annual corn planting area exceeding 12 million hectares, and a growing trend. Corn germplasm resources are the foundation of corn breeding, and the Huanghuaihai region boasts abundant resources, primarily including the Tangsipingtou and P groups. The Tangsipingtou hybrid group is a unique local germplasm in my country and one of the four classic hybrid groups, holding a prominent position in corn breeding. The inbred line LX9801, developed by the Maize Research Institute of the Shandong Academy of Agricultural Sciences, is a key Tangsipingtou inbred line. Over 30 corn hybrids have been approved and over 100 derived inbred lines have been derived from it. In 2020, it was recognized as a National Outstanding Contribution Inbred Line. The P group inbred lines possess a rich genetic base, strong resistance to biotic and abiotic stresses, and contain numerous beneficial genes required by breeders, making them another important germplasm resource in the Huanghuaihai region. Qi 319, a P-group inbred line bred by the Shandong Academy of Agricultural Sciences, is a recognized source of resistance in my country. Its improved lines have resulted in over 30 inbred lines, and it was recognized as a National Outstanding Contribution Inbred Line in 2020. The Shandong Academy of Agricultural Sciences also developed the corn variety Ludan 981, a hybrid of the Qi 319 and LX9801 lines, which won the Second Prize of the National Science and Technology Progress Award in 2009. The breeding of Ludan 981 has led to the development of the "Tangsi Pingtou × PB" hybrid vigor model.

[0003] However, with my country's rapid economic development, the degree of mechanization in agricultural production and the composition of the rural labor force have shifted, and the breeding objectives for new corn varieties are adapting to these changing circumstances. Beyond high quality and high yield, breeders must prioritize numerous traits, such as lodging resistance, tolerance to densely packed conditions, good stay-green properties, rapid grain filling for high quality, and a reduced, loose, mature bract suitable for mechanized harvesting. However, domestic corn germplasm resources are relatively lacking in genes for these specific requirements, necessitating the urgent need to import and improve targeted germplasm from abroad.

[0004] The Huanghuaihai summer corn region, a key production area in my country, currently has shortcomings such as poor lodging resistance, slow grain filling, and insufficient adaptability to mechanized harvesting. This requires the introduction of European and American germplasm for improvement. While existing technologies, such as Ludan 981 (Qi 319 × lx9801), have made progress, the narrow genetic base remains unresolved. Summary of the Invention

[0005] The Huanghuaihai summer corn region is an important corn producing area in my country, but the existing main varieties have problems such as narrow genetic basis, weak lodging resistance, poor tolerance to dense planting, and short green period. Traditional breeding methods rely on hybridization of single-group inbred lines (such as hybridization of Tangsi Pingtou group and P group), resulting in insufficient genetic diversity of germplasm. At the same time, it is difficult to integrate germplasm introduced from Europe and the United States with local germplasm, and it is difficult to construct an excellent group adapted to mechanized harvesting. The present invention aims to integrate domestic and foreign germplasm resources, establish a scientific classification system, and construct a maternal group and a paternal group with a broad genetic basis and complementary traits to break through the existing breeding bottleneck.

[0006] In order to solve the above technical deficiencies, the present invention provides a method for constructing a maternal population and a paternal population adapted to the Huanghuaihai region and its application.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention utilizes population improvement technology to amplify, improve and innovate corn germplasm resources, effectively breaking unfavorable genetic linkage and increasing the frequency of favorable genotypes.

[0009] Focusing on the breeding goals of "high and stable yield, disease resistance and lodging resistance, green and efficient", we integrate the inbred lines of the parents of approved varieties in the Huanghuaihai region with the introduction of tropical germplasm from Europe, America and Indonesia. All germplasms are genotyped and pedigree analyzed, and divided into maternal inbred lines and paternal inbred lines. Based on field performance, the synthetic maternal population and paternal basic materials are determined; single-cross varieties are formed by mutual hybridization, and further hybridization is carried out to form four-cross varieties. After two rounds of mixed pollen balance, maternal and paternal populations with a broad genetic basis adapted to the Huanghuaihai region are formed. By constructing these two major populations, the foundation for inbred line selection is broadened.

[0010] Follow these steps:

[0011] 1. Germplasm resource integration and classification

[0012] (1) Germplasm collection: 201 corn germplasm resources were collected, including:

[0013] Inbred lines of the parents of approved varieties in the Huanghuaihai region (e.g. Zhengdan 958’s parent Zheng 58, Xianyu 335’s parent PH6WC, etc.);

[0014] European and American medium-quality germplasm (Pioneer SS group PH6WC, Monsanto NSS group D1798Z, etc.);

[0015] Indonesian tropical germplasm (heat-resistant and insect-resistant resources).

[0016] (2) Genotyping and cluster analysis:

[0017] TASSEL5.0 software was used to perform genome-wide SNP typing of the germplasm;

[0018] System clustering was performed using the standard inbred line as a reference:

[0019] Domestic germplasm: Huangzaosi (Tangsipingtou group), Ye 478 (PA group), Qi 319 (PB group);

[0020] International germplasm: PH6WC (Pioneer SS group), PH4CV (Pioneer NSS group), HCL645 (Monsanto SS group), D1798Z (Monsanto NSS group).

[0021] The germplasm was divided into 8 groups: Tangsipingtou group, PA group, PB group, Pioneer SS group, Monsanto SS group, Pioneer NSS group, Monsanto NSS group and other groups.

[0022] 2. Screening of maternal and paternal population construction materials

[0023] (1) Maternal population: 23 inbred lines were selected from the PA group, Pioneer SS group, and Monsanto SS group, highlighting the characteristics of tolerance to dense planting and rapid grain filling;

[0024] (2) Male parent group: 26 inbred lines were selected from the Tang Sipingtou group, Pioneer NSS group, Monsanto NSS group, and PB group to enhance the advantages of lodging resistance and long green period.

[0025] 3. Hybridization and genetic balance treatment

[0026] (1) Preparation of single-cross and quadruple-cross varieties:

[0027] Cross the inbred lines of the maternal population with those of the paternal population to produce single-cross varieties;

[0028] Single-hybrids are further hybridized to form quadruple-hybrids, expanding the scope of genetic recombination.

[0029] (2) Two rounds of powder mixing balance treatment:

[0030] The first round of pollination: four hybrids are mixed in equal amounts as the male parent, and the female parent is emasculated and naturally pollinated (isolated area of Hainan South Breeding Base);

[0031] Second round of pollen mixing: sow 2,000 plants with seeds from the first round of pollen mixing, and mix all the seeds from the ears after natural pollination to eliminate genetic drift and ensure genetic balance of the population.

[0032] The constructed maternal and paternal populations can be used in breeding excellent maize inbred lines with lodging resistance, dense planting tolerance and staying green properties.

[0033] The beneficial effects of the present invention are:

[0034] 1. Increased genetic diversity:

[0035] The coefficient of variation of plant height in the maternal population was 8.67%, and the coefficient of variation of ear length was 18.75%;

[0036] The coefficient of variation of plant height in the male parent population was 9.79%, and the coefficient of variation of ear height was 17.04%;

[0037] The Kolmogorov-Smirnov (KS) test showed that the population traits were normally distributed (P>0.05) and the genetic structure was reasonable.

[0038] 2. Target trait enhancement:

[0039] The maternal population integrates the fast-filling characteristics of the PA group (Ye 478) and the advantages of the Pioneer SS group (PH6WC) in tolerance to dense planting;

[0040] The male parent group combines the lodging resistance of the Tangsi Pingtou group (Huang Zao Si) and the green staying advantage of the Monsanto NSS group (D1798Z).

[0041] 3. Breeding application value:

[0042] By hybridizing the maternal and paternal groups, we can efficiently select excellent inbred lines that are resistant to lodging, tolerant to dense planting (planting density ≥ 67,500 plants / hectare), and have a green period extended by more than 15%, which can meet the needs of mechanized harvesting in the Huanghuai and Haihe regions.

[0043] 4. Innovation in the germplasm classification system: For the first time, the local germplasm of the Huanghuaihai region and the European, American and tropical germplasm are uniformly divided into 8 groups, clarifying the genetic contribution direction of each group.

[0044] 5. Breakthrough in pollination balance technology: Through two rounds of natural pollination, pollination balance can solve the problem of genetic linkage drag caused by multi-parent hybridization and achieve population genetic balance.

[0045] 6. Improved breeding efficiency: Directed complementarity between maternal and paternal populations shortens trait screening cycle by more than 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a technical route constructed by two major groups.

[0047] Figure 2 This is the result of cluster analysis of maize germplasm resources.

[0048] Figure 3 It is the normal distribution diagram of agronomic traits of maternal population.

[0049] Figure 4 It is the normal distribution diagram of agronomic traits of the paternal population. DETAILED DESCRIPTION

[0050] Example 1 Method for constructing maternal and paternal populations adapted to the Huanghuaihai region and its verification

[0051] 1. Materials and Methods

[0052] 1.1 Cluster analysis of maize germplasm resources and screening of population basic materials

[0053] In 2022, 201 maize germplasm accessions were collected from breeding units within and outside the province and planted at the Nanbin Experimental Base in Hainan Province in the winter of 2022. DNA was extracted from leaves of five plants from each inbred line and sent to Huazhong Agricultural University for genome sequencing analysis. Among these accessions, Ye 478, Huang Zao Si, PH6WC, PH4CV, Qi 319, HCL645, and D1798Z were used as standard inbred lines. Huang Zao Si, Ye 478, and Qi 319 represent the Tangsi Pingtou, PA, and PB groups of domestic germplasm, respectively. PH6WC and PH4CV represent the Pioneer SS and NSS groups, respectively. HCL645 and D1798Z represent the Monsanto SS and NSS groups, respectively. Cluster analysis of these inbred lines was performed using TASSEL 5.0 software. The basal material for the maternal group was selected from inbred lines of the PA and SS groups, while the basal material for the paternal group was selected from inbred lines of the Tangsi Pingtou, NSS, and PB groups.

[0054] 1.2 Construction of two major corn groups

[0055] The two major corn groups include maternal groups and paternal groups, and the construction method is the same. The first step is to plant all the collected corn germplasm resources in the Nanbin Experimental Base in Hainan in the winter of 2022, and select the basic materials for the construction of maternal and paternal groups based on gene grouping and field performance. The second step is to hybridize the basic materials of each group with each other to form single-cross F1. The third step is to plant single-cross F1 in Jinan in the summer of 2023, plant one row of each single-cross, and hybridize two by two to form four-cross. The fourth step is to set up seed production isolation areas for maternal and paternal groups in Nanbin, Hainan and Huangliu respectively in October 2023, mix all four-cross seeds in the group in equal amounts as the male parent, and all four-cross seeds as the female parent. The female parent is castrated and naturally pollinated, and then the seeds after hybridization of all four-cross seeds are mixed in equal amounts to form the first round of group seeds. In January 2024, isolation areas for maternal and paternal groups were set up in Nanbin and Huangliu, Hainan respectively, with 2,000 plants planted in each area for natural pollination. Then, the seeds of all the fruit clusters were mixed in equal amounts to form the second round of group seeds.

[0056] 1.3 Analysis of the genetic structure of the two populations

[0057] In the summer of 2024, 500 maternal and paternal plants were planted at the Zhangqiu Experimental Base in Jinan. Four traits (plant height, ear height, ear length, and ear diameter) were investigated. SPSS software was used to plot and calculate the frequency distribution of each trait, and a normal distribution test was performed.

[0058] 2. Results and Analysis

[0059] 2.1 Cluster analysis of maize germplasm resources and screening results of inbred lines for population establishment

[0060] Based on the sequencing results and field phenotypic results, the 201 germplasms were divided into 8 categories according to the standard inbred lines, including 31 inbred lines from the domestic germplasm Tang Sipingtou group, 19 inbred lines from the PA group, and 26 inbred lines from the PB group; 27 inbred lines from the foreign germplasm Pioneer SS group, 17 inbred lines from the Monsanto SS group, 34 inbred lines from the Pioneer NSS group, and 28 inbred lines from the Monsanto NSS group; the remaining 19 inbred lines belonged to other groups (such as Figure 2 ). Among these hybrid advantage groups, the Monsanto NSS group inbred lines have the largest number, accounting for 18.91%. Due to the excellent characteristics of Monsanto NSS group inbred lines such as early maturity and suitability for mechanical harvesting, they are widely used in corn breeding in the Huanghuaihai region. The second is the Tangsipingtou group inbred lines, accounting for 15.42%. This shows that in the Huanghuaihai region, the application of Huanggai germplasm still occupies an important position. Under the condition of severe high temperature and heat damage in the Huanghuaihai region, Huanggai has the advantages of high general combining ability, high temperature and drought resistance, high density resistance, well-developed male spikes, large pollen quantity, and wide adaptability. Therefore, it is widely used by Huanghuaihai breeders.

[0061] Based on pedigree analysis and phenotypic cluster analysis, 23 inbred lines from the PA and SS groups were selected for maternal parent population construction (Table 1). These included 11 PA inbred lines, 8 Pioneer SS inbred lines, and 4 Monsanto SS inbred lines. For paternal parent population construction, 26 inbred lines were selected from the Tangsipingtou, NSS, and PB inbred lines (Table 2). These included 9 Tangsipingtou inbred lines, 5 Monsanto NSS inbred lines, 10 Pioneer NSS inbred lines, 2 PB inbred lines, and 2 other inbred lines. Most of these selected inbred lines were parental inbred lines of varieties approved after 2010.

[0062] Table 1 Inbred lines used for maternal group construction

[0063]

[0064]

[0065] Table 2 Inbred lines used for paternal group construction

[0066] Serial number Inbred line Group Typical hybrids and their certification numbers 1 KW4M029 Monsanto NSS KW3564 (New Examination No. 41, 2006) 2 F0147Z Monsanto NSS Deka 159 (Jishenyu 2015020) 3 HCL645 Monsanto NSS Decca 517 (National Examination Jade 20170005) 4 9F592 Monsanto NSS Demeya No. 3 (Jishenyu 2013001) 5 G4675Z Monsanto NSS Dika 653 (Yushenyu 201501) 6 PHB1M Pioneer NSS Xianyu 696 (National Approved Jade 2006025) 7 PH48T0 Pioneer NSS Xianyu 1729 (Liao Shenyu 20200010) 8 PHRKB Pioneer NSS Xianyu 047 (Lu Nong Shen No. 2014004) 9 PH5AD Pioneer NSS Xianyu 508 (National Approved Jade 2006043) 10 PH4CV Pioneer NSS Xianyu 335 (National Jade 2004017) 11 PH1T8W Pioneer NSS Xianyu 1466 (Hebei Shenyu 20170052) 12 PHF1J Pioneer NSS Xian 1219 (Mengshenyu 2015004) 13 DH382 Pioneer NSS Denghai 605 (National Examination No. 2010009) 14 Qi system 5102 Pioneer NSS Ludan 510 (Lu Shenyu 20210013) 15 VK22-4 Pioneer NSS Woyu 3 (Jin Shenyu 2013013) 16 lx03-2 Huang Gai Ludan 9088 (National Examination Jade 2012015) etc. 17 Beijing 2416 Huang Gai Jinghua No. 8 (Mengshenyu No. 2010031), etc. 18 Chang 7-2 Huang Gai Zhengdan 958 (National Examination Yu 20000009) etc. 19 L292 Huang Gai Liyu 37 (National Jade 2010010) 20 BA702 Huang Gai Agricultural University 372 (National Examination Jade 2015014) 21 XB1621 Huang Gai Dongdan 1331 (National Examination Jade 2016607) 22 Qi system 5232 Huang Gai Ludan 523 (Lu Shenyu 2023006) 23 M5972 PB Liangyu 99 (National Jade 2012008) 24 Qi 319 PB Ludan 981 (National Approved No. 2003011) 25 KW7M010 other KWS1568 (New Examination No. 031, 2004) 26 QR273 other Jinyu 506 (National Approved Jade 2013012)

[0067] 2.2 Genetic structure analysis results of the two populations

[0068] 2.2.1 Analysis of the genetic structure of the maternal population

[0069] like Figure 3As shown in Table 3, plant height within the maternal population varied from 179 cm to 300 cm, with a coefficient of variation of 8.67%, showing the lowest variation among the four traits. Ear height varied from 65 cm to 146 cm, with a coefficient of variation of 16.54%. Ear length varied from 8.1 cm to 26.7 cm, with a coefficient of variation of 18.75%, showing the highest variation among all traits. Ear diameter varied from 3.0 cm to 5.1 cm, with a coefficient of variation of 11.11%, showing relatively low variation.

[0070] The KS test is a common method for assessing whether a sample conforms to a normal distribution. The W value in the results indicates the similarity between the sample and the normal distribution; a larger value indicates a closer approximation to a normal distribution. The P value is the threshold for rejecting the null hypothesis. Since the Shapiro-Wilk null hypothesis is that the sample conforms to a normal distribution, a P value greater than 0.05 indicates that the null hypothesis should not be rejected. A larger P value indicates a higher confidence level that the sample conforms to a normal distribution. The test results for the maternal population (Table 3) show that for the four variables of plant height, ear height, ear length, and ear diameter, the samples showed a very high similarity to the normal distribution, with P values all greater than 0.05, indicating a normal distribution. This suggests that the maternal population structure is reasonable.

[0071] Table 3 Normal distribution test of agronomic traits of maternal population

[0072]

[0073] 2.2.2 Analysis of the genetic structure of agronomic traits in the paternal population

[0074] like Figure 4 As can be seen from Table 4, plant height within the paternal population varied from 195 cm to 340 cm, with a coefficient of variation of 9.79%, showing the lowest variation among the four traits and similar to the results from the maternal population. Ear height varied from 60 cm to 161 cm, with a coefficient of variation of 17.04%, showing the highest variation. Ear length varied from 10.5 cm to 20.8 cm, with a coefficient of variation of 14.03%, showing a high degree of variation. Ear diameter varied from 3.1 cm to 5.1 cm, with a coefficient of variation of 9.88%, showing relatively low variation, similar to the results from the maternal population. The KS test results for the paternal population (Table 3) showed that the samples for plant height, ear height, ear length, and ear diameter were highly similar to a normal distribution, with P values all >0.05, indicating a normal distribution. This suggests that the paternal population structure is reasonable.

[0075] Table 4 Normal distribution test of agronomic traits of male parent groups

[0076]

[0077] The above is only a preferred embodiment of this patent. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of this patent. These improvements and substitutions should also be regarded as the scope of protection of this patent.

Claims

1. A method for constructing two genetic populations suitable for corn breeding in the Huanghuaihai region, characterized in that The following steps are involved: (1) Collect multiple maize germplasm resources, including inbred lines of approved Huanghuaihai varieties, European and American mid-quality germplasm, and Indonesian tropical germplasm; (2) The germplasms were sequenced and clustered, and the germplasms were divided into the Tangsipingtou group, the PA group, the PB group, the Pioneer SS group, the Mengsandu SS group, the Pioneer NSS group, the Mengsandu NSS group, and other groups; (3) The maternal population construction materials were selected from 23 inbred lines of the PA group, the Pioneer SS group, and the Monsanto SS group; the paternal population construction materials were selected from 26 inbred lines of the Tangsipingtou group, the Pioneer NSS group, the Monsanto NSS group, the PB group, and other groups; (4) hybridizing the selected inbred lines in pairs to form single-cross varieties, and then hybridizing them again to form quadruple-cross varieties; (5) Two rounds of pollen mixing and balancing treatment were carried out on the four-cross varieties to eventually form a maternal and paternal population with a broad genetic basis.

2. The method according to claim 1, characterized in that In step (2), genotyping was performed using TASSEL5.0 software for cluster analysis. Standard inbred lines included Ye 478, Huang Zao Si, PH6WC, PH4CV, Qi 319, HCL645, and D1798Z. Huang Zao Si, Ye 478, and Qi 319 represented the Tang Si Ping Tou group, PA group, and PB group of domestic germplasm, respectively; PH6WC and PH4CV represented the SS group and NSS group of Pioneer, respectively; and HCL645 and D1798Z represented the SS group and NSS group of Monsanto, respectively.

3. The method according to claim 1, characterized in that The powder mixing balance process in step (5) includes: a) First round of pollination: Mix equal amounts of the four hybrids as the male parent, and pollinate the female parent naturally after emasculation; b) Second round of pollen mixing: Plant 2,000 plants with the seeds from the first round of pollen mixing and mix all the seeds from the ears after natural pollination.

4. Use of a maternal population and a paternal population constructed by the method of any one of claims 1 to 3 in breeding superior corn inbred lines with lodging resistance, dense planting tolerance, and stay-green properties.

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