Genetic Diversity and Genetic Structure of Maize Inbred Lines from Yunnan Revealed by SNP Chips

Junjiao GUAN Peng ZHANG Sheping LI Junhao LU Qingmei HUANG Xiaohong YANG Jianhua ZHANG Zhuke KANG

Abstract ［Objectives］ The genetic diversity and population genetic structure of 107 inbred lines of maize in Yunnan were analyzed, in order to provide technical support for maize germplasm innovation, genetic improvement of germplasm resources, variety management, and lay a solid foundation for exploring genes related to fine traits in the future.

［Methods］The 107 maize inbred lines generalized in Yunnan were selected, and 45 backbone inbred lines commonly used in China were used as reference for heterotic group classification. On Axiomk Maize 56K SNP Array platform, maize SNP chips (56K) were used to scan the whole maize genome, and the NJ-tree model of Treebest was used to construct a phylogenetic tree. Principal component analysis (PCA) was conducted by GCTA (genome-wide complex trait analysis) to reveal the genetic diversity and population genetic structure.

［Results］ In the 107 Yunnan local inbred lines, 5 533 uniformly distributed high-quality SNP marker sites were finally detected. Based on the analysis of these SNP marker sites, Neis gene diversity index (H) of 107 maize germplasm genes was 0.298 1-0.500 0 with an average value being 0.483 2, and polymorphism information content (PIC) values were 0.253 6-0.375 0 with an average value being 0.366 2. The minimum allele frequency value was 0.500 0-0.817 8 with an average value being 0.574 4. The analysis of population genetic structure showed that when K=6, the maximum value of △K was the maximum, which meant that the inbred lines used in this study could be divided into six groups. They were Tangsi Pingtou blood relationship group, PB blood relationship group, 335 female blood relationship group, Zi 330 and the Lüda Honggu blood relationship group, unknown group 1 and unknown group 2. No inbred lines were divided into other heterotic groups. Among them, 37 inbred lines from the 2 unknown groups could not be classified into the same group as the 10 known heterotic groups in China. The results of principal component analysis showed that the 107 maize inbred lines generalized in Yunnan could be clearly distinguished from the backbone maize inbred lines commonly used in China. Most of the maize inbred lines in Yunnan were concentrated near the reference backbone inbred lines. But some Yunnan inbred lines were far away from the reference inbred lines commonly used in China.

［Conclusions］The maize germplasm resources in Yunnan area were rich in genetic diversity, including multiple heterotic groups, and there was a rich genetic basis of breeding parents. They could be clearly distinguished from the backbone inbred lines commonly used in China, and some of them had a long genetic distance from the backbone inbred lines. The resources which have good application potential can be used to create new heterotic groups.

Key words Maize; SNP chips; Group genetic structure; Genetic diversity; Principal component analysis

Received: November 22, 2020  Accepted: January 23, 2021

Supported by Study on Maize Variety Management Based on DUS Test and SNP Molecular Fingerprint.

Junjiao GUAN (1985-), female, P. R. China, associate researcher, devoted to research about field crop variety breeding and molecular markers.

*Corresponding author. E-mail: zhjhua6748@163.com.

Maize is Chinas largest food crop with a long history of cultivation, and its germplasm resources are constantly being updated. At present, the number of approved varieties in China has reached more than 6 000［1-2］, which are mainly planted in the northeast, north and southwest regions. Yunnan is located in southwestern China, and its unique climate and ecological diversity has created distinctive and diverse maize germplasm resources［3］. Analyzing genetic structure group, evolution pattern and population genetic structure of Yunnan maize germplasms, sorting the backbone inbred lines and hybrid dominant groups in Yunnan maize inbred lines and clarifying the genetic relationship of maize varieties are of great significance to the selection of excellent varieties and regulate the market management of maize varieties in Yunnan Province［4］. SNP is the third-generation most advantageous molecular marker technique developed on the basis of RAPD, RFLP, AFLP, SSR and other molecular marker techniques［5-7］. It has been widely used in research fields such as genetic diversity analysis, QTL mapping, fingerprint construction, population structure analysis, germplasm resource analysis and molecular marker-assisted selection of crops［8-9］. Chen et al.［10］ performed a correlation analysis on plant height-related traits with 205 wheat varieties (lines) in the winter wheat region of China as materials using 243 55 SNP molecular markers distributed throughout the wheat genome, and found that 38 SNP molecular markers were extremely significantly correlated to plant height. Lu et al.［9］ conducted a  genetic relationship analysis on the parents of the maize variety Jidan 50 and 10 backbone inbred lines based on SNP molecular markers. Cai et al.［11］ used SNP molecular markers to analyze the genetic diversity and genetic structure of 92 tobacco germplasm resources. Huang et al.［12］ studied how SNP can control miRNA-related traits by affecting the complementary pattern of miRNA and target gene pairs, and performed genome-wide SNP analysis on genes encoding miRNA based on 3 000 rice genome data. Zhao et al.［6］ used 344 broadly representative and time-sensitive maize inbred lines as materials and used SNP chips to analyze their genetic diversity and population genetic structure. Cui et al.［13］ constructed a wheat genetic map based on 90K chip SNP molecular markers and performed QTL mapping for the trait of resistance to sheath blight. Yao et al.［14］ used SNP and SSR molecular markers to analyze the genetic diversity of 23 sweet corn inbred lines. So far, there have been few reports on the study of population genetic structure analysis of maize inbred lines in Yunnan area using SNP molecular markers. In this study, with 107 excellent maize inbred lines promoted locally in Yunnan as materials, the maize SNP chips (56K) were used to scan the whole genome on the Axiomk Maize56K SNP Array platform, and the SNP molecular markers were used to analyze the genetic structure and genetic diversity of their populations, so as to understand the blood relationship, evolutionary groups, heterotic populations and population genetic structure of Yunnan local inbred lines. This study provides a theoretical basis for maize germplasm innovation, genetic improvement and variety management in Yunnan Province, and also lays a foundation for further exploration of genes related to excellent traits in the future.

Materials and Methods

Experimental materials

The test materials were 107 local Yunnan maize inbred lines (No. ZBS001-ZBS107), and 45 commonly used maize backbone inbred lines in China (Table 1) were used as control checks (CK) for the division of heterotic groups, including 10 heterotic groups currently known in China. The above materials were provided by Yunnan Datian Seed Industry Co., Ltd. The maize SNP chips (56K) purchased from Affymetrix chip company were synthesized by the unique photoetching in-situ synthesis technology, and could detect 56 000 SNPs sites with a marker detection rate over 97%. The chips were 384 system chips.

SNP molecular marker analysis

Maize inbred lines were planted in spring at a planting density of 60 000 plants/hm2. New leaves were collected at the seedling stage with 3 leaves and 1 heart. Five young leaves were taken from each material and mixed and ground in liquid nitrogen. The DNA was extracted by the CTAB method, detected with an ultraviolet spectrophotometer for DNA concentration, and purified for later use. On the Axiomk Maize56K SNP Array platform, the maize SNP chips (56K) were used to scan the whole genome. Sample preparation was completed on the Backman automatic workstation, and hybridization, washing and scanning were completed on GeneTitan.

Statistical analysis

The Treebests NJ-tree model was employed to construct a phylogenetic tree, and the GCTA (genome-wide complex trait analysis) tool was applied for principal component analysis. Based on the detected 5 533 uniformly distributed high-quality SNP molecular markers, the Structure 2.2 was applied to analyze the population genetic structure of the test materials. Referring to the line graph model of △K under the change of K in Evanno et al.［15］, K was preset to 1-10, and the optimal population group number K was estimated. Each K value was independently calculated 10 times［16］. The PowerMarker v3.25 was applied to calculate polymorphic information content (PIC), minimum allele frequency and Neis gene diversity index (H), etc.［17］.

Results and Analysis

Genetic diversity and population genetic structure analysis results

Population genetic structure refers to a non-random distribution of genetic variation in a species or population. A group can be divided into several subgroups according to geographical distribution or other criteria. Different individuals in the same subgroup are closely related to each other, while the genetic relationship between subgroups and subgroups is slightly farther. Population structure analysis is helpful to understand the evolution process, and the subgroup to which an individual belongs can be determined through the correlation between genotype and phenotype. The genetic structure analysis results of the 107 Yunnan maize inbred line populations are shown in Fig. 1. There were 33 test inbred lines mainly in light blue, which belonged to Tangsi Pingtou blood relationship; 13 inbred lines were mainly blue, and belonged to PB blood relationship; and 22 inbred lines were mainly in light green, and belonged to 335 female parent blood relationship; 26 test inbred lines were dominated by green, 11 test inbred lines were dominated by pink, and because there was no reference inbred line in the classification of green and pink, they were classified as unknown groups; and finally, there were two inbred lines mainly in red, which belonged to Zi 330 and Lüda Honggu blood relationship.

Agricultural Biotechnology2021

The Structure 2.2 was applied to systematically analyze the population genetic structure of 107 Yunnan maize inbred lines. The results (Fig. 2) showed that when the number of groups (K) was 1-10, and ΔK reached the maximum value at K=6, indicating that the 107 test inbred lines and 45 reference inbred lines could be divided into six groups. According to Fig. 1, they could be divided into Tangsi Pingtou blood relationship group, PB blood relationship group, 335 female blood relationship group, Zi 330 and Lüda Honggu blood relationship group, and two unknown groups. No inbred lines were divided into 335 male blood relationship group, European male parent blood relationship 1, European male parent blood relationship 2, European female parent blood relationship, Lancaster blood relationship, and modified Reid blood relationship groups, and there were 37 test inbred lines in total in the 2 unknown groups that were divided into the current known heterotic groups in China.

From the 107 Yunnan maize inbred lines, 5 533 evenly distributed high-quality SNP molecular marker sites were detected. Based on the analysis results of these SNP molecular marker sites, it can be seen that the H of the 107 inbred lines tested was 0.298 1-0.500 0, with an average of 0.483 2; the PICs were 0.253 6-0.375 0, with an average of 0.366 2; the minimum allele frequency was 0.500 0-0.817 8, with an average of 0.574 4 (Table 2), indicating that the overall genetic diversity of the tested inbred lines was rich. The H and PICs of the six groups were all high, but there were obvious differences in different groups. Among them, the PIC of the Tangsi Pingtou blood relationship group, the PB blood relationship group, the 335 female blood relationship group and the unknown group 1 was relatively high, was 0.343 2, 0.298 3, 0.290 5 and 0.325 8, respectively, while the PICs of the unknown group 2 and Zi 330 and Lüda Honggu blood relationship group was relatively low, being 0.241 4 and 0.193 8, respectively.

Phylogenetic analysis results

With 45 commonly used maize backbone inbred lines in China as reference for the classification of heterotic groups, each heterotic group could be well distinguished, as shown in Fig. 3. All materials were clearly divided into three groups. Among them, the Tangsi Pingtou blood relationship lines belonged to group I, which did not include the tested inbred lines; the Lancaster blood relationship and the European male parent blood relationship belonged to group II, which also did not include the tested inbred lines; and the rest of the materials were divided into group III, which included the 107 test inbred lines, and could be divided into two subgroups (III-a and III-b). In the subgroup III-a, the 335 male blood relationship, 335 female blood relationship and European female blood relationship were a small group, the PB blood relationship was a small group, and the Zi 330 and Lüda Honggu blood relationship and modified Reid blood relationship were a small group. In the III-b subgroup, 72 tested inbred lines and Suwan 1611 were grouped together, and it was speculated that these 72 inbred lines were tropical germplasm materials. In group III, the blood composition of 31 Yunnan local maize inbred lines was not in the 10 currently known heterotic groups in China, accounting for 28.97%, which was different from the results of the population genetic structure analysis. It was inferred that these 31 inbred lines were a new heterotic group. Combining the above population genetic structure analysis, it can be seen that among the 107 Yunnan maize inbred lines, the number of materials mainly composed of the PB blood relationship was the largest, being 32, accounting for 29.91%; and the number of materials mainly composed of the 335 female, modified Reid and Zi 330 and Lüda Honggu blood relationship was the smallest, and each included 3 materials accounting for 2.80%.

Principal component analysis results

Based on the individual genotype SNP, the principal component analysis method was used to cluster different genotype varieties into different subgroups to clarify the genetic structure of the 107 Yunnan maize inbred lines. The first 3 dimensions of the principal component analysis diagram could explain all material variation, and were also the 3 dimensions with the most genetic variation［18］. In this study, the dimensions 1v2, 1v3, and 2v3 were extracted and drew into two-dimensional maps. The results showed that PCA1v2 best represented the genetic variation of all materials, followed by PCA1v3 and PCA2v3. It can be seen from Fig. 4 that the 107 Yunnan maize inbred lines could be clearly distinguished from the 45 commonly used maize backbone inbred lines in China. Most of the Yunnan maize inbred lines were concentrated near the commonly used maize backbone inbred lines in China, but a few Yunnan maize inbred lines were far away from the maize backbone inbred lines commonly used in China. Among them, very few inbred lines were relatively close to the three heterotic groups of the European female blood relationship, the European male blood relationship 1 and European male blood relationship 2, and there were also some inbred lines that were far away from all the 10 heterotic groups, and were not classified into the same categories with the commonly used corn backbone inbred lines in China, which was similar to the results of population genetic structure and phylogenetic tree analysis.

Discussion

In this study, 45 maize backbone inbred lines commonly used in China were selected as reference inbred lines (CK) for heterotic group classification to analyze the population genetic structure of the 107 local maize inbred lines in Yunnan. These reference inbred lines were the backbone parental resources currently or historically used in China in maize production, involving 10 heterotic groups. By constructing a phylogenetic tree, the genetic relationship between individuals can be inferred according to the common points or differences of population genetic characteristics［19］, and the earlier the branches are separated, the farther the genetic relationship is［20］. Meanwhile, combined with principal component analysis, the phylogenetic tree method can be applied to plant phenotype, resource evaluation, genetic analysis and other research and other methods for mutual verification［21-22］. In this study, principal component analysis and population genetic structure analysis were combined to divide the 107 Yunnan local maize inbred lines and 45 reference backbone inbred lines into six major groups, namely Tangsi Pingtou blood relationship group, PB blood relationship group, 335 female blood relationship group, Zi 330 and Lüda Honggu blood relationship group, and two unknown groups. No inbred lines were divided into the Lancaster blood relationship and modified Reid blood relationship groups, and the 2 unknown groups included in total 37 test inbred lines that were divided into the current known heterotic groups in China. However, according to the phylogenetic tree, 72 varieties in subgroup III-b were similar to the reference inbred line Suwan 1611, and they were divided into one group. It was speculated that the varieties in this unknown group contained tropical blood and were tropical germplasm materials, but the specific reasons should also be combined with the identity by descent (IBD) genetic background proportion for a comprehensive analysis, which is similar to the results of previous studies［23-24］. It was found that traditional heterotic groups such as the Tangsi Pingtou blood relationship, PB blood relationship and 335 female blood relationship could be clearly distinguished, but there were also differences, which might be due to differences in the number and sources of test materials.

Light blue was tested maize inbred line in Yunnan; red was 335 male parent blood relationship; orange was 335 female parent blood relationship; yellow was European male parent blood relationship 2; light yellow was European male parent blood relationship 1; green was European female parent blood relationship; light green was Lancaster blood relationship; blue was Pb blood relationship; purple was improved Reid blood relationship; pink was Tangsi Pingtou blood relationship; rose red was Zi 330 and Lüda Honggu blood relationship.

Fig. 3 Phylogenetic tree of Yunnan local maize inbred lines with 45 maize inbred lines commonly used in China as reference

The genetic improvement of maize varieties is closely related to the discovery or introduction of excellent germplasms. The introduction of inbred lines and commercial hybrid lines plays an important role, because the introduction of excellent foreign germplasm genes into domestic germplasms can effectively increase genetic variability and produce strong-heterotic groups［6］.   The earliest report on the introduction of foreign maize germplasm resources in Yunnan was the introduction of maize inbred line SSE232 through the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences in May 1976［25］. The inbred line was widely used as a parent in variety breeding. Subsequently, many scholars have obtained strong heterosis through introduced varieties, most of which were tropical and subtropical maize inbred lines［26-27］. Studies have shown that there is strong heterosis between tropical and subtropical maize germplasms and temperate maize germplasms improved with tropical germplasms, the heterosis of the temperate lines introduced with tropical germplasms and the tropical and subtropical donor systems still exists, and the heterotic groups to which they belong have not changed［28］. Therefore, it can be seen that through the improvement with foreign high-quality germplasm resources, complementary advantages can be formed with regional variety resources［6］. The introduced materials have strong heterosis on the basis of maintaining the original advantages, and are capable of playing an important role in variety breeding. At present, the introduction and innovative utilization of tropical germplasms and broadening the genetic basis of maize breeding in China have become an important direction of maize breeding in China. Therefore, in the future, we should make full use of the new heterotic groups discovered in this study to combine with the existing heterotic group in China to enhance the innovation ability of maize breeding in China.

Red was 335 male parent blood relationship (335F); blue was 335 female parent blood relationship (335M); green was European male parent blood relationship 1（EUROF1)；purple was European male parent blood relationship 2 (EUROF2); orange was European female parent blood relationship (EUROM); yellow was Lancaster blood relationship (LAN); pink was Pb blood relationship (PB); grey was improved Reid blood relationship; light blue was Tangsi Pingtou blood relationship; blue was Zi 330 and Lüda Honggu blood relationship (ZI330); brown was 107 maize inbred lines.

Fig. 4 Principal components analysis of SNP genotype in maize inbred lines

Conclusions

The maize germplasm resources in Yunnan area are rich in genetic diversity, including multiple heterotic groups, and there is a rich genetic basis of breeding parents. They could be clearly distinguished from the backbone inbred lines commonly used in China, and some of them have a long genetic distance from the backbone inbred lines. The resources which have good application potential can be used to create new heterotic groups, and there is an urgent need to make full use of local heterotic groups for combining hybrids and accelerating the generation of new genetic groups.

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