Association Analysis of the Growth of Black Poplar (Populus Nigra L.) Under Contrasting Nitrogen Levels

The European black poplar (Populus nigra L.) has been used as a germplasm resource for the breeding of new poplar varieties around the world. The identification and screening of its high nitrogen use efficiency genotypes could enable the breeding of new resource-efficient poplar varieties. The accessions were screened using MALDI-TOF MS genotyping technology for ammonium transporter (AMT) and nitrate transporters (NRT) genes against phenotypic data for seedling height and ground diameter traits, in both low and high nitrogen environments. Allele re-sequencing of seven genes related to root development was carried out using the minisequencing method. By cluster analysis, 101 accessions of black poplar were divided into 4 populations, and it was concluded that Central Europe is the origin of the evolution of low-nitrogen and highefficiency populations of European black poplar. Association study between SNP typing and seedling height and ground diameter traits showed that there were significant correlations between four SNP loci and growth traits under the contrasting N levels. We found that SNP3 and SNP4 in the PttAMT1;3 gene were significantly associated with seedling height traits, and that SNP2 and SNP7 in the PttAMT1;2 and PttAMT1;5 genes, respectively, were significantly associated with ground diameter traits. Thus, considerable allelic diversity is present within the candidate genes studied and can be utilized to develop functional markers to select for poplars with improved growth under N stress conditions.


Introduction
European black poplar (Populus nigra L.) is a riparian tree species that has a wide geographic distribution, ranging from Western Europe to Central Asia and Northern Africa. It is not only ecologically important as an indicator species for biodiversity of riparian ecosystems [1] , but is also one of the most commonly used species in poplar breeding programs, because of its adaptability to a broad range of ecological conditions, rapid growth, easy clonal propagation, and favorable crossing ability with many major poplar species [2,3]. It is currently utilized in many breeding programs as a 'parent pool' to achieve superior hybrids for biomass production [4,5].
Nitrogen (N) is a macronutrient crucial for maximizing the yield of numerous crops. The global demand for fertilizer nutrients N for 2017 was 113.6 million tons and it was expected to increase to 118.8 million tons in 2020; an annual growth rate of 1.5% [6]. To increase land yields, nitrogen fertilizer use has often been excessive, leading to environmental pollution and increased input costs for farmers [7]. Ideal plant species and clones should thus not only have high yield characteristics but also high nutrient utilization efficiencies, especially with regards to the frequently used N, Phosphorus (P), and Potassium (K) elements of most fertilizers. Genes are keys to controlling the expression of important agronomic traits, and they usually have complex multiple quantitative trait loci that can be identified by genetic mapping and molecular identification of the functional loci. It has been reported that the family genes of Ammonium transporter(AMT)and (NRT) Nitrate transporters of poplar have an important influence on the efficiency of nitrogen uptake and utilization [8,9]. Molecular markers that were not affected in the genome by environmental and genetic variations were used to identify poplar varieties for use as genetic resources.
There are relatively few reports on the association analysis between SNP markers and European black poplar phenotypes, including focusing on the wood material traits and water and high light efficiency [10,11]. Studies have shown that poplar growth is extremely sensitive to nitrogen [12], so it is important to fully exploit and mine the functional variation in the genes related to nitrogen uptake and phenotypic variation through association analysis in the germplasm resources of P.nigra.

Plant Material
Nitrogen use efficiency (NUE) was evaluated based on the growth traits of a heuristic black poplar set of 101 accessions. The black poplar cuttings were selected from countries in Europe and Asia, to represent the genetic diversity of the species (Tab. 1).

Evaluation of Growth Traits
The test site is located in Yuquanshan Nursery, Haidian District, Beijing. The N, P and K content of the test site were determined [13]. Black poplar seedlings were planted in sample plots with either high N (HN) levels, apply urea to each seedling of European black poplar twice, and apply 4 grams per plant, or low N (LN) levels, where nitrogen fertilizer was not applied; the other macro-and micro-nutrients applied were the same in both treatments [13]. The seedling heights and ground diameters in the different fertilization environments were measured and used to determine their Nitrogen use efficiency (NUE). The date of seedling height and ground diameter were surveyed before the growth of caps.

DNA Extraction and SNP Selection
The leaves from the black poplar were collected and stored at -20°C. The genomic DNA was extracted from the leaves according to the Plant Genomic DNA Kit instructions (Beijing TIANGEN). Tag SNPs were selected using a pairwise tagging algorithm and r 2 exceeded 0.8 for all SNP sites for the 7 genes. PCR primers for mass spectrometry were designed using iPLEX GOLD (Sequenom MassARRAYW) and as shown in Tab. 2 [14].

Population Structure and Association Analysis
A model-based program STRUCTURE 2.2.3 was used to infer subgroups with distinctive allele frequencies using a burn-in of 100,000 and run length of 100,000, and an individual was assigned to a group if more than 75% of its genome fraction value was derived from that group. Five runs of the structure program were performed and an average likelihood value, L (K), across all runs was calculated for each K. The model criterion for detecting the most probable value of K was DK, an ad hoc quantity related to the second-order change in the log probability of data with respect to the number of clusters inferred by the structure program [15]. The population structure matrix (Q) identified at K = 4, was used as the number of model-based populations.

Association Analysis
TASSEL 5 and SPSS were used to conduct association analysis for the growth habits of the black poplar accessions [16]. The following models were involved for association analysis: Single factor analysis of variance was performed using SPSS version 13.0, the mixed linear model pattern in TASSEL software was used to identify significant nucleotide variations associated with growth traits and contrasting levels of nitrate. A p-value less than 0.05 was considered significant.

Association Analysis Using the Generalized Linear Model (GLM)
We attempted to discover the NUE through analyzing the genotyping data of the 16 SNPs and the two traits with GLM approaches, for selected genes from the Ammonium transporter (AMT) and Nitrate transporter (NRT) gene families. The results showed that only 4 SNP markers showed significant differences in seedling height and ground diameter as shown in Tab. 3. *, **Seedling height and ground diameter among accessions significant at p < 0.01 and p < 0.05, respectively SNP 2, SNP 3 and SNP 4, SNP 7 were located in the coding region of the PttAMT 1;2, PttAMT 1;3 and PttAMT 1;5 gene. SNP 2, SNP 3 and SNP 4 were non-synonymous mutations, while SNP4 was synonymous mutations. The association study indicated that SNP3 was significantly associated (p ≤ 0.05) with the seedling height of black poplar and the contrasting levels of nitrate explained about 7.70% of the variation for seedling height in the LN field, and about 7.37% of the variation in the HN field. The seedling height in the HN and LN fields with the GG genotype was higher than that for the GT and TT genotypes, as shown in Fig. 4(a). The analysis result of SNP 4 is similar to SNP 3. The seedling height in the HN and LN fields with the TT genotype was higher than that for the GT and GG genotypes, as shown in Fig. 4(b). SNP 2 and SNP 7 had only two genotypes. The association study indicated that SNP2 was significantly associated with the ground diameter the of the black poplar and the contrasting levels of nitrate explained about 5.82% of the variation for ground diameter in the LN field, and about 9.80% in the HN field. Ground diameter in the HN and LN fields with the CT genotype was higher than that of the CC, as shown in Fig. 4(c).
During the evolution of black poplar, the nitrogen use efficiency of different provenances was different. We infer the population genetic structure by STRUCTURE, and classify black poplar into four categories. Through analysis, it is concluded that Central Europe and Western Europe STRUCTURE. Combined with the evaluation of nitrogen use efficiency of black poplar in Europe, the low-nitrogen highefficiency type was inferred to infer that Western Europe was the main origin of evolution of European black populations.
The analysis result of SNP 7 is similar to SNP 2. The association study indicated that SNP 2 was significantly associated with the ground diameter the of the black poplar and the contrasting levels of nitrate explained about 7.76% of the variation for ground diameter in the LN field, and about 10.23 in the HN field. Ground diameter in the HN and LN fields with the GA genotype was higher than that of the AA, as shown in Fig. 4(d).  Figure 4: a Genetypic effect of SNP 3 on value of seedling height in P.nigra; b Genetypic effect of SNP4 on value of seedling height in P.nigra; c Genetypic effect of SNP2 on value of groud diameter in P.nigra; d Genetypic effect of SNP7 on value of groud diameter in P.nigra

Discussion
For most plants, ammonium and nitrate are essential sources of nitrogen required for growth. In well-aerated soil, the concentration of nitrate is usually 10-1000 times higher than that of ammonium. However, when nitrate and ammonium exist simultaneously, plants seem to be more likely to absorb ammonium nitrogen [18]. The molecular basis of the low affinity transport system is unclear, but there is increasing evidence that the ammonium high affinity transporters could be members of the AMT1 family, as the AMT protein plays a major role in the absorption of soil nitrogen by plant roots. For example, in the root system of thale cress (Arabidopsis thaliana (L). Heynh.), all of the AtAMT1;1, AtAMT1;2, and AtAMT1;3 genes were fully expressed [18]. Under the condition of nitrogen deficiency, plants enhance the expression of specific ammonium transporter genes, which is one of the most important internal mechanisms utilized by plants to adapt to nitrogen stress conditions [19].
Identification of candidate genes that are responsible for variation in continuous traits or quantitative traits has been a challenge in modern genetics [20]. The seedling height and ground diameter are the most direct growth traits for evaluating seedling production for trees. Therefore, the two trait values were correlated with SNP loci in this study and analyzed by of one-way ANOVA and a general linear model. The correlation analysis between seedling height and ground diameter showed that of the 16 successful SNP markers, 4 of them were significantly or extremely significantly correlated with the two measured traits during both the low and high nitrogen treatments. Therefore, it was preliminarily concluded that these four SNP markers are related to the Nitrogen use efficiency (NUE) of European black poplar.
The AMT1;2 gene is one of the greatest contributors to plant ammonia uptake, from the AMT family of genes. Sonoda and Kumar et.al have found that OsAMT1; 2 in Oryza sativa is expressed in the roots and induced by ammonium ions, the increase of the OsAMT1;2 gene expression was 50% of the OsAMT1;1 gene when treated with low concentrations of ammonium (10 μΜ) in the ammonium nutrient solution for 2 d [21,22]. When nitrogen deficient conditions occur, the OsAMT1;2 gene expression is enhanced to three times that found during normal nitrogen supply conditions. Our genotyping results indicated that there is a C-to T-transition at SNP2 in the coding region of the PtAMT1;2 gene, which leads to an amino acid change from Thr to Ile. The different PtAMT1;2 genotypes showed significant associations with the ground diameters of the black poplar. There are CC and CT genotypes, and the CC genotype has a frequency of 0.871, making it the dominant genotype. Therefore, it could be speculated that its correlation with nitrogen use efficiency may be because the mutation affects the transcriptional regulation levels of the PttAMT1;2 gene in different individuals. It could thus be utilized as an identification marker for genotypes with high Nitrogen use efficiency (NUE).
The AMT1;5 gene is expressed in the roots; but studies have shown that it is not expressed if there is sufficient nitrogen and its expression is significantly increased in nitrogen deficient conditions [23]. In the case of the deletion of the other AMT family genes, the AtAMT1;5 gene will functionally replace them and absorb ammonium ions [23]. The presence of the AtAMT1;5 gene will help to increase the ammonium ion uptake capacity of plants in the absence of nitrogen [23]. Our genotyping results indicated that there is an A-to G-transition at SNP7, a synonymous mutation, that is in the coding region of the PtAMT1;5 gene, it could be speculated that its correlation with NUE may be due to the fact that the mutation affects the transcriptional regulation levels of the PttAMT1;5 gene in different individuals, and significant association with the ground diameter in the HN and LN fields. The ground diameter of the GA genotype was higher than that of the AA genotype.
Nitrogen deficiency leads to enhanced expression of the AMT1;3 gene. After 3 days of nitrogen deficiency in Arabidopsis, the expression of AtAMT1;1 and AtAMT1;3 genes in the roots was enhanced. After a long period of nitrogen starvation, there was a marked change in the mRNA expression profile of the AtAMT1;3 gene [23]. The expression of OsAMT1;3 was enhanced 4 folds by nitrogen starvation in comparison to when ammonium was the sole source of nitrogen [24]. Our genotyping results indicated that there were T-to G-transitions at SNP3 and SNP4 in the coding region of the PtAMT1;3 gene, causing amino acid changes from Met to Arg and Lys to Asn. The different PtAMT1;5 genotypes showed significant association with the seedling height of the black poplar. The seedling heights of the GG and TT genotypes were higher than GT genotype.
During the evolution of black poplar, the nitrogen use efficiency of different provenances was different. We infer the population genetic structure by STRUCTURE, and classify black poplar into four categories. It is inferred that Central Europe and Western Europe were the centers of evolutionary origin of the black poplar population. Combined with nitrogen use efficiency evaluation of black poplar [13], it was inferred that Western Europe is the main origin of the evolution of low-nitrogen and high-efficiency black poplar populations.
In summary, we obtained 16 SNPs of 7 genes, and the association results revealed that SNP 3 and SNP 4 were significantly associated with seedling height and that SNP 2 and SNP 7 were significantly associated with ground diameter. Here, we provide the partial preliminary molecular information of the genes related to NUE for the first time. The association results might be useful and applicable in future poplar genetic marker-assisted selection processes and breeding programs.
Disclosure statement: No potential conflict of interest was reported by the authors.