agronomy

Article
Evaluation of Yield-Based Low Nitrogen Tolerance
Indices for Screening Maize (Zea mays L.)
Inbred Lines
Zhixin Zhao, Kunhui He, Zhiqian Feng, Yanan Li, Liguo Chang, Xinghua Zhang, Shutu Xu,
Jianchao Liu * and Jiquan Xue *
Key Laboratory of Biology and Genetic Improvement of Maize in Arid Area of Northwest Region, Ministry of
Agriculture and Rural Affairs, College of Agronomy, Northwest A&F University, Maize Engineering &
Technology Research Centre of Shaanxi Province, Yangling 712100, China; [email protected] (Z.Z.);
[email protected] (K.H.); [email protected] (Z.F.); [email protected] (Y.L.);
[email protected] (L.C.); [email protected] (X.Z.); [email protected] (S.X.)
* Correspondence: [email protected] (J.L.); [email protected] (J.X.); Tel.: +86-298-708-2934 (J.L.);
+86-298-708-2934 (J.X.)




Received: 5 March 2019; Accepted: 7 May 2019; Published: 10 May 2019


Abstract: To screen the desired criterion to identify desirable genotypes and select genotypes best
suited to limited nitrogen availability in order to facilitate the practice of low-nitrogen-tolerant
breeding in maize, the response of 31 maize inbred lines, containing four control inbred lines (PH6WC,
PH4CV, Zheng58, and Chang7-2) and others selected from the Shaan A and Shaan B heterotic groups
cultivated at Northwest A&F University (Yangling, Shaanxi, China), were evaluated. The experiment
was conducted following a split plot design with two replications during three growing seasons
(2015, 2016, and 2017) under both high nitrogen (HN) and low nitrogen (LN) conditions at the Yulin
and Yangling in Shaanxi Province, China. Seven screening indices, based on grain yield under two
contrasting nitrogen (N) conditions, the stress susceptibility index (SSI), yield stability index (YSI),
mean productivity (MP), geometric mean productivity (GMP), stress tolerance index (STI), harmonic
mean (HM), and low nitrogen tolerance index (LNTI), were computed to assess the overall index that
accurately screened the desirable genotypes. The results of the correlation analyses and principal
component analysis showed that MP, GMP, HM and STI were correlated with grain yield significantly
and positively under contrasting N conditions, and were able to accurately discriminate the desirable
genotypes. Compared with the control inbred lines, many inbred lines selected from the Shaan A
and Shaan B groups showed a higher LN tolerance. This shows that we can effectively improve
the LN tolerance of maize inbred lines through LN screening. Based on the screening indices, the
three-dimensional diagram and genotype and genotype × environment (GGE) biplots are agreed with
this results, and we identified KA105, KB081, KA225, 91227, and 2013KB-47 as the desired genotypes
that have the potential to be used to breed a high yield and stable hybrid.

Keywords: maize; screening indices; low nitrogen tolerance; grain yield

1. Introduction
Maize (Zea mays L.) is not only an important cereal crop, is also an important forage, economic, and
energy crop. The production of maize affects a large proportion of the world population. As a C4 plant,
maize has a high photosynthetic efficiency and acquires considerable biomass under an appropriate
nitrogen (N) supply [1]. Since the green revolution, farmers have tended to maximize N fertilization
to increase crop yield [2]. The large application of N fertilizer is essential to the development of the
maize plant and grain yield [3]. However, the excessive application of N fertilizer has created several

Agronomy 2019, 9, 240; doi:10.3390/agronomy9050240 www.mdpi.com/journal/agronomy

Agronomy 2019, 9, 240 2 of 14

environmental problems since the 2000s [4,5]. The excessive use of N fertilizer damage the ecological
environment by causing ‘Algae blooms’ in lakes and ‘red tides’ in estuaries, as well as increasing the
NO and NH3 emissions from farmlands [5–8].
N fertilizer waste results from the excessive application of N fertilizers for maize plants which
have a lower nitrogen use efficiency (NUE) [9,10]. One possible method to ensure the NUE and meet
the needs of increasing demands for a nitrogen efficient maize cultivar in the future involves the
breeding of a low nitrogen (LN) tolerant cultivar, which could be created by crossing the LN tolerant
inbred lines of maize. Breeding of LN tolerant varieties may maintain grain yield and decrease the
amount of required N fertilizer.
Therefore, the need to screen for a LN tolerant cultivar has encouraged scholars to identify several
reliable indices as the criteria for the LN tolerant genotype [11,12]. Researchers usually choose the
relative grain yield performance genotypes under stress and normal conditions as the first selection
factor to determine the desired genotypes [13]. Screening indices are mathematical expressions that
consider the performance of plants under both stress and normal conditions. Different screening indices
reflecting stress influence have been suggested based on the relative grain yield between the stress
and normal conditions [14–16]. Rosielle and Hamblim [17] defined mean productivity (MP) as the
average yield of a genotype under contrasting stress and optimal conditions. Fischer and Maurer [18]
proposed the stress susceptibility index (SSI) for evaluating the yield stability and determining the
changes in both potential and actual yield in variable environments. Fernandez [19] defined a new
index, the stress tolerance index (STI), which can be used to identify genotype that produce a high yield
under both stress and non-stress conditions. Another yield-based estimation of drought resistance is
the harmonic mean (HM) [20,21]. Francisco et al. [16] suggested the use of the low nitrogen tolerance
index (LNTI) to understand yield and to reflect the influence of a LN fertilizer. The researches on
screening indices related to the different stressed conditions and variable test materials have been
reported. Such as, Homa et al. [22] identified the GMP, MP, STI, and HM as the most suitable resistance
indices to screen the salinity tolerance at the rice seedling stage. Khan et al. [10] researched 15 indices,
including of GMP, MP, and STI, to identify the N tolerant genotype of wheat. Ganjeali et al. [23] used to
identity a screening index to identify a drought tolerant genotype of chickpea. However, few articles
have reported screening indices for LN tolerance in maize.
Genotype and genotype × environment (GGE) biplots have been widely applied in
multi-environmental test data analysis [24–26]. GGE biplots are based on phenotype data that examine
the environment (G), genotype (E), and the effect of G × E interactions [27]. GGE biplots are drawn
based on principal component analysis (PCA) to graphically display the performance of the genotype by
phenotype value and can accurately reflect the yield and stability of test varieties. The convenient and
fast GGE system can improve research efficiency [28]. Bahrami et al. [29] used the GGE graphs to analyze
the screening indices, to choose the desirable safflowers. Han et al. [30] analyzed the performance of fatty
acids in 26 soybean cultivars by GGE biplots. Dehghani et al. [31] used the GGE biplots methodology to
analyze yield and related traits in melon. However, no reports have been published on the application of
GGE biplots to the study of screening LN tolerance in maize genotypes until now.
Herein, 31 inbred lines including four controls were planted under low nitrogen (LN) (0 kg/ha)
and high nitrogen (HN) (180 ka/ha) at two locations over three years, and the related indices were
calculated based on the yield under the two conditions. The goal was achieved by verifying the LN
tolerant genotypes from a variety of inbred lines selected using the indices above and combining the
GGE biplots. The desired genotypes were then identified for breeding a high-yield and high-NUE
hybrid in the future. This study of the screening indices analysis provides a theoretical basis for the
practice of LN tolerance breeding in maize.
Agronomy 2019, 9, 240 3 of 14

2. Materials and Methods

2.1. Plant Materials and Field Experiment

According to the theory of domestic and international maize breeding, Shaan A and Shaan B
heterotic groups were built employing the breeding strategy of two divergent heterotic groups, on the
basis of the simplifying heterotic model, following seven rounds of optimization and upgrades from
2007 to 2015. The superior varieties were the basic materials used to adapt maize production in Shaanxi
Province. [32]. Thirty-one maize inbred lines containing four elite inbred lines (PH6WC, PH4CV,
Zheng58, and Chang7-2) as controls and other inbred lines selected from the Shaan A and Shaan B
heterotic groups that were cultivated at Northwest A&F University were used to assess LN tolerance.
Detailed information about the 31 inbred lines are provided in Table 1.

Table 1. Best linear unbiased prediction (BLUP) value for grain yield under high nitrogen (HN) (Yp)
and low nitrogen (LN) (Ys) and seven screening index values for 31 inbred lines.

Code Group1 Genotype Yp (kg ha−1 ) Ys (kg ha−1 ) GMP HM MP STI YSI LNTI SSI
1 Shaan A KA008 5184.35 3705.16 4392.91 4306.59 4471.99 0.49 0.71 0.24 0.95
2 Shaan A 2012KA-1 6107.00 4214.43 5091.76 4989.91 5203.15 0.67 0.68 0.28 0.93
3 Shaan A KA064 6235.58 3636.09 4653.10 4399.80 4969.88 0.62 0.62 0.42 1.19
4 Shaan A 2012KA-58 6466.16 4400.15 5318.04 5180.47 5385.22 0.81 0.67 0.27 0.97
5 Shaan A KA103 6401.76 4393.07 5315.92 5203.67 5444.96 0.73 0.71 0.24 0.80
6 Shaan A KA203 5920.33 3626.73 4589.25 4397.79 4805.05 0.53 0.64 0.36 1.09
7 Shaan A 2013KA-34 6317.18 4047.09 4987.97 4789.97 5223.45 0.71 0.65 0.33 0.85
8 Shaan A KA105 6734.33 4923.36 5780.16 5698.23 5887.49 0.95 0.70 0.22 0.67
9 Shaan A KA227 5899.12 4370.32 5089.30 5009.21 5178.11 0.67 0.71 0.21 0.61
10 Shaan A KA225 6410.79 4740.25 5530.16 5450.85 5628.76 0.86 0.69 0.25 0.72
11 Shaan A XCA-1 6399.93 4466.13 5372.68 5278.98 5481.75 0.78 0.70 0.26 0.90
12 Shaan A KA060 6291.86 4369.85 5267.45 5170.89 5377.20 0.72 0.69 0.27 1.00
13 Shaan B KB081 6591.79 5272.05 5918.44 5869.12 5995.13 1.01 0.98 0.15 0.55
14 Shaan B KB417 5450.83 4064.38 4727.30 4661.26 4792.66 0.80 0.76 0.19 0.87
15 Shaan B KB109 6274.73 4282.33 5179.44 5055.69 5323.33 0.70 0.69 0.30 1.09
16 Shaan B 91227 6824.74 4347.14 5441.65 5269.80 5630.59 0.83 0.65 0.36 1.07
17 Shaan B KB-7 6433.13 4461.62 5368.64 5258.73 5496.27 0.76 0.68 0.27 0.84
18 Shaan B KB020 6194.17 4118.80 5043.45 4902.05 5198.03 0.64 0.69 0.25 0.63
19 Shaan B 2013KB-37 6472.23 4532.60 5406.49 5284.90 5552.75 0.81 0.70 0.26 0.92
20 Shaan B 2013KB-47 6929.63 4325.99 5500.27 5344.82 5678.26 0.83 0.66 0.38 1.37
21 Shaan B KB043 5411.19 3643.14 4411.33 4276.45 4555.11 0.49 0.66 0.29 0.88
22 Shaan B Z140588 6061.74 4016.02 4922.82 4783.60 5077.77 0.64 0.68 0.32 1.07
23 Shaan B Z140580 6382.42 4212.83 5198.92 5070.63 5342.11 0.71 0.66 0.32 1.06
24 Shaan B 2013HXB-4 5807.66 3800.68 4710.44 4585.85 4837.65 0.57 0.66 0.34 1.12
25 Shaan B 2013ZZB-6 6256.82 4484.76 5322.61 5240.09 5418.73 0.75 0.71 0.27 1.17
26 Shaan B 2014KB-54 6465.35 4276.59 5259.68 5120.53 5417.12 0.76 0.66 0.32 1.06
27 Shaan B KB215 6382.60 4463.20 5309.47 5185.88 5471.44 0.82 0.69 0.27 0.89
28 Checks Zheng58 6198.42 4003.97 4972.65 4819.70 5140.92 0.66 0.65 0.33 0.94
29 Checks Chang7-2 6378.99 4471.96 5147.36 5344.59 5244.57 0.84 0.66 0.30 1.02
30 Checks PH6WC 6950.85 4795.11 5789.19 5675.06 5931.18 0.91 0.69 0.25 0.77
31 Checks PH4CV 5782.55 3674.20 4591.15 4436.34 4759.62 0.53 0.67 0.34 1.10
1 Group indicates the population that the genotypes were selected. GMP: geometric mean productivity, HM:
harmonic mean, MP: mean productivity, STI: stress tolerance index, YSI: yield stability index, LNTI: low nitrogen
tolerance index, SSI: stress susceptibility index, the same below.

2.2. Phenotype Evaluation

These inbred lines were planted over three years (2015, 2016, and 2017) at the experimental farm
located in Yangling (34◦ 540 N, 108◦ 7E, 472 m altitude, clay soil) and Yulin (34◦ 160 N, 109◦ 450 E, 1100 m
altitude, sandy soil) at Shaanxi Province, China. The average daytime temperature was 12.9 ◦ C and
8.3 ◦ C in Yangling and Yulin, respectively. The soil nutrition, collected depths 0–20 cm, was measured
as total N 0.92 g/kg (Yangling) and 0.98 g/kg (Yulin); Available potassium was 178 mg/kg (Yangling)
and 180 mg/kg (Yulin); Available phosphorus was 14.63 mg/kg (Yangling) and 17.53 mg/kg (Yulin);
the organic-M content was 17.04 g/kg (Yangling) and 15.47 g/kg (Yulin). The experiments were designed
as a split plot design with N treatment as the main plot under each level of N fertilization conditions.
Two levels of pure nitrogen were used: 180 kg/ha for HN and 0 kg/ha for LN. For the HN treatment, the
fertilizers used before sowing, included urea (191 kg/ha), super phosphate (750 kg/ha), and potassium
Agronomy 2019, 9, 240 4 of 14

chloride (135 kg/ha). An additional 200 kg/ha of urea was used at the V9 stage. For the LN treatment,
fertilizers were used as in the HN treatment, except with no N fertilizer. The 31 inbred lines were planted
in two-row plots with two replications. Each row was 5m in length, with a 0.6-m spacing between the
rows. The planting density was 66,667 plants ha−1 . Field management was performed according to
local standards.
The grain yield of each genotype was recorded to calculate the grain yield per ha−1 (kg) with
an adjustment to a 14% moisture content. Based on the grain yield of every genotype under the LN
and HN conditions, seven indices, SSI, YSI, MP, GMP, STI, HM, and LNTI, were computed. The HN
experiment was considered to be non-stressed conditions and the LN experiment was considered to be
stressed conditions. Different screening indices were calculated as follows.
The stress susceptibility index is [18]:

1 − Ys/Yp
SSI =
SI
Yms
SI = 1 −
Ymp
where Ys is the yield of the cultivar under LN conditions; Yp is the yield of cultivar under optimal
condition; Yms and Ymp are the mean yields of all cultivar under stress and non-stress conditions,
respectively; 1-(Ys/Yp) is the stress intensity; and SI is the stress intensity. Genotypes with SSI<1 are
more resistant to low nitrogen.
The yield stability index is [33]:
Ys
YSI =
Yp
Genotypes with a high value of YSI are regarded as stable genotypes under both stressed and
non-stressed conditions.
Mean productivity is [17]:
Yp + Ys
MP =
2
The genotypes with a high value of this index are more desirable.
Geometric mean productivity is calculated as [19]:
p
GMP = Yp × Ys

Genotypes with a high GMP will be more desirable.

Stress tolerance index is calculated as [19]:

Yp × Ys
STI =
(Ymp)2

Genotypes with a high STI are tolerant to low nitrogen.

The harmonic mean is as follows [20]:

2 × Yp × Ys
HM =
Yp + Ys

Genotypes with a high value of this index are more desirable.

The low Nitrogen Tolerance Index is calculated as [16]:

Yp − Ys
LNTI =
Yp

Genotypes with a low value of this index are more desirable.

Agronomy 2019, 9, 240 5 of 14

2.3. Statistical Analysis

A combination analysis of variance (ANOVA) was performed for grain yield and the tolerance
indices using the GLM procedure of SPSS (Statistical Product and Service Solutions) 22.0 [34].
Correlation among the seven screening indices and grain yield under contrasting fertilizer conditions
was determined using Pearson analysis in SPSS 22.0. To identify the LN tolerance and high yielding
genotypes, a three-dimensional (3D) diagram was drawn using originPro2017 (OriginLab Corporation,
Northampton, MA, USA). PCA and GGE biplots based on the indices and yield of the six environments
were drawn using the Genstat 19.0 (Beijing VSNC Statistics Software Co. Ltd., Beijing, China) [35].
The PROC MIXED procedure (SAS, Statistics Analysis System) [36] was used to assess the best linear
unbiased prediction (BLUP) value of the genotypic effect for grain yield under different N treatments,
and a mixed linear model for each line applied as follows [37]:

yi = µ + Gi + Ei + ei

where yi is the phenotypic value; µ is the total mean value of the total yield in all environments; Gi is
the genotype effect; Ei is the environment effect; and ei is the random error. The genotype effect and
environment effect were random effects, with the others assumed to be fixed.
The GEE biplots model is as follows [38]:

Yij − Yi = λ1 ξi1 ηi1 + λ2 ξi2 ηi2 + εij

where Yij is the mean value of the i genotype under environment j; Yj is the mean value of all genotypes
under environment j; λ1 and λ2 is the singular value of the first principal component and the second
principal component, respectively; ξi1 and ξi2 are the score of the i-th genotype on the first principal
component and the second principal component, respectively; ηj1 and ηj2 are the score for the j
environment on the first principal component and the second principal component, respectively; and
εij represents the difference between the actual value and the first two principal component estimates.

3. Results

3.1. Phenotypes for the Seven Screening Indices and Grain Yield under HN and LN Treatments of 31 Inbred
Lines
The results of the BLUP value for grain yield and different screening indices calculated are
presented in Table 1. Compared with HN, the grain yield of all inbred lines decreased by varying
degrees under the LN treatments (Table 1). GMP, MP, STI, and HM identified that genotypes KB081,
PH6WC, KA105, and 91227 were the most tolerant. Genotypes KB081, KA227, KB020, KA105, 91227,
and PH6WC were identified as having the least susceptibility per SSI and LNTI (Table 1). The results
of the variance analysis of the combined data for the grain yield and screening indices for 31 maize
inbred lines are presented in Table 2. We found a significant different between the three growing
years and two locations. Grain yield and all screening indices were significantly different between
all genotypes (p < 0.01), which indicated that different inbred lines performed differently in grain
yield and all screening indices. That variation in all genotypes provide a basis screening indicator for
LN treatments.
Agronomy 2019, 9, 240 6 of 14

Table 2. Combined analysis of variance for the BLUP of grain yield under HN (Yp) and LN(Ys) and seven screening index values for 31 inbred lines.

F
Source of Variation Df
Yp Ys SSI STI MP GMP YSI HM LNTI
Genotype (G) 30 12.053** 7.192** 3.037** 20.545** 11.36** 13.301** 2.334** 10.524** 2.616**
Year (Y) 2 7.11** 31.34** 5.994** 16.703** 25.951** 34.471** 18.105** 30.118** 17.411**
Location (L) 1 1012.76** 4.196* 0.764 247.582** 304.328** 219.199** 261.056** 97.121** 256.633**
G×Y 60 1.65** 1.158 1.884** 1.447* 1.269 1.544* 1.435* 1.436* 1.477*
G×L 30 9.969** 5.114** 6.958** 14.068** 8.32** 8.421** 4.586** 6.957** 4.481**
G×L×Y 60 2.742** 1.726** 2.532** 2.303** 2.683** 2.843** 1.307 2.366** 1.31
* indicates difference at p < 0.05 level; ** indicates significant difference at p < 0.01 level. Df indicates degrees of freedom.
3.2. Correlation Analysis for the Seven Screening Indices and Grain Yield under HN and LN
Treatments of 312019,
Agronomy Inbred
9, 240Lines 7 of 14

To select the best and most effective index as the selection criterion for discriminating the
3.2. Correlation Analysis for the Seven Screening Indices and Grain Yield under HN and LN Treatments of 31
desirableInbred
genotypes,
Lines the correlation coefficients between the BLUP value of grain yield under HN
(Yp) and LN (Ys) treatments and seven screening indices for 31 inbred lines were determined and are
To select the best and most effective index as the selection criterion for discriminating the desirable
presented in Figure
genotypes, the 1. Correlation
correlation analysis
coefficients showed
between the BLUPthatvalue
Yp had a significantly
of grain yield under HNpositive
(Yp) and LNassociation
with MP(Ys)(r =treatments
0.92), GMP and (r = 0.87),
seven HM indices
screening (r = 0.83) and
for 31 STI lines
inbred (r = 0.77); And Ys had
were determined andaare
significant
presented positive
relationship with1. MP
in Figure (r = 0.91),
Correlation GMP
analysis (r = that
showed 0.95),
Yp STI
had a(rsignificantly
= 0.92), HM (r = association
positive 0.97), andwith YSIMP(r = 0.59),
(r = 0.92), GMP (r = 0.87), HM (r = 0.83) and STI (r = 0.77); And Ys had a
indicating that MP, GMP, HM, and STI were able to identify the most stable and productive significant positive relationship
with MP (r = 0.91), GMP (r = 0.95), STI (r = 0.92), HM (r = 0.97), and YSI (r = 0.59), indicating that MP,
genotypes under both the stressed and non-stressed conditions. The several indices were dealt with
GMP, HM, and STI were able to identify the most stable and productive genotypes under both the
two major indexand
stressed groups: (1) selecting
non-stressed conditions.high-yielding lines were
The several indices (MP,dealt
GMP, STI,
with twoand HM)
major index and (2) selecting
groups:
most stable lines (SSI,
(1) selecting YSI, andlines
high-yielding LNTI). AfterSTI,
(MP, GMP, correlation
and HM) and analysis, the most
(2) selecting GGEstable
biplots
linesgenerated
(SSI, YSI, using
Genstat and
19.0LNTI).
(Beijing VSNC
After Statistics
correlation Software
analysis, the GGECo,.Ltd, Beijing, using
biplots generated China) 19.0,19.0
Genstat showed
(Beijingthat
VSNC MP, GMP,
Statistics Software Co,.Ltd, Beijing, China) 19.0, showed that MP, GMP, STI, and
STI, and HM were associated positively Yp and Ys (Figure 2). Therefore, the analysis agreed with the HM were associated
positively Yp and Ys (Figure 2). Therefore, the analysis agreed with the results above.
results above.

Figure 1. Correlation coefficients and diagram between grain yield (Yp and Ys) and seven screening
Figure 1.index values for the
Correlation 31 inbred lines.
coefficients and Data above the
diagram diagonal
between are theyield
grain correlation
(Yp andcoefficients.
Ys) andData below
seven screening
the correlation coefficients are the confidence intervals. The pie charts below the diagonal indicates
index values for the 31 inbred lines. Data above the diagonal are the correlation coefficients. Data
the correlation size. The blue color in the clockwise pie charts indicates that the two variables are
below the correlation
positively coefficients
correlated, and the redare the
color in confidence intervals.
the counterclockwise The pie
pie charts charts
indicates thatbelow the diagonal
the variables
indicatesare
the correlation
negatively size. The blue color in the clockwise pie charts indicates that the two variables
correlated.
are positively correlated, and the red color in the counterclockwise pie charts indicates that the
variables are negatively correlated.
Agronomy 2019,
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x FOR PEER REVIEW 28 of 14
of 14

Figure 2. Drawing of the Genotype and genotype × environment (GGE) biplot based on the first (PC1)
and the2.second
Figure Drawing (PC2) principal
of the Genotype component for 31× inbred
and genotype lines and
environment different
(GGE) biplot indices.
based onTransform = 0,
the first (PC1)
Scaling = 0, Centering = 2, and SVP = 2. Numbers assigned to the genotypes are:(1)
and the second (PC2) principal component for 31 inbred lines and different indices. Transform = 0, KA008; (2) 2012KA-1;
(3) KA064;
Scaling = 0, (4) 2012KA= -58;
Centering 2, and(5)SVP
KA103; (6) KA203;
= 2. Numbers (7) 2013KA-34;
assigned (8) KA105;
to the genotypes (9) KA227;
are:(1) (10)2012KA-
KA008; (2) KA225;
(11) XCA-1;
1; (3) KA064;(12)KA060;
(4) 2012KA (13)KB081; (14) KB417;
-58; (5) KA103; (15) KB109;
(6) KA203; (16)91227; (17)KB-7;
(7) 2013KA-34; (8) KA105; (18)(9)
KB020;
KA227;(19)(10)
2013KB-37;
KA225;
(20)
(11) 2013KB-47; (21) KB043;(22)Z140588;
XCA-1; (12)KA060; (13)KB081 ; (14) (23)KB417;
Z140580;(15)(24) 2013HXB-4;
KB109; (25) 2013ZZB-6;
(16)91227; (17)KB-7; (18) (26)KB020;
2014KB-54;
(19)
(27) KB215; (28) Zheng58; (29) Chang7-2; (30) PH6WC; and (31) PH4CV. Dotted
2013KB-37; (20) 2013KB-47; (21) KB043;(22)Z140588; (23) Z140580; (24) 2013HXB-4; (25) 2013ZZB-6; vertical and horizontal
lines indicate points
(26) 2014KB-54; (27)where
KB215;the(28)
PC1Zheng58;
and PC2 (29)axes Chang7-2;
had values(30) of zero. Smaller
PH6WC; andvectors angles indicate
(31) PH4CV. Dotted
avertical
larger correlation value, a positive ◦
and horizontal lines indicatecorrelation
points where exists,
thewhen
PC1 andthe PC2
angleaxes
is less
hadthan 90 ,of
values and a negative
zero. Smaller
correlation
vectors angleswhen greaterathan
indicate 90◦correlation
larger . value, a positive correlation exists, when the angle is less
than 90°, and a negative correlation when greater than 90°.
3.3. PCA for Seven Screening Indices and Grain Yield under HN and LN Treatments of 31 Inbred Lines
3.3. PCA for Seven
The main Screening of
components Indices and Grain
attribute Yield under
performance wereHN and LN using
analyzed Treatments
grainofyield
31 Inbred Lines
and endurance
indices, both calculated based contrasting N calculated as 91.03% (Table 3 and Figure 2). As the first
The main components of attribute performance were analyzed using grain yield and endurance
two components were interpretable (value > 1) and deleting the other components that had minimal
indices, both calculated based contrasting N calculated as 91.03% (Table 3 and Figure 2). As the first
effect on the variations (value < 1), the GGE biplot was drawn based on the first two components
two components were interpretable (value > 1) and deleting the other components that had minimal
using BLUP data value for Yp, Ys, GMP, MP, STI, YSI, HM, LNTI, and SSI. The first component
effect on the variations (value < 1), the GGE biplot was drawn based on the first two components
explained 68.14% of the variations and demonstrated strong correlations with Yp, Ys, GMP, MP,
using BLUP data value for Yp, Ys, GMP, MP, STI, YSI, HM, LNTI, and SSI. The first component
STI, and HM. This component could be called the group of great performance under both two N
explained 68.14% of the variations and demonstrated strong correlations with Yp, Ys, GMP, MP, STI,
conditions which was able to discriminate the high grain yield in HN and LN conditions. The second
and HM. This component could be called the group of great performance under both two N
component explained 22.89% of the variations, having a strong correlation with SSI, YSI, and LNTI in
conditions which was able to discriminate the high grain yield in HN and LN conditions. The second
the LN condition or sensitivity to stress. The second component could be used to determine genotype
component explained 22.89% of the variations, having a strong correlation with SSI, YSI, and LNTI
sensitivity to LN condition. Therefore, selecting genotypes that have high PC1 and low PC2 are suitable
in the LN condition or sensitivity to stress. The second component could be used to determine
for both HN and LN conditions. The results showed that the six genotypes KB081, KA105, PH6WC,
genotype sensitivity to LN condition. Therefore, selecting genotypes that have high PC1 and low PC2
91227, 2013KB-47, and KA225 best reflected these qualities, and would thus have the best LN tolerance
are suitable for both HN and LN conditions. The results showed that the six genotypes KB081, KA105,
with high PC1, but low PC2 values.
PH6WC, 91227, 2013KB-47, and KA225 best reflected these qualities, and would thus have the best
LN tolerance with high PC1, but low PC2 values.

Table 3. Results of principal component analysis for grain yield under HN (Yp) and LN (Ys) and seven
screening index values for 31 inbred lines.
Agronomy 2019, 9, 240 9 of 14

Agronomy 2019, 9, x FOR PEER REVIEW 3 of 14

Table 3. Results of principal component analysis for grain yield under HN (Yp) and LN (Ys) and seven
screening index values for 31 inbred lines.
Contribution Cumulative
Yp Ys GMP MP STI SSI YSI HM LNTI
to variation
Contribution percentage
Cumulative
Yp Ys GMP MP STI SSI YSI HM LNTI
to Variation Percentage
————%———— ——kg ha−1——
————%———— −1
——kg ha ——
PC1
PC1 68.14
68.14 68.14 68.140.77 0.77
0.99
0.99
0.97
0.97
0.95
0.95
0.94
0.94
−0.50
−0.50
0.56
0.56
0.98
0.98
−0.56
−0.56
PC2 PC2 22.89 22.89 91.03 91.030.61 0.61
−0.06 −0.06
0.21 0.21
0.29 0.29
0.12 0.12
0.70 0.70
−0.64 −0.64
0.15 0.15
0.79 0.79

3.4.
3.4. 3D 3D Diagram
Diagram for thefor
STItheIndices
STI Indices and Grain
and Grain Yield under
Yield under HN andHN
LNand LN Treatments
Treatments of 31 Inbred
of 31 Inbred Lines Lines
Considering
Considering the positive
the positive correlation
correlation betweenbetween the
the STI STIYp,
and andYs,Yp,
andYs,the
and the character
character of stability
of stability in in
production
production for thefor the value
value of STI,ofa STI, a 3D diagram
3D diagram basedbased
on STIon STIdrawn
was was drawn to discriminate
to discriminate the 31the 31 maize
maize
inbredinbred lines according
lines according their performance
their performance (Figure(Figure 3). Based
3). Based on theon3Dthe 3D diagram,
diagram, KB081, KB081,
KA225, KA225,
91227,91227,
2013KB-47,
2013KB-47, PH6WC,PH6WC, and KA105
and KA105 were identified
were identified as theas the target
target genotypes
genotypes that produced
that produced a suitable
a suitable STI STI
value. These genotypes with a higher STI value were also located the first quadrant,
value. These genotypes with a higher STI value were also located the first quadrant, which indicates which indicates
a higher
a higher yield under
yield under HN and HNLN and LN conditions.
conditions. The results
The results indicated
indicated these these genotypes
genotypes are dominant
are dominant
underunder both stressed
both stressed and non-stressed
and non-stressed environments
environments and the
and have have the potential
potential to breedto breed
more more
stablestable
and and
high yielding hybrids. These results verify the evaluation function of the
high yielding hybrids. These results verify the evaluation function of the screening indices. screening indices.

Figure 3. Three-dimensional diagram for identifying LN tolerant genotypes based on Yp and Ys as

well as the STI.
Figure 3. Three-dimensional diagram for identifying LN tolerant genotypes based on Yp and Ys as
3.5. GGE Biplot for Seven Screening Indices and Grain Yield under HN and LN Treatments of 31 Inbred Lines
well as the STI.
The yield and stability of maize inbred lines are shown in Figure 4. The GGE biplot was drawn
using 3.5.
the GGE
valueBiplot
of Yp for
andSeven Screening
Ys under Indices and
all locations, all Grain
years,Yield under HN and
and containing LN Treatments(HN+LN).
12 environments of 31
Inbred Lines
The genotypes of PH6WC, KA105, KB081, KA225, 91227, and 2013KB-47 were located in front of
the direction
Theofyield
the arrow, indicating
and stability that inbred
of maize these genotypes couldin
lines are shown produce a high
Figure 4. yield.biplot
The GGE However,
was drawn
KA008 and KB043 were located behind the direction of the arrow, implying that the two
using the value of Yp and Ys under all locations, all years, and containing 12 environments (HN+LN). genotypes
wouldThe
produce the lowest
genotypes grain yield.
of PH6WC, KA105,TheKB081,
stable KA225,
inbred lines,
91227,which individually
and 2013KB-47 demonstrated
were goodof the
located in front
performance and
direction of stability,
the arrow, were observed
indicating thatinthese
XCA-1, 91227, and
genotypes could2013KB-37.
produce aThe sensitive
high genotypesKA008
yield. However,
were identified as KA103, 2013KB-34, and KA064. In conclusion, the genotypes
and KB043 were located behind the direction of the arrow, implying that the two genotypes of PH6WC, KB081,would
91227,produce
2013KB-37, and KA105 produce high values for high yield and yield stability. We
the lowest grain yield. The stable inbred lines, which individually demonstrated good discriminated
the high-yielding
performanceand andstable inbred
stability, lines
were that have
observed in the potential
XCA-1, 91227,to and
breed desirable hybrids
2013KB-37. with great
The sensitive genotypes
were identified as KA103, 2013KB-34, and KA064. In conclusion, the genotypes of PH6WC, KB081,
91227, 2013KB-37, and KA105 produce high values for high yield and yield stability. We
discriminated the high-yielding and stable inbred lines that have the potential to breed desirable
 Agronomy 2019, 9, 240 10 of 14
Agronomy 2019, 9, x FOR PEER REVIEW 4 of 14

performance
hybrids in both
with great HN and in
performance LNboth
conditions.
HN and LN Theconditions.
results of GGE biplot analysis
The results agreedanalysis
of GGE biplot with the
agreed with the analysis of the screening indices, which verified the accuracy and efficiency of theon
analysis of the screening indices, which verified the accuracy and efficiency of the assessment based
the index based
assessment selected.
on the index selected.

Figure 4. GGE biplot of high yield and stable maize inbred lines. Transform = 0, Scaling = 0, Centering = 2,
Figure 4. GGE biplot of high yield and stable maize inbred lines. Transform = 0, Scaling = 0,
and SVP = 1. The small circle represents the average environment and the straight line with an arrow is the
Centering = 2, and SVP = 1. The small circle represents the average environment and the straight line
average environment vector that points in the direction of greater performance. The vertical line drawn by
with an arrow is the average environment vector that points in the direction of greater performance.
the origin to the average environment vector represents the sensitivity of the participating inbred. From the
The vertical line drawn by the origin to the average environment vector represents the sensitivity of
point of the genotype to the average environment, the vector is perpendicular. The longer the perpendicular,
the participating inbred. From the point of the genotype to the average environment, the vector is
the more sensitive the genotype. Conversely, the shorter the projection, the more stable the variety.
perpendicular. The longer the perpendicular, the more sensitive the genotype. Conversely, the shorter
the projection, the more stable the variety.
4. Discussion

4. Discussion
4.1. Evaluation of Yield-Based Low Nitrogen Tolerance Indices
The general agreement is that the modern high yielding crop cultivars are more adaptable to HN
4.1. Evaluation of Yield-Based Low Nitrogen Tolerance Indices
conditions [38,39]. LN is regarded as an abiotic stress for the growth of maize plants, which hinders
or The
limitsgeneral agreement
the expression is that
of their the modern
genetic potential high yielding
in growth andcrop cultivars are
development morethereby
stages, adaptable to
affecting
HN theconditions
performance [38,39]. LN yield
of grain is regarded as an abiotic
[40]. Scholars stress for
have usually the growth
considered the of maize plants,
accessions which
of maize were
hinders or limits the expression of their genetic potential in growth and development
selected fittingly when maize plants were treated without or less N input [41]. In this study, seven stages, thereby
affecting
screeningtheindices,
performance
MP, GMP, of grain yield
STI, YSI, HM, [40].
SSI,Scholars
and LNTI, havewereusually
used to considered
evaluate the the different
accessions of
aspects
maize were
of the selected fittingly
performance when maize
of LN tolerance in 31plants
maizewere
inbredtreated
lines.without
Geometric or less
mean N input [41]. In (GMP)
productivity this
study, seven screening indices, MP, GMP, STI, YSI, HM, SSI, and LNTI,
indicated the mean performance of a genotype across in the two environments of with and without were used to evaluate the
different aspects
N fertilizer [19].ofThethemean
performance
productivityof LN indextolerance in 31 maize
(MP) denotes as the inbred
mean grainlines.yield
Geometric
in HN mean
and LN
productivity (GMP) indicated the mean performance of a genotype across
environments. The definitions of GMP and MP imply that the two indices reflect the mean performance in the two environments
of of
with and
yield without
under N fertilizer
different [19]. The
N conditions, whichmean productivity
could mistakenlyindexidentify (MP)
the denotes as the mean
poor production grain
and evaluate
yield
the in HN and if
genotype LN environments.
the genotype had The definitions
good production of GMP
in oneandenvironment,
MP imply thatbut thethe
twopoor
indices reflect
in the other.
theTherefore,
mean performance of yield under different N conditions, which could mistakenly
the selection of a desirable genotype using only GMP and MP is partial and could result in identify the
poor production
errors. Fernandez and[19]evaluate thethat
reported genotype if the
the stress genotype
tolerance had(STI)
index goodcouldproduction
be usedintoone environment,
evaluate genotypes
butthat
thehave
poorhigh
in the other. Therefore,
production under both the normal
selection of stressed
and a desirable genotypeThe
conditions. using
STIonly GMP
depicts and MP is
to performance
partial
underand could
stress result and
condition in errors. Fernandez
Ymp, which is the[19]
mean reported
yields ofthat the stressunder
all cultivars tolerance index
stressed and(STI) could
non-stressed
beconditions,
used to evaluate
whichgenotypes that have
was considered high production
to rectify under both
the partial results shown normal
above. and stressed conditions.
Therefore, the STI value
The STI depicts to performance under
plays an important role in selection practice. stress condition and Ymp, which is the mean yields of all
cultivars under stressed and non-stressed conditions, which was considered
The genotype KB417 has a lower value of MP and GMP due to the low performance in grain yield, to rectify the partial
results
but ashown
higher above. Therefore,
STI value, the STI that
which implies valueKB417playswould
an important
be regardedrole inasselection
a geneticpractice.
resource to provide
the genetic variation for hybrids with stable yields. The value of the harmonic mean (HM)in
The genotype KB417 has a lower value of MP and GMP due to the low performance grain
proposed
yield, but a higher STI value, which implies that KB417 would be regarded as a genetic resource to
provide the genetic variation for hybrids with stable yields. The value of the harmonic mean (HM)
Agronomy 2019, 9, 240 11 of 14

high yield and stability to some extent [20]. By considering the high positive correlation between the
GMP, MP, STI, and HM indices and grain yield under both HN and LN conditions, we confirmed that
GMP, MP, HM, and STI together reflect the character of high yield and stable yield for maize plants.
Similar results have been illustrated by other scholars in wheat, soybean, and rice [21,42–44].
Other results demonstrated a strong negative correlation between LNTI, SSI and grain yield under
the LN condition. The SSI exhibited a degree of reduction in the grain yield under LN condition when
compared to the grain yield under the HN condition [36]. The LNTI was proposed for evaluating the
LN tolerant genotypes under stressed condition [16]. These indices show the sensitivity of maize inbred
lines under LN conditions; meanwhile, there was no association with yield in optimal conditions. If the
two indices used individually, then genotype are identified that could not response the stress under
stressed conditions, the result is one-side. The SSI and LNTI could be used as supplementary indices,
the similar results have been reported in other assays [18].

4.2. Analysis of the Low Nitrogen Tolerance Evaluation System for Maize
To use high NUE materials and screen for favorable genotypes, researchers have adopted a variety
of methods to classify different maize varieties or inbred lines. Several analysis methods are available for
evaluating LN tolerance and the NUE of maize inbred lines or hybrids. Fotyma et al. [45] concluded
that crop N used could be grouped into agronomic efficiency and physiological efficiency. Whereas,
Moll [46] suggested a NUE contained the uptake efficiency (UpE) and utilization efficiency (UtE), which
reflect the ability of plants to absorb and accumulate nitrogen from soil and convert nitrogen into yield,
respectively. Good et al. [47] identified the agronomic efficiency (AE), physiological efficiency (PE),
and apparent recovery (AR) to reflect the response of plants to N fertilizer. The low nitrogen tolerant
index (LNTI) and tolerant index (TI) were proposed to evaluate the efficiency of N use in different
crops [16,18,48]. Previous studies of screening LN tolerant individuals have also focused on several
physiological traits or agronomic traits, including root yield, sucrose content, starch content, leaf area,
and above ground biomass [49]. Other researchers proposed different evaluation systems given different
priorities in the study.
Many studies have been published regarding the search for suitable screening indicators to
evaluate crop tolerance, but no agreement has been reached thus far [50]. The screening indices may
provide another way to be screening. In our study, the BLUP value of the grain yield combined with
PCA was used to select a LN tolerance index that could be used to evaluate the LN tolerant individuals,
and five LN tolerant maize inbred lines were screened. The GGE biplot was also used to analyze
the high yield and highly stable genotypes, which was completely consistent with the results of the
analysis that was used to make selection based on the screening indices. The GGE biplot also screened
out inbred lines that demonstrated special adaptability at different locations. The application of the
GGE biplot method for N evaluation in maize inbred lines is still rare. Our research strengthens the
support of the use of this method.

4.3. Selecting Low Nitrogen Tolerant Maize Accessions from Shaan A and Shaan B Groups
A good cultivar used in practice must produce both a high and stable yield to pursue economic
interests. Our group has bred under LN and other abiotic stress conditions, with the purpose of
choosing inbred lines with high and stable yields. Some inbred lines, such as 91227, have been proven
successful [28,51]. Based on these successful inbred lines, we have approved more than 10 varieties
including a national variety named Shaandan 609 (No. 2016001). These achievements show that our
breeding strategy is efficient and feasible. In this study, 31 inbred lines chosen from the Shaan A and
Shaan B groups were used to identify their LN tolerance. Using selection indices, a 3D diagram and
GGE biplots verified, we identified the KA105, KB081, KA225, 91227, and 2013KB-47 as favorable
genotypes with high quality performance under HN and LN conditions. These inbred lines can be used
to provide the basic germplasm for our high-yield and low nitrogen breeding of maize in the future.
Agronomy 2019, 9, 240 12 of 14

5. Conclusions
The results of this study suggest that the combination of the screening indices STI (stress tolerance
index), MP (mean productivity), GMP (geometric mean productivity), and HM (harmonic mean)
is suitable for determining genotypes with low nitrogen tolerance, as these indices consider the
performance under optimal and limited nitrogen conditions. Based on a three-dimensional diagram
and GGE biplots, we identified KA105, KB081, KA225, 91227, and 2013KB-47 as the desired genotypes
that will provide the basic germplasm to breed high yield and stable yield maize hybrids.

Author Contributions: J.L. and J.X. conceived and designed the experiments; K.H., L.C. and Y.L., X.Z. performed
the experiments; Z.Z., Z.F., and S.X. analyzed the data; Z.Z. wrote the paper.
Funding: This study was supported financially by the Key R&D Program of Science and Technology of Shaanxi
Province (2017ZDCXL-NY-02-04), the Innovation Project of National Key R&D Program of China (2017YFD0101203),
the National Natural Science Foundation of China (31301830), and the Innovation Project of Science and Technology
of Shaanxi Province (2015KTZDNY01-01-01).
Conflicts of Interest: The authors declare no conflict of interest.

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