The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Introduction {#s1}
============

Starch or amylum is a carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. Starch rich crops are the main source of dietary energy for the world\'s population. It has been believed that plant species share the evolutionarily conserved pathway of starch biosynthesis starting from the carbon dioxide fixation, followed by transitory starch degradation, sucrose synthesis, and starch synthesis in the storage organs [@pone.0103627-Saithong1]. Four classes of enzymes are included in the starch biosynthesis pathway, they are ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE), and starch debranching enzyme (DBE) [@pone.0103627-Yang1], [@pone.0103627-Tian1]. Among them, AGPase catalyzes the first committed and rate-limiting step in this pathway, and plays vital role in the biosynthesis of starch [@pone.0103627-Kirchberger1]. Under the catalyzing of AGPase, ADP-glucose (ADP-Glc) is synthesized in the cytosol of cereal endosperms as the main precursor for starch synthesis and has to be subsequently imported into the storage plastids [@pone.0103627-Kirchberger1]. Because of the importance of cereals in the production of storage starches for human diet and other industrial usage, the activity of the ADP-Glc transporter have gained many attentions as a key component of the starch biosynthesis pathway [@pone.0103627-Bowsher1].

One of the ADP-Glc transporters demonstrated clearly in cereal is the protein Brittle1. Brittle1 proteins are plant nucleotide transporters involved in the mitochondrial carrier family (MCF) [@pone.0103627-Bahaji1]. The proteins in MCF family transports nucleotides, amino acids, inorganic ions, fatty acids, keto acids and cofactors across the mitochondrial membrane [@pone.0103627-Laloi1]. Physiological researches on the maize *Brittle1* mutant have revealed that ZmBT1 (*Zea mays* Brittle1 protein) was involved in the ADP-Glc transport into endosperm plastids, and played critical roles in the biosynthesis of starch [@pone.0103627-Kirchberger1], [@pone.0103627-Shannon1], [@pone.0103627-Sullivan1]. The maize endosperm with *bt1* mutant is severely reduced in starch content, which results in kernels with a collapsed angular appearance at maturity [@pone.0103627-Kirchberger1]. The amyloplasts from young kernels isolated from endosperms with *bt1* mutant were only 25% as active in ADP-Glc uptake and conversion to starch as amyloplasts from normal and mutant maize endosperms, suggesting that ZmBT1 is involved in the transport of ADP-Glc into maize endosperm plastids [@pone.0103627-Shannon1], [@pone.0103627-Li1]. The researches in other cereals also revealed that the homologs of BT1 protein possessed the ability in transporting ADP-Glc. For example, the barley *lys5* mutant, a homolog of *BT1* (*HvNST1*), show a reduced capacity for ADP-Glc uptake by isolated endosperm amyloplasts [@pone.0103627-Bowsher1], [@pone.0103627-Patron1]. However, the BT1 proteins in dicots, such as AtBT1 in *Arabidosis* and StBT1 in *Solanum tuberosum* do not transport ADP-Glc, but instead transport AMP, ADP and ATP in a unidirectional mode [@pone.0103627-Leroch1].

Maize (*Zea mays* L.) is one of the most important grown cereals in the world. It provides staple food to many populations, as well as a major nutrient source for animal feed. In addition, benefitting from its unique character such as low pasting temperature and slow tendency of retrogradation, maize starch is one of the important raw materials for industrial production of food. The pasting properties of maize starch will enormously affect fabrication property, flavor characteristics and keeping in storage. Recently, the RVA profile of starch paste viscosity was widely employed to evaluate the quality of cereal crops, because this method requires only a small size and the procedure is easy to perform [@pone.0103627-Yan1], [@pone.0103627-Sanchez1]. Starch gelatinization, one of the most important and unique properties, refers to the process of the disruption of granular structure causing starch molecules to dissolve in water [@pone.0103627-Ratnayake1]. The gelatinization properties of starch are the most important indexes in many food modification including cooking, baking and extruding starch-based foods [@pone.0103627-Hasjim1]. Although the physiological roles on the starch biosynthesis of the maize *ZmBT1* gene has been illustrated, the effect of this gene in the formation of maize starch pasting and gelatinization properties reminds unknown. Moreover, there is no association analysis between the nucleotide polymorphisms of the maize *ZmBT1* gene and the physicochemical properties of maize starch. In this work, we analyzed the nucleotide polymorphism of maize *ZmBT1* locus, and investigate the association between the sequence polymorphisms of the maize *ZmBT1* gene and some starch pasting and gelatinization properties.

Materials and Methods {#s2}
=====================

Plant materials and sequencing the maize *ZmBT1* gene {#s2a}
-----------------------------------------------------

A total of 80 elite maize inbred lines were used in this study. These inbred lines were also the representative lines, including temperate germplasm from 5 heterotic groups, tropic and waxy germplasm ([Table 1](#pone-0103627-t001){ref-type="table"}). They represented most of the genetic diversity available to breeding and research programs in China. In addition, some germplasm introduced from other countries were also included in this study. The inbred lines were grown in two-row plots with an randomized block design of two repetitions in a natural environment during 2012 in Sanya, Hainan province. The mature seeds for each inbred lines were harvested in bulk for phenotypic data analysis.

10.1371/journal.pone.0103627.t001

###### List of the 80 maize inbred lines used in this study.

![](pone.0103627.t001){#pone-0103627-t001-1}

  No.    Inbred Line            Origin and Pedigree            No.   Inbred Line          Origin and Pedigree
  ----- ------------- --------------------------------------- ----- ------------- -----------------------------------
  1        QH19612            Derived from Huangzaosi          41       6819              Derived from P78599
  2       Chang7-2           (Huangzaosi×Wei95) ×S901          42      Dan988             Derived from P78599
  3        LX9801                    Ye502×H21                 43       319B              Derived from P78599
  4          107                    Dekalb×L80                 44       Qi319             Derived from P78599
  5          k12                Huangzaosi×Huaichun            45       Qi318             Derived from P78599
  6         nx335                   NF358×PH4CV                46      Shen137           Derived from P6JK111
  7         Ji853         (Huangzaosi×Zi330) ×Huangzaosi       47       11099             Tropical germplasm
  8          412                    Mo17×Jing09                48       suwan             Tropical germplasm
  9         Za107                   Dekalb×L80                 49       11118             Tropical germplasm
  10     Huangzaosi           Derived from Sipingtou           50       11200             Tropical germplasm
  11         502                 Huangzaosi×Dan340             51      10533-1            Tropical germplasm
  12      Luyuan92                Yuanqi122×1137               52      RCML15             Tropical germplasm
  13        10168                Derived from 5003             53       RBS11             Tropical germplasm
  14       Dan598      (Dan340×Danhuang11) ×(Danhuang02×599)   54        Q52        Derived from an USA crossbreed
  15        Zong3                   Zi330×Lv28                 55      KWS456              Derived from KWS
  16         4CV                 Derived from Mo17             56       QF-11                   Unknown
  17         E28          (A619Ht1×Lvjiukuan) ×Lvjiukuan       57       QF01         Derived from French germplasm
  18        S122              (YeH201×Dan340) ×Dan340          58       JND-1      Derived from a Canadian germplasm
  19       Dan340                Baigulv9×Pod corn             59       BJ-2               Derived from Mo17
  20     Danhuang25             Derived from P78599            60       QF02         Derived from French germplasm
  21       JH3372                 Shen5003×Zi330               61       QDM01              Derived from KWS
  22        Dan99            Derived from Lvdahonggu35         62        M1                     Unknown
  23        Qi232                     Unknown                  63       WT262              Derived from KWS
  24        OH43                     OH40b×W8                  64       WT26               Derived from KWS
  25        MO17                    C103×187-2                 65        Y53                    Unknown
  26        8112                    P3382×P3147                66       BJ-5               Derived from Mo17
  27        K8112                   P3382×P3147                67      QZN01F               Waxy germplasm
  28        Wu314          (302D×Huangbaoli) ×Huangzaosi       68      QKN01M               Waxy germplasm
  29        4866                    7922×Ye478                 69       QDT01               Waxy germplasm
  30        3189                  U8112×Shen5003               70      QLY01Z               Waxy germplasm
  31       Tie9206                Tie8706×Tie8708              71      QJKN20F              Waxy germplasm
  32       Benyu15                    Unknown                  72       QZN12               Waxy germplasm
  33       478Xuan                U8112×Shen5003               73      QKN01F               Waxy germplasm
  34       Zheng58              Derived from Ye478             74       QBN48               Waxy germplasm
  35        7922                Derived from P3382             75      QBN029               Waxy germplasm
  36         178                    P78599×Mo17                76        BEM                Waxy germplasm
  37       QP1721               Derived from P78599            77       QBN02               Waxy germplasm
  38        Exhan               Derived from P78599            78      QBN3186              Waxy germplasm
  39        xy35              Derived from Xianyu335           79       M1132               Waxy germplasm
  40        P138                Derived from P78599            80      QZN012               Waxy germplasm

Young plant leaves were collected at the four-leaf stage for each accession and stored at −80°C until genomic DNA extraction. Genomic DNA was extracted from the frozen young leaves of the 80 inbred lines using CTAB (cetyl trimethyl ammonium bromide) method [@pone.0103627-Fulton1] according to the modified protocol. The sequences of the *ZmBT1* gene in the tested inbred lines were sequenced using the target sequence capture sequencing technology on the NimbleGen platform [@pone.0103627-Asan1] by BGI Life Tech Co., Ltd.. The genomic sequence and position of the maize *ZmBT1* gene (GRMZM2G144081) of the inbred line B73 were used as the reference sequences for target sequence capture.

Measurement of maize starch pasting and gelatinization properties {#s2b}
-----------------------------------------------------------------

The pasting properties were measured using a rapid visco analyser (RVA) (Model No. RVA-3D, Newport Scientific, Sydney, Australia). A total of 3-g starch from each inbred line was dispersed in 25 ml of distilled water in the viscometer test canister. The sequential temperature curve for a 12.5 min test was as follows: (1) incubate at 50°C for 1.0 min; (2) increase to 95°C; (3) keep at 95°C for 2.5 min; (4) cool down to 50°C; and (5) hold at 50°C for 1.4 min. The viscosity was evaluated using a constant paddle rotation of 160 rpm. Viscosity values were recorded in centipose (cp).

The gelatinization properties of mazie starches were analyzed using a differential scanning calorimeter DSC 200F3 Maia (Netzsch, Germany). Starch samples (5 mg, dried starch basis) were precisely weighed in the sample pans, mixed with distilled water (10 µl), and sealed. The heating rate was at 10°C per min over the temperature range of 20--100°C. The gelatinization properties were recorded with a thermal analysis data station equipped in DSC.

Sequence analysis {#s2c}
-----------------

Multiple sequence alignment of the maize *ZmBT1* gene was performed using Clustal X and was further edited manually. The software DNASP 5.0 [@pone.0103627-Librado1], [@pone.0103627-Rozas1] was used to analyze sequence nucleotide polymorphism and allelic diversities. Two parameters of nucleotide diversity, and ~,~ were estimated. Where is the average number of nucleotide differences per site between any two DNA sequences, and is derived from the total number of segregating sites and corrected for sampling size. Tajima\'s D [@pone.0103627-Tajima1] and Fu and Li\'s D\* and F\* [@pone.0103627-Fu1] statistical tests were used to test the evidence of neutral evolution within the selected population and each defined region. The minimum number of recombination events [@pone.0103627-Hudson1] was estimated in the period of evolution of *ZmBT1* gene among the tested inbred lines. The linkage disequilibrium (LD) between any two polymorphic sites were estimated using TASSEL v3.0 [@pone.0103627-Bradbury1]. In addition, the decay of LD with physical distance in *ZmBT* gene was evaluated by regression analysis (PROC NLIN and REG in SAS software). The regression models, including linear, loglinear, exponential, power and Remington\'s models [@pone.0103627-Remington1], were used in this study.

Population structure and association analysis {#s2d}
---------------------------------------------

Population structure is a major bias factor leading to false-positive associations. To alleviate the effect of population structure, all inbred lines were genotyped with the SNP chips contained 3,072 random SNP markers evenly covering the maize genome. These SNP markers were selected from 49,585 SNP markers used by recently reported chips [@pone.0103627-Ganal1]. SNP genotyping was performed via the GoldenGate assay at the National Maize Improvement Centre of China, China Agricultural University. The population structure was evaluated by these SNP markers, and the resulting Q-values were obtained from the STRUCTURE program [@pone.0103627-Hubisz1]. Five independent runs were performed setting the number of populations () from 2 to 8, burn in time and MCMC (Markov Chain Monte Carlo) replication number both to 100,000, and a model for admixture and correlated allele frequencies. The value was determined by LnP(D) in STRUCTURE output and an ad hoc statistic based on the rate of change in LnP(D) between successive . The tests of significant association between the sequence polymorphisms with Minor Allele Frequency (MAF) and starch pasting and gelatinization properties in the tested population were performed using the general linear model (GLM) in the TASSEL software v3.0 [@pone.0103627-Bradbury1].

Results {#s3}
=======

Nucleotide diversity and selection of the maize *ZmBT1* gene {#s3a}
------------------------------------------------------------

The position and nucleotide sequences of the maize *ZmBT1* gene in inbred line B73, whose genome has been fully sequenced, were used as the references to capture of the sequences of this gene in 80 inbred lines. Sequence polymorphisms were detected among 80 maize inbred lines across 2,442 bp of sequence, which covers a 520 bp 5′ upstream promoter region, a 624 bp exon_1 region, a 131 bp intron_1 region, a 162 bp exon_2 region, a 128 bp intron_2 region, a 534 bp exon_3 region and a 337 bp 3\'-UTR region. Nucleotide substitutions and indels at the *ZmBT1* locus were identified, and the results were summarized in [Table S1](#pone.0103627.s001){ref-type="supplementary-material"} and [Table 2](#pone-0103627-t002){ref-type="table"}. From the putative genomic sequences in 80 maize inbred lines, a total of 30 variants were identified, including 22 SNP sites and 8 indels. Among all the SNP sites, only one belongs to singleton variable site, while the other 21 sites belong to parsimony informative sites ([Tables 2](#pone-0103627-t002){ref-type="table"} and [S1](#pone.0103627.s001){ref-type="supplementary-material"}). In addition, 2 indels were found to be singleton variations, while the other 6 indels belonged to parsimony variations. For all the 80 inbred lines, the overall nucleotide diversity () of *ZmBT1* locus was 0.00351. However, we also noticed that the polymorphic sites were unevenly distributed among 7 defined regions of maize *ZmBT1* locus. There is no nucleotide substitution in the regions of intron1, exon2 and intron2. In addition, no indel was found in exon2, while all the other regions possessed at least one indel.

10.1371/journal.pone.0103627.t002

###### Summary of parameters for the analysis of nucleotide polymorphisms of the maize gene *ZmBT1.*

![](pone.0103627.t002){#pone-0103627-t002-2}

  Parameters                                            Promoter        Exon1      Intron1   Exon2   Intron2    Exon3    3\'-UTR   Entire region
  --------------------------------------------------- ------------- ------------- --------- ------- --------- --------- --------- ---------------
  Total length of amplicons (bp)                           520           624         131      162      128       534       337         2442
  Number of all sequence variants (SNPs and indels)         8             6           3        0        1         5         7           30
  Number of nucleotide substitutions (bp)                   7             5           0        0        0         4         6           22
  Number of indels                                          1             1           3        0        1         1         1            8
  Number of indel sites                                     1             3          19        0        7         6         1           37
  Average indel length                                      1             3        6.3333     NAN       7         6         1          4.625
                                                         0.00590       0.00407        0        0        0      0.00297   0.00383      0.00351
                                                         0.00272       0.00163        0        0        0      0.00153   0.00361      0.00185
  Tajima\'s D                                          2.83337\*\*   3.32754\*\*     NAN      NAN      NAN     1.93935   0.14499    2.71716\*\*
  Fu and Li\'s D\*                                       0.36446       1.05574       NAN      NAN      NAN     0.96034   1.13780      1.37729
  Fu and Li\'s F\*                                       1.39842      2.10430\*      NAN      NAN      NAN     1.49149   0.95695     2.23468\*

The Tajima\'s D statistic is a widely used test to identify sequences which do not fit the neutral theory model at equilibrium between mutation and genetic drift. In this analysis, the estimates of Tajima\'s D in the regions of promoter and exon1 were both statistically higher than 0 at the level of 0.01. In addition, we also noticed that the Tajima\'s D statistic for the entire region of the maize *ZmBT1* gene was statistically higher than zero. Furthermore, when we combined all three exons, the estimate of Tajima\'s D was 3.23599, which was also statistically significant at the level of 0.01. These results revealed that low levels of both low and high frequency polymorphisms in maize *ZmBT1* locus, and also indicated a decrease in population size and/or balancing selection. In addition, the estimates of Fu and Li\'s F\* for both coding (2.3416) and entire regions were significant for the *ZmBT1* gene, also suggesting balancing selection on this gene.

Haplotype diversity of the maize *ZmBT1* gene {#s3b}
---------------------------------------------

According to the full length of the *ZmBT1* gene in the tested 80 maize inbred lines, a total of 11 haplotypes were detected with a haplotype diversity (*Hd*) equal to 0.7734 ([Table 3](#pone-0103627-t003){ref-type="table"}). The tested inbred lines were unbalancedly distributed in these haplotypes. Among the haplotypes identified in this analysis, 6 contained only one inbred line. The most frequent haplotype was Hap_1, which contained 29 inbred lines. In addition, we also noticed that four frequent haplotypes, including Hap_1--4, contained 90% of the tested inbred lines.

10.1371/journal.pone.0103627.t003

###### The distribution of haplotypes of *ZmBT1* gene in 80 inbred lines using both the full-length sequence and coding regions.

![](pone.0103627.t003){#pone-0103627-t003-3}

  Haplotype    Number   CDS haplotype                                                                                                      Inbred line
  ----------- -------- --------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Hap_1          29       CDS_Hap_1     Wu314, Dan988, QH19612, Exhan, Dan99, QZN12, M1132, 502, Qi319, QF01, QBN029, JND-1, QDT01, QKN01F, P138, Luyuan92, 178, Shen137, 6819, Chang7-2, QDM01, WT262, LX9801, 107, JH3372, 11200, BJ-5, Danhuang25, 412
  Hap_2          16       CDS_Hap_2                                                            nx335, RBS11, QJKN20F, xy35, M1, 11099, 7922, Tie9206, S122, QF02, Y53, 4CV, WT26, Mo17, BJ-2, BEM
  Hap_3          18       CDS_Hap_3                                               QLY01Z, RCML15, QBN02, E28, Q52, Qi232, K8112, Benyu15, 8112, QZN01F, 11118, 4866, Za107, Dan340, 319B, QF-11, Zong3, Zheng58
  Hap_4          9        CDS_Hap_4                                                                           QZN012, QBN48, KWS456, QBN3186, 10533-1, 3189, Qi318, 10168, 478Xuan
  Hap_5          1        CDS_Hap_1                                                                                                           OH43
  Hap_6          1        CDS_Hap_5                                                                                                          QP1721
  Hap_7          2        CDS_Hap_6                                                                                                      QKN01M, Dan598
  Hap_8          1        CDS_Hap_1                                                                                                        Huangzaosi
  Hap_9          1        CDS_Hap_5                                                                                                            k12
  Hap_10         1        CDS_Hap_1                                                                                                           suwan
  Hap_11         1        CDS_Hap_6                                                                                                           Ji853

In the coding region of the maize *ZmBT1* gene, a total of 9 SNPs were detected. In addition, 2 indels were also identified in the coding regions. When we used the coding sequences to identify the hapotype diversity, a total of 6 haplotypes were identified for these 80 inbred lines ([Table 3](#pone-0103627-t003){ref-type="table"}) with a *Hd* equal to 0.7440. Each of the haplotypes defined by coding sequences of *ZmBT1* gene contained at least 2 inbred lines. The most frequent CDS haplotype was CDS_Hap_1, which contained 32 inbred lines.

Among the SNPs detected in the coding regions, only one belonged to the nonsynonymous site which could cause the replacement change of amino acid sequences. In addition, the variation of 2 indels could also result in the change of ZmBT1 protein. Both of these two indels covered 3 or 6 nucleotides, which will not result in frame shift during translation. The indel7 contained three types, including no deletions, 3 and 6 nucleotides deletions. When we translated the CDS into amino acid sequences, 6 types of ZmBT1 protein sequences were found to be encoded by these inbred lines ([Fig. 1](#pone-0103627-g001){ref-type="fig"}). Three evolutionarily conserved mitochondrial carrier protein domains (Mito_carr, PF00153) were detected in maize ZmBT1 protein using the tool of Pfam. However, None of the four variants of amino acid caused by two indels and one nonsynonymous SNP was located in the regions of these three domains.

![Sequence alignment of maize ZmBT1 proteins encoded by different CDS haplotypes.\
The haplotypes defined by the coding sequences of the maize *ZmBT1* gene were used as the sequence names. Polymorphisms from inferred amino acids were indicated by boxes. Three mitochondrial carrier protein domains were indexed by lines.](pone.0103627.g001){#pone-0103627-g001}

Linkage disequilibrium and recombination events {#s3c}
-----------------------------------------------

Linkage disequilibrium was investigated between pairwise segregating sites in order to predict the expected resolution and marker density needed for candidate-gene association mapping. In this analysis, all the SNPs identified in maize *ZmBT1* gene and the values of were used and the result revealed that more than half of the pairs between any two polymorphic sites of maize *ZmBT1* gene (130 out of 231 for the tested LD are significant at ) showed significant linkage disequilibrium (LD). To test the decay of LD with increasing physical distance, some regression equations, including linear, loglinear, exponential, power and Remington\'s models, were estimated. In this analysis, the linear regression model was selected to fit the data, because this model possessed the highest coefficient of determination. Our result revealed that the LD decayed rapidly with increasing physical distance. The predicted value of declined to 0.1 within 2184 bp at *ZmBT1* locus ([Fig. 2](#pone-0103627-g002){ref-type="fig"}).

![LD patterns across the whole locus of *ZmBT1*.\
(A) LD between pairs of *ZmBT1* sequence polymorphic sites. (B) Decay of LD between pairs of *ZmBT1* sequence informative polymorphisms. The linear regression coefficient is −0.00029.](pone.0103627.g002){#pone-0103627-g002}

The polymorphic sites in the entire *Zmisa2* locus were used to detect the evidence of recombination. The patterns of the polymorphisms identified in inbred lines surveyed in this study indicated the history of recombination at *ZmBT1* locus, which contributed to the haplotype diversity and the decay of LD. However, only one recombination event has been detected according to the algorithm of Hudson and Kaplan for minimum number of recombination events, and the recombination has been detected between sites 979--1793 bp.

The phenotypic variations and association analysis {#s3d}
--------------------------------------------------

Pasting properties of various corn starches measured by RVA, including PV, TV, BD, FV, SB, PT and PTP have been summarized in [Table 4](#pone-0103627-t004){ref-type="table"}. The gelatinization temperatures (onset, ; peak, ; and conclusion, ) and enthalpy of gelatinization (), for maize starches from different inbred lines, measured using DSC are also presented. Significant difference in all the pasting and gelatinization properties among different maize inbred lines was observed through one-way ANOVA. These results suggest that the 80 inbred lines used in this study are representative in terms of maize quality and are qualified for association analysis.

10.1371/journal.pone.0103627.t004

###### The descriptive statistics and the results of one-way ANOVA for 7 starch pasting and 4 gelatinization properties of 80 maize inbred lines.

![](pone.0103627.t004){#pone-0103627-t004-4}

  Parameter                PV         TV         BD         FV         SB         PT        PTP                                      
  -------------------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
  Mean                  1317.013   926.856    390.156    2575.488   1258.563    5.336      72.759     6.225      75.591     70.912     81.266
  Standard deviation    405.135    250.758    216.227    841.476    593.455     0.391      2.641      1.008      1.037      1.219      1.989
  Minimum                577.5       422         38       892.5      −161.5     4.535      69.15      4.244      73.25      67.65      70.25
  Maximum                2321.5     1406.5      924        4066      2160.5     7.165      93.075      8.1        77.5       73.5       83.9
  *F*                    130.35     77.74      99.17      258.13     235.29     46.07      69.43       5.62      11.28      12.82      62.73
  *P*                   \<0.0001   \<0.0001   \<0.0001   \<0.0001   \<0.0001   \<0.0001   \<0.0001   \<0.0001   \<0.0001   \<0.0001   \<0.0001

To explore the relationship among 11 starch pasting and gelatinization properties, the pairwise correlation analysis was performed, and the Pearson correlation coefficients (*r*) between any two parameters were obtained ([Table 5](#pone-0103627-t005){ref-type="table"}). Interestingly, the significant correlations were found between any two pasting parameters, and only 5 pairwise correlations, including PT/PV, PT/TV, PT/FV, PT/SB and SB/PTP did not reach the significant level. Among the 6 pairwise correlations for gelatinization properties, only the pairs of and showed no significance. In addition, the correlations between the pasting and gelatinization properties were also investigated, and the results revealed that showed significant correlations with PV, TV, BD, PT and PTP, with PV and BD, and with BD. These results suggested that potentially different genetic mechanisms were responsible for these starch viscosity properties.

10.1371/journal.pone.0103627.t005

###### The pairwise correlation analysis among 7 pasting and 4 gelatinization properties of maize starch.

![](pone.0103627.t005){#pone-0103627-t005-5}

             PV           TV           BD          FV         SB         PT          PTP                            
  ----- ------------ ------------ ------------ ----------- -------- ------------ ------------ --------- ----------- -----------
  TV     0.887\*\*                                                                                                  
  BD     0.845\*\*    0.503\*\*                                                                                     
  FV     0.762\*\*    0.665\*\*    0.657\*\*                                                                        
  SB     0.398\*\*    0.337\*\*    0.355\*\*    0.897\*\*                                                           
  PT       −0.212       −0.046     −0.343\*\*     0.049     0.215                                                   
  PTP    −0.422\*\*   −0.421\*\*   −0.302\*\*   −0.259\*    −0.078   0.645\*\*                                      
         0.353\*\*    0.371\*\*     0.232\*       0.065     −0.149   −0.558\*\*   −0.593\*\*                        
           −0.157       −0.005     −0.290\*\*    −0.218     −0.203     −0.094       −0.024     0.239\*              
           0.018        0.093        −0.073       0.084     0.106      −0.069       −0.032      0.181    0.851\*\*  
          −0.250\*      −0.148     −0.298\*\*    −0.214     −0.133     −0.141       −0.084      0.103    0.468\*\*   0.342\*\*

GLM of association analysis that controlled the effects of population structure was used to identify relative association of 11 starch pasting and gelatinization properties and genotype variants in maize *ZmBT1* gene. All nucleotide polymorphisms, including SNPs and indels, with a frequency of more than 0.05 of the rare alleles were considered in the association analysis of phenotype-genotype in both genes. Only one variant (indel7 in exon2) in maize *ZmBT1* gene showed significant association with , while all the other variants had no association with starch pasting and gelatinization properties. Indel7 can cause a deletion of glutamic acid (E) in 27 inbreds or a deletion of glutamic and asparagic acids (ED) in 3 inbreds. Because the latter possessed a frequency of lower than 0.05 in the tested population, it was not used in the association analysis. According to the result of association analysis, indel7 explained 9.26% of the phenotypic variant of starch . The mean value of was 6.091 with a standard deviation 1.087 for the alleles carrying the deletion of glutamic acid (E) in protein product, and this value was statistically lower than those without deletion () based on independent samples *t* test ().

Discussion {#s4}
==========

The abundant genetic variations enable plant breeders to create novel plant gene combinations and select crop varieties more suited to the needs of diverse agricultural systems. The analysis of the genetic diversity for crop functional genes is critical for understanding the genetic background of phenotypic variation, and in turn will provide great help for crop improvement [@pone.0103627-Yang2], [@pone.0103627-Zhang1]. In this study, 30 variants, including 22 SNPs and 8 indels, were identified in the full-length sequence of the maize *ZmBT1* gene. Among these SNPs, 9 were found in the coding region, one of which were nonsynonymous and the others were synonymous. In addition, there were two indels in the coding region of this gene. The nonsynonymous SNPs and indels in the coding region would result in the changes of protein product. The SNP sites and indels in the coding region also classified the tested inbred lines into 6 haplotypes, which encode 6 deferring ZmBT1 proteins. However, lower frequency of variant was found in the intron regions of this gene. Particularly, none of the SNPs was identified in two introns of this gene. This may be the result of that the intron regions in this gene are much shorter than the coding region.

In this study, significantly positive statistics were obtained for promoter, coding and entire regions of the maize *ZmBT1* gene through Tajima\'s neutrality tests. Thus, a decrease in population size and/or balancing selection was suggested for this gene. Balancing selection refers to a number of selective processes by which multiple alleles are actively maintained in the gene pool of a population at frequencies above that of gene mutation. Balancing selection usually happens when the heterozygotes for the alleles under consideration have a higher adaptive value than the homozygote [@pone.0103627-Wang1]. Thus, potential high heterozygosity at the *ZmBT1* locus in the tested population is suggested.

LD is the non-random association between allelic polymorphisms at two loci. It was suggested that recombination and selection were the main determinants of LD [@pone.0103627-Myers1]. Maize is an outcrossing crop with extensive morphological variation, genetic diversity and high effective frequency of recombination [@pone.0103627-Jiao1]. Recent researches revealed that the rapid breakdown of LD in diverse sets of maize germplasm [@pone.0103627-Remington1], [@pone.0103627-Tenaillon1]. In this analysis, we found that the decay of LD in the maize *ZmBT1* locus was slower than the expected value. This may be the result of low frequency of recombination in this gene, because only one recombination event was detected in the *ZmBT1* gene.

Gelatinization temperature and enthalpy of maize starch plays an important role in grain quality. The enthalpy of gelatinization gives an overall measure of crystallinity and may be indicative of the loss of molecular order within the granule [@pone.0103627-Tan1]. Previously, some genes in starch biosynthesis pathway were found to affect the phenotypic variation of starch gelatinization properties. Based on the strategy quantitative trait loci (QTL) mapping, Tan et al. demonstrated that the *Wx* gene and two loci including starch-branching enzyme (SBE) genes in rice controlled the starch gelatinization properties [@pone.0103627-Tan1]. According to the results of association analysis, the sequence variations of rice genes *wx*, *SSI*, and *SSII-3* were found to be associated with gelatinization properties , and in waxy rice. In addition, the enthalpy of gelatinization () of rice starch is controlled by *wx* and *SSII-3* [@pone.0103627-Xu1]. The cereal protein BT1 is involved in the ADP-Glc transport into endosperm plastids, and played vital roles in the biosynthesis of starch. In this study, we showed that the maize *ZmBT1* gene possessed abundant nucleotide polymorphism. Further evidence based on association with pasting and gelatinization properties revealed that an indel in coding regions of this gene was associated with gelatinization enthalpy () of maize starch. Although showed correlations with other pasting and gelatinization properties, no association was found between the polymorphic sites and these traits. In addition, the formation of starch pasting and gelatinization properties is a complex process, and all these properties are quantitative traits influenced by multiple genes. Thus, these results obtained needs further verification owing to that only one gene *ZmBT1* in the starch biosynthesis was used.

Supporting Information {#s5}
======================

###### 

The positions of nucleotide polymorphism of *ZmBT1* gene among 80 maize inbred lines.

(XLSX)

###### 

Click here for additional data file.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: ZY CX. Performed the experiments: SX EZ YJ LP QC. Analyzed the data: ZY EZ ZX. Contributed reagents/materials/analysis tools: EZ ZY ZX. Contributed to the writing of the manuscript: SX ZY.
