Breeding potential of maize germplasm line GEMS-0067 for high amylose proportion

: Maize varieties with high amylose proportion are more valuable for starch industry. The SBE Ⅱ b gene encodes one of the starch branching isozymes (SBE Ⅰ, SBE Ⅱ a, and SBE Ⅱ b). Its recessive mutant amylose-extender ( ae/sbe2b ) decreases the total activities of SBEs and increases amylose proportion up to 60%. Here, the breeding potential of introduced germplasm line GEMS-0067 was eval-uated by genotyping and phenotyping. The deletion of the ninth exon of the SBE Ⅱ b gene, high amylose proportion, and the typical irregular granules suggested that this germplasm line was de-rived from the same resource of high amylose line AE11. The gelatinization and thermal properties, and degree of polymerization of starch chain showed its advantages used for high amylose breeding. However, the negative correlation between amylose proportion and starch content, as well as ker-nel filling characteristics should be overcome during breeding process.


Introduction
Maize is one of the staple crops in the world [1]. The kernels provide large amount of starch, consisting of essentially linear amylose and highly branched amylopectin [2]. The content and proportion of amylose and amylopectin determine the quality and usage of maize [3]. Native starch granules in cereal endosperm normally comprise 20%-30% amylose and 70%-80% amylopectin [4]. In waxy maize, which are usually planted as vegetable varieties for favorite food, the proportion of amylopectin can be as high as 100% almost [5,6]. Whereas, high amylose varieties with amylose proportion more than 50% are more valuable for starch industry and beneficial to control dietary intake [7][8][9], because it has high level of resistance to gelatinization and hydrolysis and cannot be digested in the upper gastrointestinal tract [8,[10][11][12][13]. Therefore, maize improvement for high amylose proportion has attracted more considerable attention in recent years [14][15][16].
The amylose proportion in maize endosperm is controlled by a series of starch synthesis genes [4-6, 12, 17]. One of the critical is the SBEⅡb gene that encodes starch branching enzyme (SBE) Ⅱb (one of the SBE isozymes: SBEI, SBEIIa, and SBEIIb). The full length of its genomic sequence is 23,449 bp and contains 22 exons [4-6, 12, 16-19]. The amylose proportion of its recessive mutant amylose-extender (ae/sbe2b) is over 60% [20][21][22][23][24]. For example, the total activities of SBEs in an ae mutant AE11 were decreased to about 71% of its wild control because of the deletion of the ninth exon of the SBEIIb gene containing 84 bases. Under the modification of other modifier genes, such as sbe1, the amylose proportion of this line was raised as high as 70% [5].
In the present study, the introduced germplasm line GEMS-0067 released as parent of maize breeding for high amylose by Truman State University [25], together with its wild type (WT) control, was genotyped for the ae/sbe2b mutation, and phenotyped for morphology of starch granules, starch content and amylose proportion, gelatinization and thermal properties, degree of polymerization, and major agronomic traits, to evaluate its breeding potential for high amylose proportion.

The ae/sbe2b gene
A 750 and a 1576 bp specific band was separated from the amplified products of GEMS-0067 and its WT control ( Figure 1A). The results of sequencing and alignment showed that the full length of the genomic sequence of the ae/SBEⅡb gene was 23449 bp in the WT control. The whole 84 bases of the ninth exon were completely deleted in GEMS-0067, but not causing a frame shift ( Figure 1B).

Morphology of starch granules and amylose proportion
The scanning electron microscopy images showed that the starch granules isolated from WT endosperm were spherical and elliptical with smooth surface, whereas the starch of GEMS0067 contained individual or aggregate elongated granules with irregular depressions, as well as small subgranules ( Figure 2). The starch content and amylose proportion of GEMS0067 were 72.63% and 72.95%, respectively, significantly different from 74.21% and 26.26% of WT (Table 1).

Gelatinization properties
Rapid viscosity analysis showed obvious difference of viscosity curves between GEMS0067 and WT starch ( Figure 3). Along with the increasing of temperature, the viscosity of WT starch gradually increased caused by gelatinization and presented a typical double-peak curve, whereas GEMS0067 demonstrated almost a horizontal straight line with a small peak at the beginning. The peak viscosity, through viscosity, break viscosity, final viscosity, and setback viscosity of GEMS0067 starch were significantly lower, while its peak time and gelatinization temperature were higher than those of WT starch ( Table  2).

Thermal properties
Differential scanning calorimetry showed that the transition temperatures, including onset temperature (To), peak temperature (Tp) and conclusion temperature (Tc), of GEMS0067 starch were significantly higher than those of WT starch, and the gelatinization enthalpy of GEMS0067 starch was significantly lower than that of WT starch (Table 3). Along with the increasing of temperature, the differential scanning calorimetry thermogram of WT starch presented an obvious absorption peak at 70°C, whereas the absorption peak of GEMS0067 starch almost became a straight line ( Figure 4) because its gelatinization enthalpy was six times lower than WT (Table 3).  1 Values are presented as mean ± standard deviation (n = 3). Double asterisk (**) indicates significant difference at P < 0.01.

Degree of polymerization
The result of high-performance anion-exchange chromatography (HPAEC) showed that the degree of polymerization heavily depended on the proportion of amylose and amylopectin of starch. Referring to Hanashiro et al. (1996) [26], the percentage of short chains (6 ≤ DP ≤ 24) was less in GEMS0067 starch than that in WT starch, whereas the percentage of medium chains (25 ≤ DP ≤ 36) and long chains (DP ≥ 37) was more in GEMS0067 starch than that in WT starch ( Figure 5).

Figure 5.
Degree of polymerization of GEMS0067 and WT starch. The percentage of short chains (6 ≤ DP ≤ 24) was less, and the percentage of medium chains (25 ≤ DP ≤ 36) and long chains (DP ≥ 37) was more in GEMS0067 starch than that in WT starch.

Agronomic traits
During all the growth period, the two lines grew normally and was not infected by any pathogen. The result of paired t-test showed that the differences of anthesis stage, silking stage, growth period, plant height, ear height, and row number per ear were nonsignificant between GEMS-0067 and WT (Table 4). Whereas, the GEMS0067 kernels were a little shrunk and dull, and its endosperm was stained much darker by I2/KI solution than WT kernels (Figure 6), resulting in the significant decrease of its 100-kernel weight and kernel weight per plant, although its kernel number per row was significantly more than WT (Table 4).

Discussion
The SBEⅡb gene encodes one of the starch branching enzyme (SBE) isozymes (SBEI, SBEIIa, and SBEIIb) that play critical role in synthesis of amylopectin [4-6, 12, 16-22]. Its recessive mutant amylose-extender (ae/sbe2b) decreased the total activities of SBEs to about 71% and increases amylose proportion of maize kernel up to 60% [23][24][25]. From the SBEⅡb gene of the inbred germplasm line GEMS-0067, the whole 84 bases of the ninth exon were completely deleted ( Figure 1B), suggesting that this germplasm line was derived from the same resource of high amylose inbred line AE11 [5].
The gelatinization temperature of GEMS0067 starch was recorded as >95°C because it was higher than the upper limit of heating temperature of the differential scanning calorimeter. This property of maize amylose was also observed under confocal laser scanning microscopy and scanning electron microscopy [30]. The viscosity of starch was increased along with its gelatinization during gradual heating due to the absorption of water and loss of starch structure [31][32][33]. The significantly lower peak viscosity, through viscosity, break viscosity, final viscosity and setback viscosity, and gelatinization enthalpy ( Figure 3, Table 2, 3), and significantly higher peak time, gelatinization temperature, and transition temperatures (To, Tp, and Tc, Table 2, 3), as well as the inconspicuous absorption peak (Figure 4) of GEMS0067 starch implied that gelatinization initiation of GEMS0067 starch needed much more energy to heat, because of its high proportion of amylose (Table 1) and high percentage of medium and long chains ( Figure 5).
Negative correlation between amylose proportion and starch content, as well as kernel filling characteristics, was found in most of high amylose inbred germplasm lines [34]. In some high amylose lines with recessive mutation of some other modifier genes, the kernel filling characteristics were not much affected, but the amylose proportion was not as high as the ae/sbe2b mutant lines [35]. In the present study, the shrinkage of GEMS0067 kernels resulted in the significant decrease of its 100-kernel weight ( Figure 6, Table 4). However, the significant increase of kernel number per row recovered some of the loss. The kernel weight per ear of GEMS0067 was only 8.3% lower than its WT (Table 4). Therefore, GEMS0067 was suggested as an elite germplasm line for maize breeding of high amylose proportion.

Genotyping of ae/sbe2b mutation
The genomic DNA was extracted from the leaf samples of the inbred germplasm line GEMS-0067 and its WT control with the CTAB buffer [36]. The quality and quantity of DNA samples were detected on Nano Photometer™P-Class (Implen, Schatzbogen, Germany) and visualized by 1% agarose gel electrophoresis. The reference sequence of the WT SBE Ⅱ b gene (GenBank: AF072725.1) was downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/gene/?term=AF072725.1). Referring to Kim et al. (1998) [20], the genomic sequence from the 8th to 10th exon of the SBEⅡb gene was amplified with a pair of specific primers (5′-CCAGCCTGGATCAAGTACTC-3′/5′-CTTGGATA-CAATGCAGTGCAA-3′), separated by 1.5% agarose gel electrophoresis, subcloned into pMD-19-T vector, and sequenced at Sengon Biotech (China). The possible mutation of the ae/SBEⅡb gene was genotyped by alignment between the inbred germplasm line GEMS-0067 and its WT control by using the SnapGene software (https://www.snapgene.com/).

Scanning electron microscopy
As described by He et al. (2020) and Lin et al. (2016) [5,8], starch was isolated from mature endosperm, dried completely at 40°C, mounted on aluminum specimen holder using double-sided adhesive tape and sputtered with a gold in a vacuum evaporator, and viewed with a scanning electron microscopy (Hitachi, Tokyo, Japan). The images of starch granules were obtained at an accelerating voltage of 10 kV and a magnification of 4000 ×. Amylose proportion of three replicates of each line was determined by using an amylose/amylopectin assay kit (Megazyme, Bray, Ireland) according to the manufacturer's instructions. After eliminating the possible lipids, proteins and amylopectin by using ethanol and concanavalin A, respectively [38], the remained amylose was enzymatically hydrolyzed to D-glucose, oxidized with glucose oxidase/peroxidase reagent, treated with the GOPOD reagent, and read for absorbance at 510 nm in a spectrophotometer (UV5Nano, Mettler Toledo, Switzerland).

Determination of physical properties
As described by   [39], the starch samples were homogenized and sieved through a 200-mesh sifter, determined for moisture content in a rapid moisture meter (HR83-P, Mettler Toledo, Switzerland), accurately weighted for 5 g (error < 0.05 g), transferred onto the aluminum foil, mixed well with 25 ml sterile water, and determined the viscosity in a rapid viscosity analyzer (RVA Super 4, Newport Scientific, Australia).
As described by   [39], the homogenized and sieved starch samples were accurately weighted for 10 mg, add with 30 µL sterile water, balanced at room temperature for 24 h, heated from 30℃ to 95℃ at a heating rate of 10 ℃/min and determined for transition temperatures (To, Tp, and Tc) in a differential scanning calorimeter (TA Instruments, New Castle, USA). The gelatinization enthalpy (ΔH) was calculated by the software of the instrument.
As described by Hanashiro et al. (1996) [26], the starch samples were dissolved in 50 mM sodium acetate and debranched with isoamylase (Sigma, Darmstadt, Germany). The degree of polymerization (chain length distribution) was determined and analyzed by HPAEC (Thermo Fisher Scientific, Waltham, USA) via pulsed amperometric detection.

Phenotyping of agronomic traits
The inbred germplasm line GEMS-0067 and its WT control were planted in field randomized complete block design with three replicates. Twenty-one plants were cultivated in three rows (230 cm long and 30 cm apart) per plot. Growth status and disease resistance were observed during all the growth period. All the plants in the middle row of each plot were investigated for anthesis stage, silking stage, growth period, plant height, and ear height. At mature stage, the ears were harvested from the five middle plants in the middle row of each plot, air dried, photographed and investigated for row number per ear, kernel number per row, 100-kernel weight, and kernel weight per plant. And then, their kernels were cut transversely and longitudinally, stained with I2/KI solution (5% [w/v] I2 / 10% [w/v] KI,) as described by Hunt et al. (2013) [40], and photographed under optical microscope (Mshot, Guangzhou, China) with 2 × magnification.
The data was analyzed by Microsoft Excel 2013 (https://www.microsoft.com/) and Origin 8.0 software (https://www.originlab.com/), and presented as means ± standard deviations (SD). The significance of the difference between the two lines was tested with the method of paired t-test by using SPSS software 11.2 (https://www.ibm.com/analytics/spssstatistics-software).

Conflicts of Interest:
The authors declare no conflict of interest.