Roles of Waxy and Soluble Starch Synthase Iia Alleles in Determining Different Type Resistant Starch Contents of Rice

investigate effects


Abstract Background
Resistant Starch (RS) is a functional starch that has functions of regulating diabetes, hypertension and obesity. The effects of most starch synthesis-related genes (SSRGs) on RS content and their relationships are largely unknown.

Results
In current study, ninety-nine lines from a recombinant inbred line were selected to investigate the effects of SSRGs on the RS content in different process status. Results revealed that RS content decreased dramatically after cooking, but it did not increase signi cantly after cooling for 7 days. And RS was closely related to many indexes of physicochemical properties, but was not correlated with granule size. Waxy (Wx) played an important role in controlling RS content and Wx a could elevate RS content in raw milled rice, cooked rice and retrograded rice. Soluble starch synthase IIa (SSIIa) had an impact on RS2, and RS2 content of indica SSIIa were signi cantly higher than that of japonica SSIIa (SSIIaj). Moreover, interaction of Wx and SSIIa was responsible for variations of RS content in three sample types, RS2 and volume proportion of different size starch granules.

Conclusions
Wx and SSIIa together signi cantly regulate different types content of RS in rice, but SSIIa only affects RS2. Wx a -SSIIaj is favorable to forming large-diameter starch granules.

Background
Resistant starch (RS) is a sum of starch or starchy food products that cannot be digested in the small intestine of healthy individuals but fermented in the large intestine by microbial ora (Asp 1992).
According to the botanical origin and nature, RS can be furtherly classi ed into ve subtypes: RS1, RS2, RS3, RS4 and RS5 (Xia et al. 2018). RS2 and RS3 are the main types found in raw and cooked rice, respectively. RS2 is made up of native starch granules whose formation and structure protects it from digestion and is usually present in raw potato, green banana and uncooked starch (Hernández et al. 2008). The physiological functions of RS2 are few because of its rapid digestion after being thermally processed and cooked. Because of the higher starch gelatinization temperature and lower viscosity, RS2 is often used to improve the gluten structure of biscuits, bread and other pasta products, to increase the product moisture content, and has certain commercial value (Korus et al. 2009). RS3 consists of retrograded starch, mainly the recrystallized amylose, formed during cooling of gelatinized starch and cooked foods that are stored at room or low temperature (Sharma et al. 2008), which is widely used in food processing industry.
With the development of living standards and changes of life style, the number of people with type-2 diabetes are increasing. Consumption of food with high RS can help to prevent from diabetes, and there is a decrease in postprandial glucose and insulin responses because of its slow digestion and absorption by the small intestine (Raigond et al. 2015). Meanwhile, RS can be decomposed into short chain fatty acids such as acetic acid, propionic acid and butyric acid in the large intestine, which can improve the health of large intestine and prevent the occurrence of intestinal in ammatory diseases and colon cancer (Robertson et al. 2000;Topping et al. 2008). Consumption of RS can increase satiety and reduce calorie intake, which helps weight management (Raigond et al. 2015). Therefore, it is important to elevate the content of RS in food.
Rice (Oryza sativa L.) feeds more than half of the world's population as staple food and is the main source of nutrition and carbohydrates for many people. RS content is generally under 3% in hot cooked rice cultivars, which is not enough to confer the associated health bene ts (Frei et al. 2003;Hu et al. 2004). Therefore, many scholars have focused on the promotion of RS content in rice varieties. In the past decades, many mutants or varieties with elevated RS have been identi ed, such as RS111 (Yang et al. 2006 Starch biosynthesis is a sophisticated process involving multiple enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SS), starch branching enzymes (SBEs), isoamylase (ISA) and pullulanase (PUL) (Tian et al. 2009). Among them, GBSSI is encoded by the Waxy (Wx), which plays an important role in determining the synthesis of amylose. SS, SBEs, ISA and PUL can control the synthesis of amylopectin. RS content can be affected by the ratio of amylose to amylopectin and the ne structure of starch (Eggum et al. 1993;Hu et al. 2004).
Thus, these starch synthesis-related genes (SSRGs) naturally have great in uence on the synthesis of RS.
A high-RS mutant has been developed by in uencing the expression of starch branching enzyme 3 (SBE3) (Yang et al. 2012). Zhou et al. (2016) believed that soluble starch synthaseIIIa (SSIIIa) can regulate RS content and further revealed that RS yield is dependent on the high expression of Wx a allele, which is prevalent in indica varieties. Krishnan et al. (2019) analyzed the correlation between PUL activity and inherent RS (RS1, RS2, RS5) and found that high PUL activity contribute to inherent RS. In other cereals, the down-regulation of soluble starch synthaseII-3 (SSIIa) and SBE could increase RS content in barley (Topping et al. 2003;Bird et al. 2004) and wheat (Hazard et al. 2014;Ahmed et al. 2015). Due to the molecular basis regulating the RS synthesis is largely unclear, to identify the RS genes is crucial for clarifying the RS synthesis and for the breeding of high-RS varieties.
It is generally considered that eating and cooking qualities (ECQs) are the most crucial rice qualities, which are mainly comprised of three physicochemical properties: apparent amylose content (AAC), gel consistency (GC), and gelatinization temperature (GT) (Tian et al. 2009). In recent years, because of its advantages of simple and rapid, the rapid visco analyzer (RVA) has been widely employed to evaluate the rice ECQs. The AAC shows a signi cantly positive correlation with RS content (Kong et al. 2015). Most of rice varieties with eminent RS content tend to have high AAC and poor taste, which is not acceptable to most consumers. Therefore, it is an important goal to study the relationships between RS and physicochemical properties in rice, which can make a foundation for obtaining rice varieties with elevated RS content and good taste. In this study, the recombinant inbred lines (RILs) were employed to study the effects of Wx and SSIIa on RS content, and the relationships between RS and physicochemical properties were analyzed, aiming to provide useful information for molecular breeding of rice RS.
Glutinous identi cation showed that the second exon of their waxy gene had nucleotides insertions of 23 bp, namely, there was recessive wxwx genotype for them (You et al. 2019). The alleles of two parents differed in ADPglucose pyrophorylase small unit (AGPsma), ADPglucose pyrophorylase large unit (AGPlar), Wx, SSIIa, soluble starch synthaseIIIb (SSIIIb) and soluble starch synthaseIV-2 (SSIV-2) ( Fig. 1). At seventh generation, a total of 99 lines with homozygous genetic background of SSRGs were identi ed from 142 lines and then a single plant was harvested from each line and taken as experimental materials when they were mature. All the materials were grown in an experimental agricultural eld of the Southwest University of Science and Technology (Sichuan, China) and maintained normal management.

Determination of resistant starch and physicochemical properties
Harvested rice grains were air-dried and stored at room temperature for 3 months to balance water before analyses. Rice was dehulled by using a Dehuller (TR-200; Kett, Tokyo, Japan). The brown rice was polished by using polisher (Pearlest; kett, Tokyo, Japan) and subsequently ground to our in a Laboratory Mill (LM3100; Petern, Malmö, Sweden) with a 100 mesh sieve for measuring RS content and physicochemical properties.
Cooked rice was prepared according to the Zhou's method (Zhou et al. 2016) and taken a part of cooked rice stored at 4℃ for a week to product retrograded rice. In this work, RS content was measured in three sample types, including raw milled rice, cooked rice and retrograded rice by using RS Rapid Assay Kit (Megazyme, Bray, Co. Wicklow, Ireland), according to the manufacture's assay procedure. Each starch sample was measured in triplicate.
AAC and GC were measured according to the standard of Chinese Ministry of Agriculture, NY/T 2639-2014, and Chinese national standards, GB/T 17891-1999, respectively. GT was estimated by differential scanning calorimetry (DSC) according to the methods reported previously . In order to measure sample percentage of retrogradation (R%), the gelatinized starch was stored at 4℃ for 7 days.
Afterwards, it was equilibrated to room temperature for 1 h and then with the same thermal program as the measurement of GT. R% was calculated by the following formula: R%=∆Hr (enthalpy of retrogradation)/∆Hg (enthalpy of gelatinization) Each index of per sample was measured in duplicate.

Measurement of RVA pro le characteristics
A RVA (RVA4500, NewPortSci. Co. Warriewood, Australia) was used to estimate RVA pro le characteristics in accordance with the American Association of Cereal Chemists (AACC) operational procedure. Three original parameters obtained from the RVA pro le as following: peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV); and three secondary parameters including: breakdown viscosity (BDV=PKV-HPV), setback viscosity (SBV=CPV-PKV), consistence viscosity (CSV=CPV-HPV). Besides that, the pasting temperature (PaT) and pasting time (PeT) also were recorded. Each starch sample was measured in duplicate.
Analysis of starch granule shape and particle size distribution Rice starch granules were isolated according to the procedures of peng et al. (1999). The shape of starch granule was observed using scanning electron microscopy (SEM)H, Oberkochen, . Particle size distribution of starch was determined with a laser diffraction particle size analyser (Model LS 13320, Beckman Coulter, USA). Granule size was represented with mean diameter automatically calculated by the instrument software.  Table S1). PCR-ACCI was designed to detect the rst intron G/T polymorphism of Wx and Wx M1 was used to distinguish glutinous rice varieties from the others. And then, the molecular markers of genes with polymorphism between two parents were selected to detect the genotypes of 99 lines. All the primers were synthesized by Sangon Biotech (Shanghai, China). Polymerase chain reactions (PCR) ran on an Eppendorf Thermal Cycler (Mastercycler® nexus GSX1, Germany). 5 µL of each ampli cation products of CAPS primers was digested with corresponding restriction endonuclease in a total of 15 µL enzymatic system including 1.5 µL10 × buffer, 8 µL ddH 2 O and 0.5 µL restriction endonuclease. The digestion reactions were performed by using an Eppendorf Thermal Cycler at 37℃ for 3-4 h. All the ampli ed products were detected on a 3% agarose gel in 0.5 × Tris-Borate EDTA (TBE) buffer using GreenView (Applied BioProbes, Rockville, MD, USA).

Statistical analyses
Analysis of variance (ANOVA) and multiple comparisons of the Duncan method were performed with the Statistical Product and Service Solutions software version 22 (SPSS, https://www.ibm.com/analytics/cn/zh/technology/spss/) after the data being classi ed into groups according to the genotyping results. In addition, the correlation analysis between RS content and physicochemical properties was conducted by using SPSS 22.

Results
Analysis on genetic background of materials and grouping of RIL Polymorphism of eighteen SSRGs had been detected between two parents and they differed in 6 genes (AGPsma, AGPlar, SSIIIb, SSIV-2, Wx and SSIIa). Genetic analysis on SSRGs of the 99 lines came from the RIL showed that there were different allelic genotypes for the 6 genes ( Fig. 1). Among the experimental population, statistically signi cant differences on RS were only detected in different Wx alleles and SSIIa alleles (P<0.05) (Additional le 2: Table S2). So we only focused on the effects of Wx and SSIIa alleles for RS content. Wx genotypes of the lines were identi ed by utilizing molecular marker PCR-AccI and results indicated that the lines could divided into two groups: Wx a Wx a genotype and Wx b Wx b genotype. And then, glutinous identi cation showed that all lines with the background of Wx b were glutinous rice, which were recessive wxwx genotype ( Fig. 1, Additional le 1: Fig. S1). Because SSIIa alleles ampli ed by SSII-3 M1 were haplotype 1 (G/G/GC, indica-type) and 4 (A/G/TT, japonica-type) (You et al. 2020), the detected result by SSII-3 M1 showed that haplotype of SSIIa of Javanica 22 and CG133R belonged to haplotype 1 and haplotype 4, separately ( Fig. 1). Diverse groups were obtained according to different alleles among the 99 lines, and were designated as follows: to Wx alleles: Wx a type (came from CG133R), wx type (came from Javanica 22), respectively; to SSIIa locus: SSIIa-I type (same as Javanica 22) and SSIIa-II type (same as CG133R), respectively.

Phenotypic variability of RS content in rice RIL and their parents
A t-test was implemented in order to compare the difference of parents on RS content. The result showed that the parents differed signi cantly from each other on the RS content in raw milled samples (RSm), the RS content in cooked rice (RSc) and the RS content in retrograded rice (RSr) (P<0.05) ( Table 1). RS content in three sample types showed a wide variations among the RIL, especially RSm, with an average of 10.61% and ranging from 0.03% to 29.42% (Table 1). Compared with the RSm, the average of RSc and RSr decreased by 9.88% and 9.55%, respectively. The difference of RS content between raw milled samples and cooked rice was signi cant in RIL (P<0.01), but there was no statistical difference on RS content between cooked rice and retrograded rice at 0.05 level (Additional le 1: Fig. S2). This fact indicated that RS contents of the different lines were decreased sharply after cooking, but no distinct variation was observed in RS content after retrogradated process. Therefore, the subtype of RS in the RIL was mainly RS2, and less RS3.

Correlation analysis between RS and physicochemical properties in RIL
In this study, the physicochemical properties of the RIL and two parents were tested, which showed a wide variations among the tested materials ( Table 2). The correlation analyses between physicochemical properties and RSm, RSc and RSr showed that the AAC, all DSC thermal parameters, PKV, PaT and CSV had a positive correlation with the RSm ( Table 3). The AAC and all RVA pro le parameters, except for PKV, BDV and PaT, were signi cantly positively correlated with RSc, while all DSC thermal parameters showed signi cantly negative correlation with RSc (P<0.05), and the same result also showed in RSr. Besides that, all the correlation coe cients of RSr were lower than that of RSc, except for PKV. These data suggested that RSm, RSc and RSr would increase along with the rise of AAC, and higher RSm along with higher GT. However, RSc and RSr would decrease when GT increased. AAC, apparent amylose content; GC, gel consistency; T0, gelatinization start temperature; TP, gelatinization peak temperature; Tc, gelatinization end temperature; R%, percent of retrogradation; PKV, peak viscosity; HPV, hot paste viscosity; CPV, cool paste viscosity; PeT, peak time; PaT, pasting temperature; BDV, breakdown value; SBV, consistence value; SCV, setback value.  (Fig. 2a). The average of RSm, RSc and RSr of Wx a type increased by 9.54%, 0.85% and 1.21%, respectively, compared with that of wx type.
The result suggested that Wx a could improve RS content in three sample types.
Furthermore, the differences between RSm and RSc, RSr and RSc were calculated. RSa= RSm -RSc, RSb= RSr -RSc. RSa mainly was RS2 and RSb mainly was RS3. Result of ANOVA revealed that RSa varied signi cantly under different alleles of Wx, as did RSb (P<0.01) (Fig. 2b). RSa and RSb of wx type (2.53% and 0.05%, respectively) also were extremely signi cantly lower than that of Wx a type (11.21% and 0.41%) (P<0.01). This result indicated that Wx alleles could affect RS2 and RS3 and Wx a could promote RS2 and RS3 content.
In order to further con rm the above results, a sub-population with total of 16 Fig. 2c and d). It could be furtherly con rmed that Wx had a great in uence on rice RS content that could be increased by Wx a .

Effects of SSIIa alleles on RS in the RIL
Ninety-nine lines were divided into two groups by SSIIa alleles: SSIIa-I type and SSIIa-II type, and their RS content distinctly differed from each other in the three sample types (P<0.01) (Fig. 3a). RSm of SSIIa-I type was 17.83%, and that of SSIIa-II type only 5.57%. However, the RSc and RSr of SSIIa-I type were lower than that of SSIIa-II type. It showed that SSIIa allelic polymorphism had an important impact on rice RS, but the speci c role needed further con rmation. Moreover, the difference of RSa was signi cant under different SSIIa alleles by ANOVA (P<0.01) (Fig. 3b). The RSa of SSIIa-I type was 17.27%, which also differed from SSIIa-II type (4.74%). In fact, SSIIa played an important role in controlling rice RS, except for RS3.
Furthermore, 18 lines that only had polymorphisms in SSIIa locus were selected from RIL under the CG133R genetic background for SSRGs, of which 6 lines belonged to SSIIa-I type and 12 lines belonged to SSIIa-II type. Signi cant differences were detected in RSm or RSa under different SSIIa alleles by t-test. RSm of SSIIa-I type (21.33%) was obviously higher than that of SSIIa-II type (5.56%) (Fig. 3c). RSa of SSIIa-I type (20.44%) was signi cantly higher than that of SSIIa-II type (4.52%) (P<0.01) (Fig. 3d) Table S4). The combined effects of Wx and SSIIa alleles on rice RS must be further studied.
There were four combinations among the tested rice lines: wx-I, wx-II, Wx a -I and Wx a -II, which came from the combination of Wx and SSIIa alleles (Fig. 4). RSm of wx-I (7.79%) was similar to that of Wx a -II (5.59%), which was signi cantly higher than that of wx-II (0.67%), but distinctly lower than that of Wx a -I (21.33%) (P<0.01), and RSa with same effect. The signi cant differences on RSm and RSa between homozygotes of Wx a and wx were detected under the same background of SSIIa alleles. In addition, there were statistical differences on RSm and RSa between homozygotes of different SSIIa alleles under the same background of Wx. RSc of Wx a -I (0.89%) and Wx a -II (1.04%) was signi cantly higher than that of wx-I (0.06%) and wx-II (0.04%). Likewise, the effects of Wx alleles on RSc were signi cant different under the same SSIIa alleles. However, the differences on RSc between homozygotes of different SSIIa alleles were not signi cant under the same background of Wx. And the same result also showed in RSr. These results indicated that Wx had an important in uence on RS under the same background of SSIIa alleles, and SSIIa only played a signi cant role in regulating RSm and RSa under the same background of Wx alleles.
Characterization of starch granule shape and particle size distribution in different Wx and SSIIa allelic combinations A single plant that were selected from each combinations (wx-I, wx-II, Wx a -I and Wx a -II), respectively, was used to determine its starch granule shape and particle size distribution (Additional le 2: Table S5).

Figures of scanning electron microscopy revealed that starch granules of the single plant with different
genotypes were variable in shape and size. And most of starch granules in tested plants were polygonal and a few were rounded (Fig. 5). Results of starch granule distribution demonstrated that particle size had a wide variations in the samples with different genotypes, and most of them belonged to medium size starch granules (4 μm ≤ granular diameter ≤ 8 μm) (Table 4). Moreover, there were statistical differences in particle size distribution among different combinations. The combination Wx a -II had the lowest proportion of medium size starch granules and the highest proportion of larger size starch granules (granular diameter >8 μm), compared with the other three combinations. And the mean diameter of Wx a -II (7.56 μm) was signi cantly higher than that of other three combinations (P<0.05). In addition, effects of Wx and SSIIa alleles on starch granule distribution were further analyze by a split block design (Additional le 2: Table S6). The results indicated Wx and SSIIa alleles had an effect on particle size of starch, especially SSIIa alleles could signi cantly affect the volume of starch granules of different sizes (P<0.01). Furthermore, a correlation analysis was implemented in order to investigate the relationships between starch granule volume and RS content, and the result showed that it was not correlated with granule volume to the RSm, RSc, RSr, RSa and RSb (Additional le 2: Table S7). To sum up, particle size of the four combinations of Wx and SSIIa alleles were different, but there was no correlation between RS content and starch granule volume.  Fig. S2). This result revealed that RS3 in these materials was low. Brie y, the subtype of RS was mainly RS2 in this RILs, while RS3 was less.
Relationships between RS andrice quality indices AAC and amylopectin ne structure of rice starch also determines RS content in addition to affecting the physicochemical properties. Therefore, it is necessary to analyze the relationship between RS content and physicochemical properties of rice. Zhang (Fig. 2). Wx a is one of the vital allele among the multiple alleles of Wx, which produces a large amount of amylose (Itoh et al. 2003). Raigond et al. (2015) found that high amylose content could contribute to RS content. Bao et al. (2017) utilized the genome-wide association study to select the candidate genes affecting RS and identi ed that Wx was one of the candidate genes, as well as the mean RS content of indica was higher than that of japonica. Furthermore, Zhou et al. (2016) revealed that RS production was dependent on the high expression of Wx a allele, which was consistence with our conclusion. It furtherly indicate that Wx affects RS through controlling amylose content.
SSIIa plays a vital role in the amylopectin synthesis pathway, and it can control the degree of crystallinity (Kong et al. 2015). SSIIa is present in a large complex including ADP-glucose pyrophosphorylase Our study showed that SSIIa had an in uence on rice RS except for RS3 in RIL ( Fig. 3a and b), but only affected RS content in raw starch and RS2 in 18 lines (Fig. 3c and d). The RSm and RSa of 18 lines with SSIIa allele from Javanica 22 (21.33% and 20.44%, respectively) were signi cantly higher than that from CG133R (5.56% and 4.52%, respectively) ( Fig. 3c and d). The results of genetic analysis demonstrated that genotype of SSIIa-I type (came from Javanica 22) belonged to indica SSIIa (SSIIai) and that of SSIIa-II type (came from CG133R) belonged to japonica SSIIa (SSIIaj) (Fig. 1). It is reported that SSIIa played a critical role in elongating short A and B1 chain (DP 6-12) to long type B1 chains (DP 13-24) of amylopectin. And the activity of SSIIa in indica rice was higher than that in japonica rice, which resulted in higher accumulation of B1 chains (Umemoto et al. 2002 effects of Wx and SSIIa alleles on RS content in rice have not been reported so far, the interaction of the two genes on RS were therefore analyzed. Apart from RS3, the combined effects of Wx and SSIIa greatly affected RSm, RSc, RSr and RS2 (Additional le 2: Table S4). In the 24 lines, the RSm of wx-I (7.79%) was similar to that of Wx a -II (5.59%), which was signi cantly higher than that of wx-II (0.67%), but distinctly lower than that of Wx a -I (21.33%) (P<0.01), and RSa with same effect. The RSc and RSr of Wx a -I (0.89% and 1.283%, respectively) and Wx a -II (1.04% and 1.49%) were higher than that of wx-I (0.06% and 0.15%) and wx-II (0.04% and 0.05%). There were signi cantly statistical differences on RS content in three sample types and RS2 between Wx a and wx alleles under the same SSIIa allele, and the differences on RSm and RS2 between homozygotes of different SSIIa alleles were signi cant under the same background of Wx (Fig. 4). These results suggested that Wx had a great impact on RSm, RSc, RSr and RS2 under the same SSIIa alleles, and SSIIa alleles only had effect on RSm and RS2 under the same Wx alleles.
It is generally considered that the granules in rice were polygonal starch granules with an irregularly shape and small size (3-8 μm) (Patindol et al. 2007). Previous studies reported that the majority of starch granules from mutants with elevated RS were oval or rounded shape, such as RS111 (Yang et al. 2006), b10 (Zhou et al. 2016) and MR4 (Shu et al. 2014). Moreover, Shu et al. (2014) found that the size of starch granule of MR4 (high-RS mutant) was smaller than that of MR7 (low-RS mutant). But in the present study, the combinations of Wx and SSIIa alleles with different RS content, whose starch granules mostly were polyhedral and sharp-edged (Fig. 5). There were statistical differences in particle size distribution among different combinations. Wx a -II had the lowest proportion of medium size starch granules and the highest proportion of larger size starch granules, compared with the other three combinations (Table 4). It implied that a high proportion of AAC and S-type amylopectin was in favor of the amyloplasta of the endosperm cells to forming large-diameter starch granules. Unfortunately, there was no correlation between RS content and starch granule diameter (Additional le 2: Table S7). Thus, we speculated that RS in the rice RIL may not be affected by starch phenotype, which regulated by Wx and SSIIa alleles. Rice starch granule belongs to a kind of semi crystalline granule, which has a lamellar structure of alternating crystalline and amorphous layers. Within these lamellae, the crystalline layers are mainly composed of amylopectin chains, while the amorphous layers contain the amylopectin branching points and the disordered conformation of amylose and amylopectin molecules (Copeland et al. 2009). Li et al. (2019) pointed that the regulation of RS mainly focused on altering branch-chain lengths, amylose to amylopectin ratio, as well as the amorphous and crystalline structure of lamellae. The Wx is crucial for amylose synthesis, and the SSIIa plays a vital role in the amylopectin synthesis. Therefore, Wx and SSIIa alleles may manipulate the RS by controlling the crystalline structure of starch granules, which needs further veri cation. Based on our results and literature review, the genetic regulation of Wx and SSIIa alleles on different type RS has a distinct pathway (Fig. 6).
In the present study, the subtype of RS in present study is mostly RS2 and less RS3, and RS content is closely relative with many indexes of physicochemical properties. Wx has a great effect on RS content  Figure 1 Polymorphism gene loci of starch synthesis-related genes between two parents and partial tested materials. J and I indicate genotype of Janavica 22, and C and II indicate genotype of CG133R.

Figure 1
Polymorphism gene loci of starch synthesis-related genes between two parents and partial tested materials. J and I indicate genotype of Janavica 22, and C and II indicate genotype of CG133R.    Scanning electron micrographs of starch granules from two parents and different Wx and SSIIa combinations.

Figure 6
The schematic diagram of Wx and SSIIa regulating RS content in rice. We found that Wx affects RS in different processing rice through controlling amylose content, and SSIIa affects RS content in raw starch and RS2 by regulating the amount of intermediate-size chains of amylopectin. The combine effects of Wx and SSIIa can affect the starch particle size distribution, but not the granule shape. Unfortunately, there is no correlation between RS content and starch granule diameter. Thus, it imply that Wx and SSIIa alleles may manipulate the RS content by controlling the crystalline structure of starch granules, which needs further veri cation. The solid line indicates the pathways of Wx and SSIIa regulating RS content in rice; the dotted line represents a hypothetical path, which needs further con rmation. RSm, resistant Page 30/31 starch content in raw milled sample; RSc, resistant starch content in cooked rice; RSr, resistant starch content in retrogradation.

Figure 6
The schematic diagram of Wx and SSIIa regulating RS content in rice. We found that Wx affects RS in different processing rice through controlling amylose content, and SSIIa affects RS content in raw starch and RS2 by regulating the amount of intermediate-size chains of amylopectin. The combine effects of Wx and SSIIa can affect the starch particle size distribution, but not the granule shape. Unfortunately, there is no correlation between RS content and starch granule diameter. Thus, it imply that Wx and SSIIa alleles may manipulate the RS content by controlling the crystalline structure of starch granules, which needs further veri cation. The solid line indicates the pathways of Wx and SSIIa regulating RS content in rice; the dotted line represents a hypothetical path, which needs further con rmation. RSm, resistant