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Article

Development of the PARMS Markers of the Waxy Gene and Utilization in Discriminating Wild Accessions, and Cultivated Rice (Oryza sativa L.) with Different Eating and Cooking Quality

by
Guibeline Charlie Jeazet Dongho Epse Mackon
1,
Enerand Mackon
2,
Yafei Ma
1,
Yitong Zhao
1,
Yuhang Yao
1,
Xianggui Dai
1 and
Piqing Liu
1,*
1
State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530005, China
2
State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Guangxi University, Nanning 530005, China
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(6), 1294; https://doi.org/10.3390/agronomy12061294
Submission received: 15 April 2022 / Revised: 25 May 2022 / Accepted: 26 May 2022 / Published: 28 May 2022
(This article belongs to the Special Issue Marker Development in the Genomics Era)
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Versions Notes

Abstract

:
Amylose content (AC) is the major indicator of rice eating and cooking quality (ECQ). Its synthesis in rice endosperm is mainly regulated by the protein, granule-bound starch synthase 1, which is encoded by the waxy gene (Os06g0133000, LOC_Os06g04200). The diversity of AC is largely attributable to the allelic variation at the Wx locus and the development of effective and accurate functional molecular markers to target rice variant alleles is crucial in the breeding strategy. In the present study, we developed six pairs of penta-primer amplification refractory mutation system (PARMS) markers to distinguish between Wxlv, Wxa, Wxin, Wxb, Wxmp, and Wxop, hp. These markers were successfully used to screen the genotype of large assets of genetic resources including 98 wild accessions, 55 cultivars, and 22 parental lines. Our results showed that Wxb in a low AC type was predominant in Guangxi cultivated rice as a result of cultural preference, while Wxlv in the wild accessions. Moreover, our findings surprisingly revealed the presence of Wxb in wild accession, which is a new outcome that may contribute to understanding the origin, selection and domestication processes of rice. These functional markers could be effectively used in marker-assisted breeding to improve selection efficiency of cultivars with desired AC in the early generation.

1. Introduction

Rice (Oryza sativa L.) is the second most important cereal crop after maize that plays an important economic role, feeding half of the world’s population and more than 60% in China [1,2]. In developing countries, rice is closely associated with energy demand, food security, and political strategies [3,4]. The economic value of white rice as a commodity is affected by its appearance (grain transparency, shape) and eating and cooking quality (ECQ) which guide selection and cultivar releasing [5]. Rice ECQ is considered the primary indicator of rice grain quality and rice flour product [6,7,8]. It is influenced by physicochemical properties such as apparent amylose content also called amylose content (AC), gel consistency (GC), gelatinization temperature (GT), and viscosity [9]. Rice ECQ is associated with the level of AC in the sense that generally, rice with high AC is dry, fluffy, separated when cooked, and hard upon cooling, while rice with low AC is moist, sticky, soft, and glossy after cooking [10,11]. In the southeast of Asia, mainly in China, the lower AC is much preferred [12]. GC is a fluid property of rice starch gel measuring the cold paste viscosity of cooked rice. The cooked rice with high GC (≥60 mm) is softer and more elastic [13]. GT is evaluated based on alkali spreading value or alkali digestion (AlkD), referring to the extent of dispersal of milled rice grains in a dilute alkali solution (1.7% potassium hydroxide), and a value of less than two is considered a high GT. Practically, it is associated with rice cooking time [13]. In the past, farmers from different regions tracked rice quality during the growing season, followed by the selection and spreading according to cultural consumption. For instance, the phylogenetic analysis revealed that sticky or glutinous rice is distributed throughout the mountainous regions of the Indochina peninsula in South and Southeast Asia because of its culinary qualities required for ceremonial, medicinal, and unique products purposes [14].
Studies have been conducted over the past 20 years to understand the genetic basis underlying rice eating and cooking attributes. Several studies pointed out the waxy (Wx) gene (Os06g0133000, LOC_Os06g04200) located on chromosome 6, encoding the protein granule-bound starch synthase 1 (GBSS1) as a principal determinant controlling AC and GC in rice endosperm, and subsequently ECQ [7,9,15,16,17]. The activities of GBSS1 potentially determine the amylose synthesis [18]. In the early classification based on AC, five major categories have been reported, including, waxy (0–2%), very low (3–9%), low (10–19%), intermediate (20–24%), and high (>25) [19,20]. More recently, with new allelic variants, a correlation was established between the AC variation and allelic diversity of the Wx locus [21,22]. New classification was released following the decreasing order: Wxlv (>25%), Wxa (24–25%), Wxin (18–22%), Wxb (15–18%), Wxmw (10–14%), Wxmp (7–11%), Wxop (5–10%), and wx (AC<2%) [23]. Wxlv was reported to be ancestral from which the three main Wx alleles Wxa, Wxin, and Wxb originated by the substitution of functional residues [22]. The main difference between Wxa and Wxb resides at the G/T substitution at the 5′ splicing donor site of intron 1-1 (Int1-1). The improper splicing of the pre-mRNA caused by the mutation produces 10-fold less GBSS1 protein, resulting in reduced AC and, consequently a slightly sticky texture of cooked rice [24,25]. Most non-glutinous indica rice has Wxa, while most non-glutinous japonica rice has Wxb. Meanwhile, the C/T SNP (Pro415Ser) in exon 10-115 (Ex10-115) is essential in non-glutinous rice responsible for the extra-long chains (ELC) of amylopectin in rice endosperm [26]. The Wxin and Wxop, hp carry an A/C SNP (Tyr224Ser) in exon 6-62 (Ex6-62), leading to intermediate AC and an A/G transition (Asp166Gly) in exon 4-77 (Ex4-77), resulting in the opaque seed with very low AC, respectively [27,28,29]. The Wxmp which is derivative of the Wxb harbor G/A SNP (Arg158His) in exon 4-53 (Ex4-53), leading to low AC. The wx is glutinous rice with 23 bp deletion in exon 2, leading to a premature termination codon within its coding sequence, resulting in no GBSS1 protein; hence, opaque seeds with null amylose in the starch, and cooked rice with elasticity [30,31]. These alleles could explain 99.9% of the AC variation in rice [23]. Recently a novel Wx allele called Wxla, which combined Wxb, and Wxin was discovered [9].
The breeding strategies aimed to combine different alleles to obtain a satisfactory ECQ. For instance, the Wxmw, which combine two natural Wx allele Wxin and Wxb, was reported with a favorable AC, improving ECQ and grain transparency [12]. Thus, the identification of genome sequence variants and efficient high-throughput markers are required for genetic analysis and molecular breeding. Conventional breeding methods are time-consuming and more expensive [32]. Recently with a series of Wx alleles identified, several gel-based markers, including simple sequence repeat (SSR), cleaved amplified polymorphic sequence/ derived cleaved amplified polymorphic sequence (CAPS/dCAPS), have been developed for genotyping [33,34,35,36,37,38,39]. The greatest inconvenience with the existing CAPS/dCAPS markers is that two pairs of primers need to be combined in a single PCR reaction. Some are unable to detect heterozygote genotypes accurately. During our recent investigation we highlighted some potential error-prone genotyping with those markers in which the gel results showed discrepancies with the sequencing (data not shown). This inconsistency was reported in similar studies applying allelic specific PCR (AS-PCR) gel-based genotyping in fragrant rice [40,41]. In addition, regarding the slight difference between the molecular weight of two samples (SNPs, indels), the visualization of the gel after electrophoresis is challenging, leading to reading error in genotyping, and often requires multiple assays. Such features limit the number of samples at a time, slow down the research, increase the experiment’s cost, and delay the selection process.
High-throughput automated molecular systems such as TaqMan, Kompetitive allelic specific PCR (KASP), and penta-primer amplification refractory mutation system (PARMS) have been widely integrated to breakdown this bottleneck in breeding program. PARMS is a KASP-like SNP genotyping technique that associates AS-PCR also known as amplification refractory mutation system (ARMS) with universal energy transfer-labeled primers [42]. It is a potential gel-free SNP genotyping technology conducted by fluorescence scanning based on five primers, including universal fluorescent primers, allele-specific primers, and reverse shared primers [43,44,45]. The advantage of PARMS over other fluorescent methods, such as KASP, is that it shows a denser genotype cluster and is less sensitive to PCR inhibitors in the alkaline lysis [45]. A single-step PARMS genotyping combining amplification and diagnostic steps is an efficient and low-cost platform system that has been used successfully to accelerate assays in maize [45,46], rapeseed [47], wheat [48], rice [43,44,49], and other crops.
The present study developed PARMS markers to discriminate between Wxlv, Wxa, Wxin, Wxb, Wxmp, and Wxop, hp and validated their accuracy through the genotyping of significant assets of genetic resources including wild accessions, cultivars, and parents of hybrid rice. The aim was to assess the potential application of this technology in the trait with multiple alleles and develop new usefulness markers for rice ECQ optimizing the selection of cultivars with desired eating quality.

2. Materials and Methods

2.1. Plant Materials

The present study used 98 wild rice accessions with significant phenotypic differences, 55 representative rice including cultivars, breeding lines, and 23 as the control materials including sterile line, restorer line, and hybrid combinations. Wild accessions were selected from the core collection of Guangxi common wild rice provided by the national germplasm Nanning wild rice nursery. The other materials were collected from different locations in Guangxi province, including: Guangxi zhaohe seed corporation LTD, Guangxi Quanhong seed Corporation Ltd., Guangxi Jinka Agriculture Corporation Ltd., Nanning Liangnong Agriculture Corporation Ltd., Guangxi Hengmao Agriculture Corporation Ltd., Guangxi Guoliang seed Corporation Ltd., Guangxi Boshiyuan seed Corporation Ltd., Guangxi Ivhai seed Corporation Ltd. The ECQ parameters, including AC, GC, AlkD, and grain morphology data of the 55 cultivars were obtained from the previous studies through the database (Table 1) (http://www.ricedata.cn/variety/index.htm; accessed on 30 June 2021), and by other measurements under Super depth three-dimensional (3D) microscopic imaging system. Microsoft Excel version 2010 was used for data preparation. The pair-wise analysis of ECQ parameters and the relation between AC, GC, and AlkD, and genotypes were performed with ggplot2 from R package version 3.6 [50].
The control group included Nipponbare, IR24, Gui99, and ZHN42 for Wxb; Zhong A, Ce 64-7, BoA, and ZHN16 for Wxa; Tianyouhuazhan for WxaWxb; Kasalath and R402 for Wxlv; Basmati, IR64, and ZHN79 for Wxin; Nangeng 46, Huzaoxiang, ZHN57, 9108, and MYP12 for Wxmp; Haopi and Haomuxi for Wxop; Huangbansuo and Mowangu for Wxmw. These rice materials were collected from Guangxi Academy of Agricultural Sciences in Nanning, and the Chinese Academy of Agricultural Sciences, in Beijing. All materials for genotyping except for wild accessions were germinated in the greenhouse at Guangxi University, Nanning, P.R. China.

2.2. DNA Extraction and Quality Analysis

Rice was grown, and genomic DNA (gDNA) was isolated by alkaline lysis [44], and cetyltrimethylammonium bromide (CTAB) [51] methods with slight modification. Briefly, for alkaline lysis the 3–4 cm leaf samples from 20-day-old seedlings were collected into a 96-deep-well plate. A 75 μL volume of 0.3 M NaOH solution and two stainless balls (4 mm diameter) were added to each sample well for grinding at 50 Hz using Tissue Lyser (JINGXIN, China) for 1 min. The well plate was placed into a water bath at 96 °C. Further, 200 μL of 0.75 M Tris-HCl (pH 7.5–7.8) were added to the sample and centrifuged at 3000 × g for 1 min. The supernatants were then transferred to a new 200 μL 96-well PCR plate, diluted 10 times, and store at −20 °C for PARMS genotyping analysis.
For the CTAB method, 0.1 g of leaf tissue were collected and mixed, ground in liquid nitrogen into a powder form in the 2-mL centrifuge tube using a grinder. Then 800 μL 2% preheated CTAB extraction buffer (CTAB-4 g; NaCl-16.34 g; 1 M Tris-HCl-20 mL (PH 8.0); 0.5 M EDTA-8 ml; PVP-360-2 g volume to 200 mL (pH 8) re-sterilization, preheated in a water bath to 65 °C for 30 min was added and incubated in Mary’s bath at 65 °C for 40 min with intermittent shaking each 10 min. Then one volume (400 μL) of chloroform-isoamyl alcohol (24:1) was added, thoroughly mixed by inverting the tube for 5 min before centrifugation (12,000 × g, 5 min). The suspension was aspirated carefully and transferred to a new 1.5-mL centrifuge tube. Afterward, twice the volume of isopropyl-alcohol was added, mixed gently, and stood at −20 °C for over 30 min. The nucleic acid in the aqueous phase was pelleted after centrifugation (12,000 × g, 5 min). A 0.5 mL volume of 70% ethanol was added to the precipitate, and centrifuged (12,000 × g, 5 min) after 5 min at room temperature. The above wash was repeated. The sample was air-dried, dissolved in 50 μL sterilized deionized water, and stored at −4 °C for further analysis.

2.3. Identification of the SNP Sites and Primer Design

To develop potential PARMS markers to target Wx alleles, we identified the relationship between the different alleles through the recently established evolutionary map [9,52], which also revealed the type of mutations corresponding to Wxa, Wxb, Wxlv, Wxop, Wxin, and Wxmp (Figure 1A). Further, the Wx locus (LOC_Os06g04200) on chromosome 6 spanning 1765622-1770574 (Figure 1B) was reconstituted from our data obtained in the previous sequencing (data not shown) and aligned with the Nipponbare genome and RiceVarMap (http://ricevarmap.ncpgr.cn/v2/two_cultivars_compare/; accessed on 28 November 2021). The flanking sequences were obtained from the Nipponbare V6 genome through the rice Gbrowse program RiceVarMap (http://ricevarmap.ncpgr.cn/cgi-bin/gb2/gbrowse/ricevarmap/; accessed on 9 December 2021 ). The fingerprint SNP primers were designed through the online tools snpway (http:/www.snpway.com/, accessed on 10 January 2022) according to the SNP positions in Figure 1C.

2.4. PARMS Genotyping Analysis

Genotyping tests were carried out with PARMS primer sets commercially synthesized by Gentides Biotech Co., Ltd. (Wuhan, China). The composition of the reaction and PCR profiling were performed similarly to that of KASP described at LGC genomics, UK (https://www.lgcgroup.com/services/genotyping/projects/; accessed on 6 January 2022) and reported by Lu et al. [44] as follows: All reactions were set in 384-well PCR plates for genotyping. An aliquot of 5 µL PCR reaction system contained 2 × PARMS PCR reaction mix (containing two common fluorescent primers, PCR buffer, dNTP, Taq enzymes, and internal standard ROX), 150 nM of each allele-specific primer, 400 nM locus-specific primer, and 1.4 µL alkali lysis DNA template were mixed. Mineral oil (5µL) was added to each well of the PCR plate to prevent evaporation of the mixture during the PCR reaction. The optimized thermal cycler run for the PARMS reaction was as follows: first denaturing at 95 °C for 15 min, followed by a touchdown phase of 10 cycles of denaturation at 95 °C for 20 s, and at annealing 65–57 °C (dropping 0.8 °C per cycle) for 1 min, then decreasing per cycle to the annealing temperature at 57 °C. This reaction was followed by 32 cycles of denaturation at 95 °C for 20 s and annealing at 57 °C for 1 min. Once the thermal cycling was complete, the PCR reactions’ well plates were read using a TECAN Infinite M1000 plate reader. SNP calling and plots were carried out using SNP-decoder, the online software (http://www.snpway.com/snpdecoder/; accessed on 25 January 2022) combining manual modification.

2.5. Validation of Genotyping Results through Sanger Sequencing

PCR amplification primers for Sanger sequencing purposes were designed through the online primer design tool Primer3Plus (https://primer3plus.com; accessed on 4 Febraury 2022) and synthesized by Bioengineering Co., Ltd. The primer sequences can be found in Table S1. PCRs were performed in a reaction volume of 20 µL consisting of 1 µL of rice genomic DNA as a template, 10 µL 2× PCR mix, 1 µL PCR enhancer, 0.5 µL of each of forward and reverse primer (10 µM), and 7 µL ddH2O. The thermal cycle of PCR reaction was programmed as follows: pre-denaturation at 95 °C for 15 min, followed by 32 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, and final extension of 72 °C for 3 min. All fragments were separated in 1.5% agarose gel. According to the BigDye™Terminator v3.1 Cycle Sequencing Kit manufacturer’s manual, the sequencing reactions were performed on the ABI3130 XL sequencer following the manufacturer’s protocols (https://biosci-batzerlab.biology.lsu.edu/Genomics/genomics_services_ABI-3130XL.php; accessed on 10 Febraury 2022). The sequence alignment was performed through CLUSTALW software (https://myhits.sib.swiss/cgi-bin/clustalw; accessed on 15 Febraury 2022).

3. Results

3.1. Development of PARMS Marker of the Wx Alleles

A total of six markers were developed from different SNPs (Table 2). Two allelic-specific forward primers (primer X-FAM and primer Y-HEX) and a common reverse primer (primer C) were designed from 100-bp left and 100-bp right flanking the SNP sequence. The PARMS marker primers were constructed with fluorescent markers at the tail. The oligonucleotides labeled with the FAM/HEX fluorophore were annealed to the forward primer.

3.2. Validation of PARMS Markers through the Genotyping of Rice Cultivars and Accessions

To assess the efficiency of the six developed markers and validate their potential use in marker-assisted breeding (MAB), we performed PARMS genotyping using 55 cultivars, 98 wild accessions, and 17 as the controls. The PARMS assay comprises two forward and one reverse primer with a linker specific to the allele, the high-quality gDNA, and the 2 × PARMS master mix, which consists of fluorescent probe FAM and HEX attached to the allele-specific tail in the primer. The sample genotype was identified based on the scatter spots shown by the fluorescent probes. While the homozygote samples formed one spot on the top left close to the Y-axis or bottom right near the X-axis, the heterozygote SNPs formed a spot right in the mid-line of the X-Y diagonal. All the samples formed a cluster corresponding to the homozygote and heterozygote SNPs identified (Figure 2).
Our results revealed for Guangxi rice cultivars that three belonged to WxaWxa, 39 WxbWxb, and 9 WxaWxb (Table 3). Among the 98 Guangxi wild accessions, 82 were homozygote WxlvWxlv, one was WxaWxa, six were WxbWxb, five heterozygote WxlvWxa, and four had no amplification. Other SNPs for Wxmp, Wxin, and Wxop, hp were fully distinguishable among all samples. All genotyping results can be found in Table S2.

3.3. Validation of PARMS Genotyping Results from Wild Accessions

Wxlv, Wxa, and Wxb are discriminated based on the Int1-1 Exon 10 SNPs. Genotyping with PARMS marker showed the presence of WxaWxa (W186), WxbWxb (W48, W168, W173, W261, W291, W326), and WxlvWxa (W37, W66, W69, W72, W189) in wild accessions, which has not been previously identified. To confirm these genotypes, we performed Sanger sequencing (Figure 3A,B). Primer sequences were designed to amplify the sequence containing the T/C transition in Exon 10-115 and G/T transition in Int 1-1, which are the characteristics of Wxa, Wxb, and Wxlv. Eight accessions were amplified (W18, W37, W48, W168, W173, W186, W261, W326 through PCR, and five representative accessions for Wxa (W186), Wxb (W48, W326), Wxlv (W18), WxaWxlv (W37) were chosen for sequencing. The result showed that PARMS genotyping was consistent with Sanger sequencing (Figure 3C), suggesting that wild accessions could harbor Wxa and Wxb alleles.

4. Discussion

Rice grain essentially comprises starch composed of amylose, a linear polysaccharide and amylopectin, a highly branched polysaccharide. The amylose in rice is the main determinant of rice taste and appearance after cooking, which are important factors for consumers and market price. The proportion of amylose in rice varies from 0 (Wx rice) to 30% of starch, referred to as glutinous and non-glutinous rice, respectively. The amount of amylose present in the starch affects rice flour’s physicochemical properties (gelatinization and retrogradation) when processing. Thus, improving rice yield and other agronomic traits while maintaining the desired AC has been a driving force for crop breeding and biotechnology. The quality cultivar selection was challenging because the determination of the AC was required for the validation. Various processes have been used for AC quantification, among which iodine binding was the most effective [53]. The breakthrough of functional genomics, which allowed the identification of Wx gene coding for GBSS1 responsible for amylose synthesis, opened a new era for understanding rice eating and cooking quality. Recently, high-throughput genome resequencing revealed SNPs and indels across the Wx locus unraveling new allelic variants. To facilitate the breeding through marker-assisted selection we developed PARMS markers, an automated direct PCR-reading genotyping system which allowed a rapid and inexpensive analysis of many samples regarding the rice ECQ.
In this study, the flanking sequences of different allelic variants were used to develop PARMS markers for G/T in Int1-1, G/A in Ex4-53, A/G in Ex4-77, A/C in Ex6-62, and T/C in Exon 10-115 facilitating the simultaneous identification of Wxa, Wxb, Wxop, hp, Wxmp, Wxin, and Wxlv. Real-time PCR showed that these markers could successfully differentiate the homozygous and heterozygous types in parental lines, cultivars, and wild accessions. By examining the 55 cultivars, we found that two dominant alleles (Wxa and Wxb) and three genotypes (WxaWxa, WxbWxb, and WxaWxb) existed in the selected Guangxi popular rice, South China, with a respective genotype frequency of 5.46%, 75.74%, and 18.18% (Table S3). The 55 cultivars could be grouped based on their AC into low AC ranging from 11.5 to 18.6, intermediate AC ranging from 20.4 to 24.9, and high AC from 25.4 to 26.6 according to the early classification [19,20,54]. We investigated the correlation between the genotype observed and the physicochemical trait AC, GC, and AlkD, which showed that all GG, GT, and TT were low, intermediate, and high AC, respectively, with significant differences between different groups (Figure 4A–C). This result showed the effectiveness of the developed PARMS markers. The phenotypic pair-wise correlation between the ECQ parameters (Figure 4D), highlighted that AC is inversely correlated (R2 = 0.77) with GC, positively correlated with AlkD (R2 = 0.16). The high AC class had a high value of AlkD. This result was consistent with previous studies reporting a correlation between AC and GC [55,56,57]. In general, GC measured the cold paste viscosity of cooked rice and was so far reported as the test that complements the amylose [58]. In conformity with Camgampang et al. [55] classification, only medium and soft gel consistency were found in these cultivars. The softer gel is long and corresponds to low AC, while the hard gel is short and corresponds to very low AC.
In wild accessions, three allelic combinations Wxa, Wxlv, and Wxb were identified in different genotype frequency WxaWxa (1%), WxbWxb (6.25%), WxlvWxlv (87.5%), WxaWxlv (5.2%). They were reported as the major allele involved in the male sterile lines of three-line hybrid rice (Wxa), restorer lines (Wxb), and male sterile lines of early two-line hybrid rice (Wxlv), respectively [23]. Hence, it was suggested to be important in MAB. Wxin, which includes many high-quality rice varieties with an intermediate AC were closely similar to Wxlv regarding the Int1-1 and Ex10-115 SNPs. At the same time, Wxop, hp and Wxmp, the rare alleles, were similar to Wxlv, and Wxb, respectively. This result was in line with Shao et al. [23], who reported three allelic combinations, WxaWxlv, WxbWxb, and WxlvWxlv, in 35 parental lines in Hunan province, South China [23]. Our results suggested that wild accessions were mostly Wxlv, and decreasing the AC happened through the selective sweep of Wxlv and Wxa and introducing new allele with preferred low AC. Recently, disputes arose among Wxa and Wxb concerning the origin and domestication of the Wx allele. In the current study, we found that wild rice had Wxb. However, whether the wild rice harboring the Wxb is not the hybrid or offspring between the cultivated rice and the wild rice is unclear. Zhou et al. [9] reported that new Wx alleles could easily be generated by crossing genotypes with different Wx alleles. So far, Hirano and co-authors, as well as other researchers, suggested that the Wxb allele in japonica rice originated from the Wxa allele of O. rufipogon, its wild progenitor, after the G/T substitution at the 5′ splicing donor site of Int1-1 [24,25,59]. Other studies proposed that the Wxb and Wxin alleles appeared in early domesticated rice in the area north of the Yangtze River [60,61]. Huang and Han [62] showed that japonica rice was first domesticated and indica emerged from japonica. Recently, with the discovery of Wxlv it was revealed that the three major cultivated Wx alleles in cultivated rice (Wxb, Wxa, and Wxin) differentiated from Wxlv haplotypes (Figure 1B), which originated directly from wild rice [9,22]. Considering the allelic frequency in the cultivar and accession (Table S3) and the evolutionary recently established [9,12], it can be concluded that the haplotype Wxlv-I is dominant (Figure 1B). Our findings will re-center the debate across the origin and domestication of cultivated rice.

5. Conclusions

This study developed and validated PARMS markers by screening different Wx alleles in wild accessions and cultivated rice of Guangxi popular rice in South China. Conversion of the detected polymorphisms to molecular markers is essential to establish the applicability of such polymorphisms to breeding. Our markers were tightly linked to Wx allelic variations in rice and can be used to simplify and accelerate the selection of the desired eating quality for AC. This work revealed the potential application of this technology in the trait with multiple alleles.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12061294/s1, Table S1: Primer sequence used in the PCR for Sanger sequencing; Table S2: PARMS genotyping results of Wx alleles; Table S3: Allele and genotype frequencies from PARMS genotyping.

Author Contributions

Author Contributions: Conceptualization, G.C.J.D.E.M., E.M. and P.L.; Data curation, G.C.J.D.E.M., E.M. and Y.Z.; Formal analysis, G.C.J.D.E.M., E.M. and Y.M., Funding acquisition, P.L.; Investigation, G.C.J.D.E.M., E.M., X.D. and Y.Z.; Methodology, G.C.J.D.E.M., E.M., Y.M.; Project administration, P.L., E.M.; Resources, P.L.; Software, G.C.J.D.E.M., E.M., Y.Y. and Y.M.; Supervision, P.L.; Validation, G.C.J.D.E.M., E.M. and P.L.; Visualization, P.L., G.C.J.D.E.M. and E.M.; Writing—original draft, G.C.J.D.E.M., E.M.; Writing—review and editing, G.C.J.D.E.M., E.M., Y.M. and P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work and APC were funded by the National R&D Priority Program-Breeding New Rice Varieties for Southern China Area (2017YFD0100100) and The Guangxi R&D Priority Program —Research and Application of Rice Resistance Breeding to Bacterial Blight (Guike AB 16380124).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw data can be provided to researchers upon request via correspondence with the first author and corresponding author.

Acknowledgments

We thank Guangxi Academy of Agricultural Science (GAAS), Chinese Academy of Agricultural Science in Beijing (CAAS), and Guangxi province seed corporations for providing us with the materials used for our experiment. We thank Moses Elleason and Wambura M. Mtemi for proofreading.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AACApparent Amylose Content
ACAmylose Content
AlkDAlkali Digestion
AS-PCRAllelic Specific PCR
CAPSCleaved amplified polymorphic sequence
CTABCetyltrimethylammonium Bromide
ECQEating and Cooking Quality
GBSS1Granule Bound-Starch Synthase 1
GCGel Consistency
GrLGrain Length
INDELInsertion–Deletion
MASMarker-Assisted Selection
MABMarker-Assisted Breeding
SNPSingle Nucleotide Polymorphism
SSRSimple Sequence Repeats
KASPKompetitive Allele-specific
PARMSPenta-primer amplification refractory mutation system
PCRPolymerase Chain Reaction

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Figure 1. Evolutionary and SNP identification in Wx locus. (A) Evolutionary scenario for different Wx alleles; (B) gene structure of Wx locus; purple rectangles denote the untranslated regions, and blue ones the different exons in the coding region; gray lines indicate the introns; the red arrows indicate the position of the SNPs on Wx locus; the gray arrows show the start of mRNA and protein GBSS1. (C) SNP identified for PARMS development in Wx locus and their origin; AC% represent the amylose content of the cultivar with each allelic type.
Figure 1. Evolutionary and SNP identification in Wx locus. (A) Evolutionary scenario for different Wx alleles; (B) gene structure of Wx locus; purple rectangles denote the untranslated regions, and blue ones the different exons in the coding region; gray lines indicate the introns; the red arrows indicate the position of the SNPs on Wx locus; the gray arrows show the start of mRNA and protein GBSS1. (C) SNP identified for PARMS development in Wx locus and their origin; AC% represent the amylose content of the cultivar with each allelic type.
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Figure 2. PARMS genotyping plots. Samples are clustered on the X-FAM (abscissa) and Y-HEX axes (ordinate). Alleles were classified based on genotype. Green and blue dots represent the homozygote alleles, red dots represent heterozygote allele, grey dots denote the non-template control (NTC), and the very few black dots in some plots denote the non-amplified samples. (A) PARMS genotyping for the exon 10 SNP (T/C), T:T = green, C:C = blue, C:T = red; (B) PARMS genotyping for the intron 1 SNP (G/T), G:G = green, T:T = blue, G:T = red; (C) PARMS genotyping for the exon 6 SNP (A/C), C:C = green, A:A = blue; (D) PARMS genotyping for the exon 4 SNP (G/A), A:A = green, G:G = blue; (E) PARMS genotyping for the exon 4 SNP (A/G), G:G = green, A:A = blue, A:G = red.
Figure 2. PARMS genotyping plots. Samples are clustered on the X-FAM (abscissa) and Y-HEX axes (ordinate). Alleles were classified based on genotype. Green and blue dots represent the homozygote alleles, red dots represent heterozygote allele, grey dots denote the non-template control (NTC), and the very few black dots in some plots denote the non-amplified samples. (A) PARMS genotyping for the exon 10 SNP (T/C), T:T = green, C:C = blue, C:T = red; (B) PARMS genotyping for the intron 1 SNP (G/T), G:G = green, T:T = blue, G:T = red; (C) PARMS genotyping for the exon 6 SNP (A/C), C:C = green, A:A = blue; (D) PARMS genotyping for the exon 4 SNP (G/A), A:A = green, G:G = blue; (E) PARMS genotyping for the exon 4 SNP (A/G), G:G = green, A:A = blue, A:G = red.
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Figure 3. Sanger sequencing results compared to PARMS test; (A) sequencing diagram of amplified samples; (B) gel electrophoresis of nine samples in exon 10 and intron 1; (C) comparison of PARMS genotyping with sequencing result.
Figure 3. Sanger sequencing results compared to PARMS test; (A) sequencing diagram of amplified samples; (B) gel electrophoresis of nine samples in exon 10 and intron 1; (C) comparison of PARMS genotyping with sequencing result.
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Figure 4. Distribution of ECQ parameters and pair-wise analysis in the 55 cultivars; (A–C), box plots of AC, GC, and AlkD, respectively, observed in the cultivars divided into different genotypes; data represent mean ± S.E.M. Student’s t-tests were used to generate p-values, whiskers represent standard error of least squared means; endpoints of upper and lower whiskers represent maximum and minimum values, respectively; upper and lower edges of boxes represent third and first quartiles, respectively; line inside box represents median. The level of significance was analyzed with a t-test and are indicated with stars, * (p < 0.05), ** (p < 0.005), *** (p < 0.005), and **** (p < 0.0005). (D) heatmap of the correlation between AC, GC, and AlkD. The color and the stars indicate the degree of significance.
Figure 4. Distribution of ECQ parameters and pair-wise analysis in the 55 cultivars; (A–C), box plots of AC, GC, and AlkD, respectively, observed in the cultivars divided into different genotypes; data represent mean ± S.E.M. Student’s t-tests were used to generate p-values, whiskers represent standard error of least squared means; endpoints of upper and lower whiskers represent maximum and minimum values, respectively; upper and lower edges of boxes represent third and first quartiles, respectively; line inside box represents median. The level of significance was analyzed with a t-test and are indicated with stars, * (p < 0.05), ** (p < 0.005), *** (p < 0.005), and **** (p < 0.0005). (D) heatmap of the correlation between AC, GC, and AlkD. The color and the stars indicate the degree of significance.
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Table
Table 1. ECQ parameters and grain characteristics of the cultivars studied.
Table 1. ECQ parameters and grain characteristics of the cultivars studied.
CultivarHybrid (H) or Inbred (I)ACGel Consistency (GC)Alkali Digestion (AlkD)Grain Length (GrL) (mm)Length–Width Ratio (L/W)1000-Grain Weight (TGW) (g)
Guangxin 5113H20.461.06.07.03.224.0
Hualiangyou 338H2478.06.69.13.424.0
Zhaofengyou 9958H20.778.05.97.33.423.5
Yongfengyou 9802H14.683.07.010.23.424.3
Guang8youhuahzhanH16.155.53.19.13.420.2
Teyou 2278H26.640.07.08.52.726.1
YouxianglongsimiaoH15.578.06.98.74.422.1
Hlianyou 6839H12.386.04.510.53.230.1
Hengfengyou 666H13.176.04.69.73.527.3
Hengfengyou 426H11.578.03.09.53.326.26
Hengfengyou 7166H19.258.05.66.63.226.7
HengfengyoujinsimiaoH12.576.04.36.73.423.6
HengfengyouhuazhanH15.176.53.07.83.524.1
GuangheyouhuazhanH12.782.06.86.53.423.6
Shenliangyou 8386H11.578.03.27.53.525.4
Guang8youxiangsimiaoH15.476.06.79.14.220.8
HengfengyouyuxiangH14.680.07.010.23.825.6
Guang8you 165H15.465.06.89.03.421.7
H liangyou 5872H16.280.03.09.23.228.9
Wuyou 305H13.474.45.56.73.123.7
Shenyou 9569H12.583.06.66.53.026.4
Teyou 6811H25.943.06.19.42.225.3
Hengfengyou 777H11.682.03.36.83.125.6
WushansimiaoI17.566.76.89.13.122.4
Quanxiangyou 822H15.186.06.38.53.624.0
Teyou 582H21.638.06.79.52.224.9
Teyou 831H25.450.06.08.32.426.9
QuanyousimiaoH1672.07.06.73.226.4
QuanxiangyoumeizhanH13.676.04.810.23.823.9
Quanyou 123H16.982.04.99.93.328.8
Hexin 5 haoI13.680.04.910.23.720.9
Fengtianyou 553H1578.05.49.23.522.4
Shenliangyou 1173H15.254.06.99.33.221.7
Y liangyou 1173H13.380.05.07.23.220.6
Fengtianyou 089H14.970.05.68.53.622.2
Zhuangxiangyoubaijin 5H15.276.06.16.64.120.8
Taiyou 2068H14.476.04.58.53.824.2
Qianliangyou 8 haoH15.867.06.89.23.324.6
Changliangyou 8 haoH17.269.06.38.83.424.1
Shenyou 9516H18.677.04.68.13.227.5
Heliangyou 713H1576.05.59.23.023.7
Y liangyou 087H15.366.06.26.73.025.8
QianliangyouhuazhanH1580.55.49.13.222.9
Changliangyou 6 haoH15.778.07.09.13.625.2
Heliangyou 1 haoH1573.55.59.03.023.6
Guoliangyou 633H15.872.06.58.93.719.6
Teyou 7671H23.348.06.58.52.727.6
Teyou 7571H21.955.06.48.62.527.5
T You 682H19.960.06.010.12.927.7
Teyou 679H20.745.06.08.02.327.3
GuihefengI14.278.06.16.83.520.9
Y liangyou 5806H13.485.05.66.83.026.7
Teyou 913H21.972.06.38.52.629.4
Teyou 986H20.436.06.16.92.128.7
Naide 606I13.876.04.89.33.121.5
These data were obtained from the database http://www.ricedata.cn/variety/index.htm; accessed on 30 June 2021.
Table
Table 2. PARMS marker sequences for genotyping of Wx alleles.
Table 2. PARMS marker sequences for genotyping of Wx alleles.
Marker NameSNP TypeSequences
Primer X-FAM (Forward 1, 5’-3’)Primer Y-HEX (Forward 2, 5’-3’)Primer C (Reverse, 5’-3’)
PM-Wxlv(T/C)5′GAAGGTGACCAAGTTCATGCTCTGGAGGAACAGAAGGGCC-3′5′GAAGGTCGGAGTCAACGGATTGCTGGAGGAACAGAAGGGCT-3′5′GAGCTCCGGGATGGCG-3′
PM-Wxa(G/T)5′GAAGGTGACCAAGTTCATGCTATCAGGAAGAACATCTGCAAGG-3′5′GAAGGTCGGAGTCAACGGATTCATCAGGAAGAACATCTGCAAGT-3′5′GATCTGAATAAGAGGGGAAACAAA-3′
PM-Wxin(A/C)5′GAAGGTGACCAAGTTCATGCTACAACCCATACTTCAAAGGAACTTA-3′5′GAAGGTCGGAGTCAACGGATTCAACCCATACTTCAAAGGAACTTC-3′5′AATTAGTCTGATCATCATGGATTCC-3′
PM-Wxb(G/T)5′GAAGGTGACCAAGTTCATGCTATCAGGAAGAACATCTGCAAGG-3′5′GAAGGTCGGAGTCAACGGATTCATCAGGAAGAACATCTGCAAGT-3′5′GATCTGAATAAGAGGGGAAACAAA-3′
PM-Wxop, PM-Wxhp(A/G)5′GAAGGTGACCAAGTTCATGCTCTCCAGGAATGACGGATGGT-3′5′GAAGGTCGGAGTCAACGGATTCCAGGAATGACGGATGGC-3′5′AGCGTGGAGTCGACCGTG-3′
PM-Wxmp(G/A)5′GAAGGTGACCAAGTTCATGCTTGAACACACGGTCGACTCCAC-3′5′GAAGGTCGGAGTCAACGGATTTGAACACACGGTCGACTCCAT-3′5′GGTTGCAGACAGGTACGAGAGG-3′
The marker at each SNP position has two allelic specific forward primers (X and Y) and one common reverse, and the underlined sequences indicated in blue and green color at 5′ forward primer represent the fluoro-labeled oligos. The red nucleotides indicate the SNPs in the primer sequence.
Table
Table 3. Representative results of genotyping with PARMS markers.
Table 3. Representative results of genotyping with PARMS markers.
TypesNameHybrid (H) or Inbred (I)Wxa and Wxb Int1-1 (G/T)Wxmp Ex4-53 (G/A)Wxop Ex4-77 (A/G)Wxin Ex6-62 (A/C)Wxlv Ex10-115 (C/T)Wx Alleles/Genotypes
CultivarsTeyou 2278HGGAATWxa
Hengfengyou 7166HG/TGAAC/TWxa/Wxb
Shenliangyou 8386HTGAACWxb
Teyou 6811HGGAATWxa
Yliangyou 286HTGAACWxb
Teyou 831HGGAATWxa
Quanyou 123HTGAACWxb
Fengtianyou 553HTGAACWxb
Guangxin 5113HG/TGAAC/TWxa/Wxb
Zhuangxiangyoubaijin 5HTGAACWxb
Changliangyou 8 haoHTGAACWxb
Guoliangyou 633HTGAACWxb
Teyou 7671HG/TGAAC/TWxa/Wxb
Teyou 913HG/TGAAC/TWxa/Wxb
Zhaofengyou 9958HG/TGAAC/TWxa/Wxb
wild accessions (*)W72 GGAAC/TWxlv/Wxa
W165 GGAACWxlv
W186 GGAATWxa
W171 GGAACWxlv
W173 TGAACWxb
W189 GGAAC/TWxlvWxa
W284 GGAACWxlv
W290 GGAACWxlv
W321 GGAACWxlv
W326 TGAACWxb
W334 GGAACWxlv
W338 GGAACWxlv
Control samples (sterile, hybrid, and restorer linesNipponbareITGAACWxb
IR24ITGAACWxb
Zhong AHGGAATWxa
Ce 64-7IGGAATWxa
TianyouhuazhanHG/TGAAC/TWxa/Wxb
Kasalath,IGGAACWxlv
R402IGGAACWxlv
BasmatiIGGACCWxin
IR64IGGACCWxin
Nangeng 46ITAAACWxmp
HuzaoxiangITAAACWxmp
ZHN63IGGGACWxop
HaomuxiIGGGACWxop
(*): Wild accessions were taken from the wild habitat, and cannot be determined to be a hybrid or an inbred.
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Jeazet Dongho Epse Mackon, G.C.; Mackon, E.; Ma, Y.; Zhao, Y.; Yao, Y.; Dai, X.; Liu, P. Development of the PARMS Markers of the Waxy Gene and Utilization in Discriminating Wild Accessions, and Cultivated Rice (Oryza sativa L.) with Different Eating and Cooking Quality. Agronomy 2022, 12, 1294. https://doi.org/10.3390/agronomy12061294

AMA Style

Jeazet Dongho Epse Mackon GC, Mackon E, Ma Y, Zhao Y, Yao Y, Dai X, Liu P. Development of the PARMS Markers of the Waxy Gene and Utilization in Discriminating Wild Accessions, and Cultivated Rice (Oryza sativa L.) with Different Eating and Cooking Quality. Agronomy. 2022; 12(6):1294. https://doi.org/10.3390/agronomy12061294

Chicago/Turabian Style

Jeazet Dongho Epse Mackon, Guibeline Charlie, Enerand Mackon, Yafei Ma, Yitong Zhao, Yuhang Yao, Xianggui Dai, and Piqing Liu. 2022. "Development of the PARMS Markers of the Waxy Gene and Utilization in Discriminating Wild Accessions, and Cultivated Rice (Oryza sativa L.) with Different Eating and Cooking Quality" Agronomy 12, no. 6: 1294. https://doi.org/10.3390/agronomy12061294

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