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Article

A New SNP in AGPL2, Associated with Floury Endosperm in Rice, Is Identified Using a Modified MutMap Method

by
Long Zhang
1,2,
Ran You
1,2,
Hualan Chen
1,2,
Jun Zhu
1,2,
Lingshang Lin
1,2 and
Cunxu Wei
1,2,*
1
Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, China
2
Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province, Joint International Research Laboratory of Agriculture & Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou 225009, China
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(5), 1381; https://doi.org/10.3390/agronomy13051381
Submission received: 26 March 2023 / Revised: 13 May 2023 / Accepted: 14 May 2023 / Published: 15 May 2023
(This article belongs to the Special Issue Research on the Mechanisms and Functional Properties of Crop Starch Synthesis)
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Abstract

:
The floury endosperm mutants of rice can not only be used to uncover the molecular mechanisms involved in regulating starch synthesis and grain development but are also suitable for dry milling to produce rice flour of good quality. In this study, we identified and characterized a rice floury endosperm mutant, M10, from a mutant pool induced by EMS. The total starch content in the M10 seeds significantly decreased, while the soluble sugar content demonstrably increased. The grain hardness of M10 was lower than that of the wild type because of the spherical and loosely packed starch granules. The modified MutMap analysis demonstrated that AGPL2 on chromosome 1 is most likely to be the candidate gene causing a floury endosperm. The genome sequences of AGPL2 in M10 carried a single nucleotide substitution of guanine (G) to adenine (A) in the seventh exon, leading to a missense mutation from glycine (Gly) to glutamic acid (Glu) at the 251st amino acid. Allele test confirmed that AGPL2 is the gene responsible for the M10 phenotype. Both transcriptional and protein levels of AGPL2 in M10 were obviously higher than those in the developing endosperm of wild type, indicating a positive feedback regulation is caused by AGPL2 mutation. Together, our results suggest that AGPL2 plays a critical role in starch synthesis and that the modified MutMap method is feasible for identifying floury endosperm mutant genes in rice.

1. Introduction

Rice is one of the most important cereal crops and the staple food of over half of the world’s population, especially in Asian countries [1]. Rice is mainly consumed whole as cooked rice, although recently, rice flour is becoming more and more popular due to the advantages of its high nutritional value and that it is gluten-free [2,3]. Dry milling is a traditional and relatively simple process to produce rice flour and yields a highly nutritious flour rich in protein, ash, and lipids [3]. However, dry milling also produces a high proportion of damaged starch content, due to the rice’s densely packed starch granules and high grain hardness [4]. Rice grains with a floury endosperm have low grain hardness and small starch granules, which are more suitable for dry milling than the grains of normal rice [5]. Therefore, it is necessary to analyze the physicochemical properties and molecular characteristics of the flour endosperm to reduce the milling costs.
Starch consists of amylose and amylopectin and accounts for more than 90% of the endosperm weight. Due to the abundant genetic resources and small genome, over the last few decades, rice endosperm has been considered an ideal model for gene cloning and for analyzing the molecular mechanism of starch biosynthesis [6]. Numerous rice endosperm-defective mutants have been created using artificial mutagenesis methods (e.g., chemicals or irradiation) [4]. These endosperm mutants reveal waxy, dull, sugary, shrunken, chalky, and floury characteristics [7]. In recent years, some of the genes that cause a floury endosperm after mutations have been cloned, some of which encode the enzymes for starch synthesis. The ADP-glucose pyrophosphorylase large subunit 2 (OsAPL2) and the small subunit AGPS2 participate in the synthesis of ADP-glucose. Loss-of-function mutations of AGPL2 and AGPS2 produce floury and shrunken endosperms with round and small-sized starch granules [8]. Starch synthase IIIa (SSIIIa) is responsible for the elongation of amylopectin and its mutation produces spherical starch granules [9]. The starch branching enzyme IIb (BEIIb) is involved in the creation of the α-1,6-glycosidic bond of amylopectin and participates in the synthesis of amylopectin clusters. The BEIIb-deficient mutant causes a floury endosperm with highly resistant starch [10]. In addition, a series of rice floury endosperm (flo) mutant-causing genes have been identified, including flo6, flo7, flo8, flo10, flo12, flo13, flo14, flo15, flo16, flo18, flo19, and flo20 [6,7,11,12,13,14,15,16,17,18,19,20].
Several methods have been used for gene isolation in rice mutants; the most conventional method is map-based cloning (positional cloning). To overcome the costly and time-consuming characteristics of map-based cloning, the MutMap method has been explored and used successfully for rapid gene isolation [21]. MutMap is based on the whole-genome re-sequencing of pooled DNA from a segregated population of plants. In addition, MutMap+ and MutMap-Gap were developed to expand the applications of MutMap [22,23]. Until now, some rice genes have been isolated using the MutMap method, including the grain-size-related genes OML4 (Mei2-like protein 4), OSH15 (Oryza sativa homeobox 15), PPKL1 (protein phosphatase with kelch-like domains 1), SG2 (small grain 2) [24,25,26,27], the salt-tolerant-related gene RR22 (B-type response regulator gene 22) [28], and the chalkiness-related gene WB1 (white belly 1) [29]. Despite the fact that many genes have been mapped, the application of MutMap in the gene identification of floury mutants has not been reported.
In this study, we identified a floury endosperm mutant, known as M10. The modified MutMap analysis revealed that the G-to-A transition of AGPL2 in M10 resulted in a missense mutation from Gly to Glu at the 251st amino acid. An allelic test confirmed that AGPL2 was the causative gene for the floury endosperm. The qRT-PCR and Western blot assays suggested that positive feedback regulation was caused by the AGPL2 mutation in the M10 mutant. These results indicate that AGPL2 plays a crucial role in starch synthesis and endosperm development. In addition, it also provides a reference for the exploration of a new approach to gene mapping.

2. Materials and Methods

2.1. Plant Materials

The M10 mutant was initially identified from an EMS-irradiated mutant pool of the japonica rice cultivar, Kitaake (wild type (WT)). A homozygous plant with a floury endosperm was obtained after self-pollination. All plants were grown in a paddy field of Yangzhou University in May and were harvested in October (2019–2022).

2.2. Microscopy Observation

Mature brown rice (dehulled) was transversely broken to observe the cross-sections using a scanning electron microscope (S-4800, Hitachi, Tokyo, Japan), following a method described previously [6].

2.3. Characterization of Starch and Soluble Sugar Contents and Grain Hardness

The total starch content in rice flour was determined with a Megazyme Total Starch Assay kit (K-TSTA), according to the manufacturer’s instructions. The soluble sugar content was measured following the anthrone-H2SO4 method, as described by Man et al. [30]. The grain hardness of brown rice was analyzed using the TA.XT. Plus Texture Analyzer (Stable Micro Systems, Godalming, Surrey, UK), as described by Lin et al. [31].

2.4. Whole-Genome Sequencing of Bulked DNA

The M2 plants of M10 were hybridized with WT to produce an F2 population for MutMap (Figure 1). For the whole-genome sequencing, 30 translucent endosperm seeds and 30 floury endosperm seeds from the F2 population were selected for germination under sterile conditions. Genomic DNA was extracted from the resulting 7-day-old seedlings using a DNAsecure plant kit (Tiangen, Beijing, China). The concentration of each seedling’s DNA was measured using a One Drop spectrophotometer (OD-1000, Wins, Nanjing, China). The DNA from each seedling was equally mixed to construct a bulked translucent endosperm DNA pool (pool 1) and a bulked floury endosperm DNA pool (pool 2). Subsequently, the DNA samples of pool 1 and pool 2 were used for the re-sequencing library construction. The libraries were re-sequenced using an Illumina HiSequation 2500 (Novegene, Tianjin, China) to generate the short sequence reads as raw reads.

2.5. Re-Sequencing Analysis and Calculation of Δ (SNP Index)

The quality of raw reads was evaluated using the FastQC program (https://github.com/s-andrews/FastQC/releases, accessed on 22 October 2021). The FASTX toolkit program and SolexaQA software were used to filter out the sequences of Illumina paired-end adapters and the low-quality reads with a quality score of Q < 20 and reads of < 40 bp, respectively [32,33]. The cleaned reads were submitted to the NCBI SRA database for aligning with the reference Nipponbare genome sequence and obtaining the unique mapped reads [34]. The alignment files were converted to SAM/BAM files using SAMtools and were applied to the GATK Pipeline to identify SNPs, based on the reference genomic sequence. The SNP index of each SNP was estimated via the sliding window method [29]. The Δ (SNP index) was obtained by subtracting SNP-index (1) from that of SNP-index (2) (Figure 1). A Manhattan plot was produced via a custom script written in R version 4.1.1, based on the Δ (SNP index) (https://www.r-project.org, accessed on 25 October 2021).

2.6. Mutation Site Sequencing and Allele-Specific PCR (AS-PCR)

The DNA in fresh leaves of WT and M10 was extracted according to the hexadecyl trimethylammonium bromide (CTAB) method [35]. The fragments containing mutation sites were amplified via PCR for sequencing. The allele-specific mismatch primers for the AS-PCR assay were designed as described by Jiang et al. [36].

2.7. Allelic Test of M10 and M37

The plants of M10 and M37 were hybridized with each other to obtain F1 hybrids. The resulting F1 plants were self-pollinated and F2 populations were obtained. The allelic test was conducted on M10 and M37, according to the endosperm phenotype of F1 hybrids.

2.8. RNA Isolation and qRT-PCR

Total RNA was prepared from the endosperm of WT and M10 at 12 days after flowering (DAF), using an RNAprep Pure Plant kit (Tiangen, Beijing, China). First, 1 μg of total RNA was reverse-transcribed with a cDNA synthesis kit (Novoprotein, Suzhou, China). qRT-PCR was performed on a Bio-Rad CFX Connect real-time PCR device using a NovoStart®SYBR qPCR SuperMix kit (Novoprotein, Suzhou, China). The qRT-PCR primers of AGPL2 and UBQ are listed in Table S1 in the Supplementary Materials.

2.9. Protein Extraction and Western Blot Analysis

The total proteins of WT and M10 endosperm at 12 DAF were extracted for SDS-PAGE and Western blot analysis, according to a method described previously [37]. The PVDF membranes were incubated with antibodies against AGPL2 and HSP82 and visualized using a chemiluminescence analyzer (Tanon, Shanghai, China).

3. Results

3.1. Phenotypes of the M10 Mutant

The M10 with a floury endosperm was identified from an EMS-irradiated mutant pool (in the japonica cultivar Kitaake background) and showed no significant differences from WT plants throughout the vegetative growth phase. The mature grains of M10 were comparable with WT (Figure 2A), while the endosperm of brown rice was opaque in M10 (Figure 2B). Transverse-sectional observation of the brown rice grains showed that the endosperm of M10 was floury-white, except for a thin peripheral area (Figure 2C,D). Furthermore, scanning electron microscopy revealed that the floury endosperm of M10 was packed with spherical and loosely arranged starch granules with large spaces between them, in contrast to the densely packed, irregular polyhedral starch granules in the transparent endosperm of WT (Figure 2E,F). The brown rice length, width, and thickness of M10 were significantly decreased compared with those of WT (Figure 2G–I), which caused the 1000-brown rice grain weight of M10 (16.80 g) to be 75.4% of WT (22.27 g) (Figure 2J). The total starch content in M10 seeds showed a significant decrease, while the soluble sugar content significantly increased compared with those of WT (Figure 2K,L). The grain hardness of M10 (3278 g) brown rice was only about 62% of the WT (5244 g).

3.2. Genetic Mapping of the Floury Endosperm Locus Using the Modified MutMap Method

The F2 population produced by hybridization between M10 and WT was used for genetic analysis. All F1 hybrids displayed a transparent endosperm, and the separation ratios of transparent endosperm to floury endosperm seeds in the F2 population were nearly 3:1 in the cross and reciprocal cross (Table 1). Therefore, the floury endosperm phenotype in M10 was controlled by a recessive nuclear gene.
To identify the causal gene for the M10 phenotype, the modified MutMap method was adopted (Figure 1). Both pool 1 and pool 2 were re-sequenced over 30 Gb of total bases, corresponding to more than 40× coverage of the rice genome (approximately 370 Mb). A total of 107,724,282 and 105,412,036 cleaned reads were identified from the re-sequencing data of pool 1 and pool 2, respectively. After these cleaned reads were aligned separately to the Nipponbare reference sequence using the BWA software (v0.7.8-r455), 104,760,974 and 102,326,402 unique mapped reads were obtained from pool 1 and pool 2, respectively. Then, we calculated Δ (SNP index), based on the sliding window of the whole genome scan, followed by plotting the Δ (SNP index) for all 12 chromosomes of rice. The absolute Δ (SNP index) analysis indicated that a significant peak nearly reaching 1.00 is located on chromosome 1 (Figure 3A, red arrow). In this region, four SNPs with high Δ (SNP index) were predicted to be candidate sites with possible responsibility for causing a floury endosperm (Figure 3B). Four SNPs were G-to-A transitions, presumably caused by EMS mutagenesis [38]. In addition, four SNPs in four genes (LOC_Os01g43820, LOC_Os01g44220, LOC_Os01g47150, and LOC_Os01g48620) were missense mutations causing amino acid changes (Table 2). The first (nucleotide 25109152) and third SNPs (nucleotide 26950694) caused amino acid changes, from proline (Pro) to serine (Ser) in LOC_Os01g43820 and from valine (Val) to isoleucine (Ile) in LOC_Os01g47150, respectively. Both LOC_Os01g43820 and LOC_Os01g47150 encode two retrotransposon proteins. The second SNP (nucleotide 25359718) caused the glycine (Gly) at the 251st amino acid replacement by glutamic acid (Glu) in LOC_Os01g44220. The fourth SNP (27875985) caused an amino acid change in an expressed protein with an unknown function, encoded by LOC_Os01g48620. This SNP altered the 116th amino acid from serine (Ser) to asparagine (Asn). Functional annotation revealed that LOC_Os01g44220 is the gene previously reported as AGPL2, which encodes a glucose-1-phosphate adenylyltransferase large subunit [39]. We suspected that LOC_Os01g44220 was the most likely candidate gene and, thus, focused on the second SNP for further analysis.
To verify whether this SNP was a natural variation between the two parent plants, we amplified and sequenced the mutation site in WT and M10. Sanger sequencing showed that this SNP at position 25,359,718 is G in WT and A in the M10 mutant (Figure 3C). Furthermore, an AS-PCR assay was used to identify the 1-bp replacement in LOC_Os01g44220, according to the mutation sequences. One forward primer (F) and two specific mismatch reverse primers (WT-R and M10-R) were designed and amplified, using the DNA of WT and M10 as templates (Figure 3D). The amplification products using F and WT-R were only detected in WT, while the amplification products using F and M10-R were only detected in M10, which further confirms the accuracy of the genotype (Figure 3E). AGPL2 harbors an N-terminal nucleotidyltransferase domain and a left-handed parallel beta-helix (LbH) domain at the C-terminal end. The single nucleotide substitution in M10 was located in the LbH domain of AGPL2 (Figure 3F).

3.3. AGPL2 Is the Causative Gene for Floury Endosperm in M10

The M37 is an allelic mutant of AGPL2 that was previously identified using a map-based cloning approach [40]. To test whether AGPL2 was the causal gene for M10 phenotypes, an allelic test was performed on M10 and M37. M10 was crossed reciprocally with M37 to produce F1 hybrids, which displayed opaque seeds (Figure 4A). The cross-section of F1 hybrids in the cross and reciprocal cross showed a floury endosperm phenotype, compared with the self-pollinated samples of WT (Figure 4A). All the F2 seeds from the two crosses appeared to have a floury endosperm, compared with the transparent seeds of WT (Figure 4B). The allelic analysis indicated that M10 is a novel allele of AGPL2.
Next, qRT-PCR and an immunoblotting assay were used to investigate the transcriptional and protein levels of AGPL2 in the developing endosperm, respectively. The relative expression level of AGPL2 in M10 was significantly higher than that in the WT (Figure 5A). As is consistent with the qRT-PCR result, the level of AGPL2 protein was dramatically increased in M10 (Figure 5B). Altogether, these data suggest that AGPL2 is the causative gene for the floury endosperm in M10.

4. Discussion

Rice endosperm can be used as a good system for elucidating how gene networks regulate starch biosynthesis and grain development. The most effective way to identify the genes regulating grain development is to generate floury endosperm mutants through chemical- or physical irradiation mutagenesis. For example, various causal genes of floury endosperms have been functionally characterized in rice, such as FLO10, FLO14, FLO15, and FLO18 from MNU (N-methyl-N-nitrosourea)-induced mutants [12,15,16,18], FLO19 from EMS (ethyl methyl sulfone)-treatment mutants [19], and FLO4 and FLO16 [17,41] from 60co-irradiation. Of these, almost all FLO genes are indirectly involved in starch synthesis.
In this study, we identified a rice floury endosperm mutant, M10, from an EMS-irradiated mutant pool. MutMap analysis, Sanger sequencing, and an allelic test suggested that AGPL2 is the causal gene for the floury endosperm seen in M10. A substitution of G to A in the seven exons of AGPL2 resulted in the Gly-251 being replaced by Glu in M10. To date, five allelic mutants of AGPL2 have been identified in rice, including gif2, M37, eb6, eb7, and w24 [31,40,42,43]. The multiple mutation sites of these mutants result in different changes to the endosperm. Both eb6 and eb7 exhibit a chalky phenotype with a white-cored endosperm. The eb6 mutant has a point mutation on C485 to T485 and alters the corresponding leucine at the 162nd amino acid to threonine in the nucleotidyltransferase domain. In the eb7 mutant, the AGPL2 has a single nucleotide transition from C802 to T802 and causes the proline at the 268th amino acid replacement by serine in the LbH domain. Both M37 and w24 display a floury and shrunken endosperm. Two single-nucleotide substitutions in the fourth exon of M37 and w24 led to the changes of alanine and leucine in the nucleotidyltransferase domain, respectively. The 2-bp deletion of AGPL2 in gif2 generates a protein of 80 aa, which has lost the nucleotidyltransferase and LbH domains of the full-length protein of 518 aa. The loss-of-function mutation of AGPL2 in gif2 results in an extremely severe phenotype exhibiting a sugary and shrunken endosperm. Thus, M10 is a new allele of AGPL2, compared with the mutation sites that have been reported above.
Rice floury endosperm is a valuable material for dry milling because of its spherical and loosely packed starch granules and low grain hardness. Mo et al. [4] have reported that the dry milling of the floury endosperm mutant Suweon 542, with low grain hardness, can produce good-quality rice flour compared with other rice cultivars. Similar results are found for another floury endosperm mutant, Hokuriku 166, which produces finer flour than its parental wild type, Koshihikari, by dry milling [44]. The grain hardness of M10 (3278 g) is largely comparable with that of Suweon 542 (3300 g) but is significantly lower than that of the WT (5244 g). Likewise, the floury endosperm of M10 is packed with spherical and loosely packed starch granules. Both M10 and M37 are isolated from the same genetic background, while the decrease in the grain thickness and weight of M10 is not as severe as in those of M37. Therefore, M10 may be more suitable when cultivating rice varieties with appropriate dry-milling adaptability than M37.
ADP glucose pyrophosphorylase (AGPase) is responsible for producing the glucosyl donor ADP-glucose (ADPG); the large subunit AGPL2 plays a key role in the catalytic and allosteric regulatory properties of the enzyme [43]. In this study, the missense mutation in AGPL2 resulted in the enhanced expression of AGPL2 itself at the transcriptional and protein level, suggesting a positive feedback regulation of AGPL2 in the M10 mutant. Wang et al. [5] have reported that the relative expression level of FLO4 in Suweon 542/flo4 showed a significant increase compared with that in the wild type during the grain-filling stage, which shows a similar feedback regulation to that observed in the M10 endosperm. The AGPase coding genes can be induced by sugar, e.g., the expression of Shrunken2 (AGPL3) and Brittle2 (AGPS2) in maize increases significantly due to the high levels of soluble sugars in the endosperm [45]. In addition, the gene expression of AGPase in rice is regulated by sucrose [46]. In the M10 endosperm, the reduction in starch content might be due to a decrease in AGPase activity, which leads to the accumulation of soluble sugar. Therefore, the significant increase in the expression of AGPL2 might be related to the high sugar content in the M10 endosperm.
MutMap is a new method for gene identification that has the advantages of rapidity, accuracy, and convenience compared with traditional map-based cloning. The MutMap method is based on second-generation sequencing technology, using it to only sequence the DNA pool of recessive individuals in the F2 population, which are derived from a cross between the mutant and its parental wild type. The re-sequencing data are aligned to the assembled whole-genome sequence of the wild type. Thus, in addition to the DNA pool, the whole-genome sequence of WT also needs to be sequenced and assembled as the reference sequence. The application of the MutMap method requires attention to several key factors, including the number of recessive individuals to be bulked (20–50), the average coverage (depth) of genome sequencing (>10×), and the accurate classification of phenotypes between the wild and mutant types [29]. In this study, a modified MutMap method (Figure 1) was applied to successfully isolate the AGPL2 gene related to endosperm development in rice. The modified MutMap method shows some differences compared with the original MutMap method. Firstly, the mutant form of M2, instead of the M3 or M4 generations in MutMap, was used to cross with the WT, which shortened the time needed to obtain the F2 population. Secondly, the elevated number of recessive individuals (30 vs. 20) and sequence depth (40× vs. 10×) ensured relatively high coverage of the genome (98% vs. 88%). Thirdly, we sequenced the DNA pool from dominant individuals instead of from the wild type. The whole-genome sequences of Nipponbare can be directly used as a reference sequence for alignment due to the small genomic differences between Kitaake and Nipponbare. If the mutants are identified from Oryza indica rice cultivars, the reference sequences should be replaced by published genome sequences of Oryza indica rice cultivars, including 9311, Zhenshan97, Minghui63, and Shuhui498 [47,48,49]. Overall, the modified MutMap method, with its relatively low cost and high efficiency regarding gene identification, can promote the development of rice genetics.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13051381/s1, Table S1. Primers used in this study.

Author Contributions

L.Z. and C.W. designed this research. R.Y., H.C., J.Z. and L.L. carried out the experiments. C.W. and L.Z. analyzed the results. L.Z. wrote the manuscript and C.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by grants from the National Natural Science Foundation of China (31901427), the Talent Project of Yangzhou University and the Qinglan Project of Yangzhou University (awarded to Long Zhang), the Innovation Program for Graduates of Jiangsu Province (Grant No. KYCX22_3470), the Innovation Program for College Students (Grant No. X20220731), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors. The data are not publicly available due to ongoing research.

Conflicts of Interest

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analysis, or interpretation of the data; in the writing of the manuscript, and in the decision to publish the results.

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Figure 1. The scheme of the modified MutMap method. The red color represents the steps of constructing a mapping population. Here, 30 recessive (floury endosperm seeds) and 30 dominant (normal seeds) from the F2 population were selected for gene mapping. The green color represents the steps of DNA library construction, re-sequencing, and candidate gene screening. The seeding DNA of 30 dominant and 30 recessive genes are mixed in an equal ratio to form DNA pool 1 and pool 2, respectively. A DNA library was constructed for re-sequencing, with 40× coverage. The cleaned reads were aligned with the reference sequence (Nipponbare rice genome sequence) followed by single nucleotide polymorphism (SNP) calling. For each identified SNP, SNP index (1) and SNP index (2) were obtained from pool 1 and pool 2, respectively. Note: Δ (SNP index), obtained from calculating SNP index 2 minus SNP index 1, was used for the Manhattan plot. From the Manhattan plot, we can establish the candidate region, followed by SNP annotation.
Figure 1. The scheme of the modified MutMap method. The red color represents the steps of constructing a mapping population. Here, 30 recessive (floury endosperm seeds) and 30 dominant (normal seeds) from the F2 population were selected for gene mapping. The green color represents the steps of DNA library construction, re-sequencing, and candidate gene screening. The seeding DNA of 30 dominant and 30 recessive genes are mixed in an equal ratio to form DNA pool 1 and pool 2, respectively. A DNA library was constructed for re-sequencing, with 40× coverage. The cleaned reads were aligned with the reference sequence (Nipponbare rice genome sequence) followed by single nucleotide polymorphism (SNP) calling. For each identified SNP, SNP index (1) and SNP index (2) were obtained from pool 1 and pool 2, respectively. Note: Δ (SNP index), obtained from calculating SNP index 2 minus SNP index 1, was used for the Manhattan plot. From the Manhattan plot, we can establish the candidate region, followed by SNP annotation.
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Figure 2. Phenotypic analyses of WT and M10 seeds. (A,B) Morphologies of the grain (with hulls) and brown rice (dehulled) of WT and M10. (C,D) Transverse sections of WT (C) and M10 (D) endosperm. (E,F) Scanning electron microscope analysis of the transverse sections of WT (E) and M10 (F) endosperm. (G–I) The brown rice length (G), width (H), and thickness (I) of WT and M10. (J) 1000-brown rice weight of WT and M10 (n = 3). (K,L) Contents of starch (K) and soluble sugar (L) in rice flour. (M) The grain hardness of mature brown rice. Scale bars = 1 mm (A–D) and 10 μm (E,F). Data are means ± SD from three biological replicates (Student’s t-test, * p < 0.05, ** p < 0.01).
Figure 2. Phenotypic analyses of WT and M10 seeds. (A,B) Morphologies of the grain (with hulls) and brown rice (dehulled) of WT and M10. (C,D) Transverse sections of WT (C) and M10 (D) endosperm. (E,F) Scanning electron microscope analysis of the transverse sections of WT (E) and M10 (F) endosperm. (G–I) The brown rice length (G), width (H), and thickness (I) of WT and M10. (J) 1000-brown rice weight of WT and M10 (n = 3). (K,L) Contents of starch (K) and soluble sugar (L) in rice flour. (M) The grain hardness of mature brown rice. Scale bars = 1 mm (A–D) and 10 μm (E,F). Data are means ± SD from three biological replicates (Student’s t-test, * p < 0.05, ** p < 0.01).
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Figure 3. Identification of the genomic regions harboring causal mutations using the modified MutMap method. (A,B) The Manhattan plots of Δ (SNP index) on the rice 12 chromosomes (A) and chromosome 1 (B). Red arrow indicates that a significant peak nearly reaching 1.00 is located on chromosome 1. (C) Sequence chromatograms of the M10 mutation. (D) The mismatch primers used for AS-PCR. (E) Genotype detection of WT and M10 using AS-PCR. (F) Structure of the AGPL2 and the point mutation site of M10.
Figure 3. Identification of the genomic regions harboring causal mutations using the modified MutMap method. (A,B) The Manhattan plots of Δ (SNP index) on the rice 12 chromosomes (A) and chromosome 1 (B). Red arrow indicates that a significant peak nearly reaching 1.00 is located on chromosome 1. (C) Sequence chromatograms of the M10 mutation. (D) The mismatch primers used for AS-PCR. (E) Genotype detection of WT and M10 using AS-PCR. (F) Structure of the AGPL2 and the point mutation site of M10.
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Figure 4. The allelic testing of M10 and M37. (A) The morphologies of WT self-pollinated seeds and the F1 hybrids from M10 and M37 reciprocal crosses. The insets represent the transverse section of representative seeds. (B) The floury endosperm phenotype of the F2 population from the M10 and M37 reciprocal crosses. Scale bars = 1 mm (A) and 1 cm (B).
Figure 4. The allelic testing of M10 and M37. (A) The morphologies of WT self-pollinated seeds and the F1 hybrids from M10 and M37 reciprocal crosses. The insets represent the transverse section of representative seeds. (B) The floury endosperm phenotype of the F2 population from the M10 and M37 reciprocal crosses. Scale bars = 1 mm (A) and 1 cm (B).
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Figure 5. Expression analysis of AGPL2 in WT and M10. (A) qRT-PCR analysis of AGPL2 expression in the developing endosperm at 12 DAF. UBQ is used as an internal control. The expression level of AGPL2 in WT is set at 1.0. Data are means ± SD from three biological replicates (Student’s t-test, ** p < 0.01). (B) Immunoblot analysis of AGPL2 expression in the developing endosperm at 12 DAF. The HSP82 is used as a loading control.
Figure 5. Expression analysis of AGPL2 in WT and M10. (A) qRT-PCR analysis of AGPL2 expression in the developing endosperm at 12 DAF. UBQ is used as an internal control. The expression level of AGPL2 in WT is set at 1.0. Data are means ± SD from three biological replicates (Student’s t-test, ** p < 0.01). (B) Immunoblot analysis of AGPL2 expression in the developing endosperm at 12 DAF. The HSP82 is used as a loading control.
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Table
Table 1. Segregation of the floury endosperm mutation in F2 populations.
Table 1. Segregation of the floury endosperm mutation in F2 populations.
TotalNo. of Normal SeedsNo. of Floury Endosperm SeedsX23:1 a
M10(♀) × WT(♂)242185570.270
WT(♀) × M10(♂)226173530.289
a The value for significance at p = 0.05 is 3.84.
Table
Table 2. The four candidate genes on chromosome 1.
Table 2. The four candidate genes on chromosome 1.
Δ (SNP Index)Gene IDNucleotide Location (bp)Reference Base (WT)Altered Base (M10)Type of MutationGene Annotation
0.744LOC_Os01g4382025109152GAMissense (Pro451Ser)Retrotransposon protein
0.789LOC_Os01g4422025359718GAMissense Gly251GluGlucose-1-phosphate adenylyltransferase large subunit
0.833LOC_Os01g4715026950694GAMissense (Val1093Ile)Retrotransposon protein
0.827LOC_Os01g4862027875985GAMissense (Ser116Asn)Expressed protein
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Zhang, L.; You, R.; Chen, H.; Zhu, J.; Lin, L.; Wei, C. A New SNP in AGPL2, Associated with Floury Endosperm in Rice, Is Identified Using a Modified MutMap Method. Agronomy 2023, 13, 1381. https://doi.org/10.3390/agronomy13051381

AMA Style

Zhang L, You R, Chen H, Zhu J, Lin L, Wei C. A New SNP in AGPL2, Associated with Floury Endosperm in Rice, Is Identified Using a Modified MutMap Method. Agronomy. 2023; 13(5):1381. https://doi.org/10.3390/agronomy13051381

Chicago/Turabian Style

Zhang, Long, Ran You, Hualan Chen, Jun Zhu, Lingshang Lin, and Cunxu Wei. 2023. "A New SNP in AGPL2, Associated with Floury Endosperm in Rice, Is Identified Using a Modified MutMap Method" Agronomy 13, no. 5: 1381. https://doi.org/10.3390/agronomy13051381

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