LITERATURE REVIEW
Evolution and domestication of ricecultivar
Rice has been known as the world‟s largest food crop, providing the caloric needs of millions of people daily Rice belongs to genus Oryza of family
OryzaGramineae that consists of 21 wild-types of the domesticated rice varieties
(Vaughan et al 2003) The O.sativa genus is divided into four species complexes, namely O sativa, O.officialis, O.ridelyi and O.granulata
The O sativa complex contains two domesticated species, including
O.sativa and O.glaberrima, are the only cultivated species grown worldwide, and in some parts of West and Central Africa, then, six wild-types, including
O.rufipogon, O.nivara, O.barthii, O.longistaminata, O.meridionalis and
O.glumaepatulaare fit into O.sativa complex O.sativa is distributed globally with a high concentration in Asia, while O.glaberrima is grown in West Africa
O.rufipogon can be found throughout Asia and Oceania Oryza barthii and
O.longistaminata are African species, O.barthii endemic in West Africa and
O.longistaminata is found throughout Africa Oryza meridionalis is native to Australia and O.glumaepatula is endemic in Central and South America Based on these distributions, it is easy to locate the ancestral pools from which modern rice was extracted The African cultivars were domesticated from
O.breviligulata and O.sativa was domesticated from O.rufipogon
Researchers have demonstrated that independent domestications of indica and japonica rice led to the creation of hybrids between these two subspecies The resulting offspring are expected to exhibit a segregation of wild alleles at various loci, resulting in the re-emergence of wild characteristics within sub-specific populations Notably, traits such as dormancy and shattering are influenced by intra-specific crosses; when parents with low dormancy and shattering are crossed, the progeny tend to display higher levels of these traits than either parent.
Research indicates that the levels of dormancy and shattering in crosses between indica and japonica rice are lower than those found in wild accessions This suggests that these subspecies may share some domestication alleles or that independent mutations occurred within the same domestication loci, which do not complement each other when crossed Additionally, QTL studies provide further evidence that different domestication genes were selected in various subpopulations, as populations derived from crosses between a single wild accession and diverse cultivars often identify distinct QTLs for domestication traits.
Septiningsih et al., 2003; Thomson et al., 2003; McCouch et al., 2006; Xie et al., 2006)
Figure1.1.The evolution of rice (Purugganan 2010)
Research by Cao et al (2006) indicates that rice cultivation dates back to 4000 BC, as evidenced by rice grains found in the oldest known paddy fields in the lower Yangzi River Valley Identifying the genetic changes that transitioned rice from wild to domesticated forms is more complex, with mutations that reduce grain shattering being essential for successful domestication.
Wild grasses possess unique adaptations, such as barbed awns on their seeds that deter seed-eating animals and aid in seed dispersal In species like Triticum spp, humidity changes cause these awns to flex, assisting in seed burial Over thousands of years, domestication has led to cereal crops with reduced or absent awns, simplifying grain harvesting and storage Additionally, the domestication of rice (Oryza sativa) involved significant changes in various traits, including growth habit, seed shattering, panicle structure, grain size, and hull color (Sang and Ge, 2013).
Figure1.2 Awns of wild and cultivated rice
The comparison between wild rice and domesticated rice reveals distinct differences in their panicles, seeds, and awns Panel (A) illustrates the panicles of wild rice on the left and domesticated rice on the right, with a scale of 10 cm Panel (B) presents the seeds, showcasing wild rice at the top and domesticated rice at the bottom, with a scale of 1 cm Lastly, panel (C) features the surface of a wild rice awn on the left and a domesticated rice awn on the right, observed under a scanning electron microscope with a scale of 200 µm (Hua et al 2015).
The wild progenitors of cultivated rice were O.nivara and
O.rufipogon, two most closely related wild species with the current distribution from southeastern Asia to India.O nivara was often regarded as an annual ecotype of O rufipogon in the literature partly because the correct identification of the species was not always possible or reliable for seeds distributed from the germplasm centers Nevertheless, the two taxa are morphologically, physiologically, and ecologically distinct in the natural habitats, experimental field, and green houses.O rufipogon, adapted to the stable, deep-water habitat, is perennial, predominantly out crossing, and photoperiod sensitive O.nivara was derived from O.rufipogon by adapting to the seasonally dry habitat in regions with a clear monsoon season In order to promptly complete the life cycle in the unstable habitat, O.nivara went through a series of adaptive steps and became an annual, self- fertilized, and photoperiod insensitive species (Sang and Ge 2013), (Grillo, Li et al 2009).
Purple sticky rice
Purple sticky rice, a glutinous variety of Oryza sativa L., is primarily cultivated in Asia and is rich in nutrients Its deep purple hue, attributed to a high anthocyanin content, resembles the color of blueberries and can appear almost black in its raw form Upon cooking, the rice transforms into a vibrant deep purple or violet shade The intense color is primarily due to anthocyanins, which are flavonoids known for their antioxidant properties.
Purple sticky rice, with over 200 varieties, has a rich history of cultivation in Southeast Asia, particularly in China, India, and Thailand China dominates global production, accounting for 62% and developing more than 54 high-yield varieties Other notable producers include Sri Lanka, Indonesia, India, and the Philippines, while Thailand ranks ninth The growing interest in purple rice is reflected in germplasm collections, with China holding 359 accessions Nutritionally, purple rice surpasses brown and white rice in protein, minerals, and dietary fiber Its rising demand in the USA and Europe is driven by its health benefits and appealing organic color, making it a unique and increasingly popular choice for consumers seeking nutritious food options.
In Vietnam, purple sticky rice is a traditional variety that thrives in short daylight conditions, allowing for only one annual harvest This unique rice is primarily cultivated in the northwestern mountainous regions, including Hoa Binh, Son La, and Yen Bai, with additional growth in areas like Phu Tho, Ninh Binh, and the Mekong Delta.
2 provinces are Long An and Can Tho.
Overview of anthocyanin
Most rice (Oryza sativa) globally has a white pericarp, but varieties with brown, red, and purple pericarp also exist Two key genes, Rc and Rd, influence proanthocyanidin synthesis in red and brown rice Notably, the Rc gene is essential for the development of red pericarp in rice, highlighting its role in rice domestication.
Anthocyanins, a small group of pigments within the flavonoid family, play a crucial role in providing red-blue hues to many plant species globally Their presence in fruits and flowers is beneficial for attracting pollinators and facilitating seed dispersal.
Anthocyanins are the largest and most significant group of water-soluble phenol pigments in plants, responsible for the red, purple, and blue colors in various fruits, vegetables, cereal grains, and flowers These pigments are generally odorless and nearly flavorless, contributing a moderately astringent taste While anthocyanins are widely present in higher plants across approximately 30 families, they are typically absent in liverworts, algae, and other lower plants, with some exceptions noted in mosses and ferns In most cases, anthocyanins are concentrated in the skin of fruits, although certain red fruits may differ Specifically, in purple rice, anthocyanin compounds are primarily associated with the bran layer.
Figure 1.4 Basic anthocyanin structure (Khoo, Azlan et al 2017)
Proanthocyanidins, the red pigments found in rice grains, are also referred to as concentrated tannins (Oki et al., 2002) These compounds are part of the anthocyanin pathway and share several biosynthetic genes (Winkel-Shirley, 2001) They exhibit significant inhibitory effects on various pathogens and predators The diversity in anthocyanin anthocyanidins arises from the different R radicals present (Figure 1.5).
Anthocyanins are produced in purple rice and the entire plant from the amino acid phenylalanine and malonyl coenzyme A These substrates combine to create chalcones, the foundational material for anthocyanins, which are synthesized through a series of enzymatic reactions The precursor compounds for anthocyanins are anthocyanidins or proanthocyanidins, which do not contain sugar molecules.
Figure 1.6 Metabolic pathways for the synthesis of pro‐ anthocyanidins and anthocyanins (Chaves-Silva, Dos Santos et al 2018)
Genetic analysis allows for the easy identification of mutations that exhibit novel expression patterns of anthocyanins and proanthocyanidins The anthocyanin biosynthetic pathway (ABP) is extensively studied within plant secondary metabolism, with most genes encoding enzymes for this pathway already isolated The regulation and determination of gene activities involved in anthocyanin biosynthesis primarily occur at the transcriptional level.
Anthocyanin is a natural colorant increasingly utilized in the food industry and medicine due to its valuable properties It works alongside other natural colorants like carotenoids and chlorophyll to restore and enhance the original colors of food products, making them visually appealing With its antioxidant capacity, anthocyanin also stabilizes fats, making it a significant source of color in food production This vibrant, water-soluble compound is non-toxic and can be safely used in products like candies and soft drinks Beyond its aesthetic benefits, anthocyanins contribute to health by combating diseases, including cancer, inflammation, and oxidative stress.
Roles and characteristics of the Rc gene
Plant coloration is primarily influenced by three types of pigments: anthocyanins, betalains, and carotenoids, in addition to chlorophyll, playing vital roles in photosynthesis, defense, and reproduction Anthocyanins, a significant subclass of flavonoids, are the key secondary metabolites responsible for the coloration of rice tissues The accumulation of anthocyanins in plants serves various functions, including enhancing resistance to environmental stressors.
UV radiation, participating in hormone regulation, and responding to biotic and abiotic stress, and is beneficial to human health (Koes, Quattrocchio et al 1994)
In rice, anthocyanins color tissues and give them various functions (Figure1.7)
Figure 1.7 Genetic diversity of rice color
Rice exhibits a remarkable diversity in pericarp color, ranging from white and light red to red, brown, and black Additionally, the variation in rice seed color is notable, with distinct differences in seed morphology depicted on the left and corresponding grain morphology on the right Furthermore, the diversity extends to other plant parts, including leaves, apiculus, stigma, sheath, stem, and overall plant color.
Rice (Oryza sativa) is predominantly grown and consumed in its white form, but it can also yield brown, red, and purple rice bran kernels, which are visible upon shelling The red pericarp is common among wild rice ancestors, and in certain regions, red rice is preferred for its flavor and medicinal properties The Rc gene, prevalent in most white rice genotypes, indicates that its absence in modern white rice varieties suggests a historical dispersal during domestication, likely due to strong selection for this allele Additionally, two genes, Rc and Rd, have been identified as influencing proanthocyanidin synthesis in red and brown rice, with classical genetic analysis revealing that their combined presence results in red seed coloration.
&Ishikawa, 1921) Rc in the absence of Rd produced brown seeds, while Rd alone had no phenotype (Figure 1.8)
Figure 1.8 Rc allele phenotypes (Sweeney, Thomson et al 2006)
Rc is a domestication-related gene required for red rice bran husk in rice
Oryza sativa is linked to seed breakage and dormancy, with red rice bran husk lacking a 14 bp deletion in the Rc locus The Rc gene encodes a basic helix-loop-helix (bHLH) protein, which has been precisely mapped to an 18.5 kb region on rice chromosome 7 through a cross between Oryza rufipogon (red rice bran husk) and O sativa Jefferson (white rice bran husk).
Research indicates that the red dominant allele in rice differs from the white recessive allele due to a 14 bp deletion in exon 6, which affects the bHLH protein (Sweeney, Thomson et al 2006) An early stop codon was found in a mutant herd with light red shells, and RT-PCR confirmed the Rc gene's expression in both red and white rice, although a truncated version was present in the white varieties The color of rice husk, whether red or brown, is influenced by the interaction of two genes, Rc and Rd Specifically, the RcRd and Rcrd genotypes yield red and brown rice bran husks, while the rcRd and rcrd genotypes result in white pods (Nagao 1947).
Top row: seeds from cv Jefferson (A) and O rufipogon (B)
Seeds from Surjamkuhi and H75 have been identified in rice bran husks RcRd and Rcrd, indicating that the Rd and Rc loci play a role in proanthocyanidin synthesis This suggests that the rd mutation may not be a simple loss of function but rather a conditional mutation, as intermediates of proanthocyanidin synthesis appear to accumulate in Rcrd granules, leading to a brown coloration.
The pericarp of rice exhibits various colors, including purple, brown, red, and white, with two key genes identified that influence proanthocyanidin synthesis in red and brown rice Genetic analysis indicates that the Rd and A loci are the same, both encoding dihydroflavonol-4-reductase (DFR) Introducing the DFR gene into a Rcrd mutant transformed its color from brown to red, confirming that the Rd locus encodes the DFR protein Additionally, proanthocyanidin accumulation was noted in transgenic plants after introducing the Rd gene into the rice Rcrd line (Furukawa, Maekawa et al 2007) Protein blot analysis revealed that the DFR gene undergoes translation in seeds through alternative initiation.
A search for the Rc gene, responsible for encoding a transacting regulatory factor, utilized DNA markers and the Rice Genome Automated Annotation System, leading to the identification and cloning of three candidate genes from a rice RcRd line These genes were introduced into a rice rcrd line, resulting in transgenic plants with brown-colored seeds, confirming that the Rc gene encodes a basic helix–loop–helix (bHLH) protein Analysis of the Rc locus revealed a 14-bp deletion exclusive to the rc locus (Furukawa, Maekawa et al 2007) Additionally, Brooks et al (2008) discovered the spontaneous mutant red pericarp rice cultivar Wells in the USA, which features a G-base deletion at the 20-bp site upstream of the absent 14-bp fragment in the seventh exon, restoring the reading frame and enabling proanthocyanidin accumulation in pigmented rice Lee et al (2009) further confirmed that the Italian red pericarp rice variety PerlaRosso is a spontaneous mutant with a G-base deletion at the 44-bp site upstream of the absent 14-bp fragment in the seventh exon, designating this new Rc allele.
Red pericarp rice, commonly grown in Africa, undergoes a transverse mutation (A to T) in the seventh exon, which changes its pericarp color from red to white (Li et al 2014; Gross et al 2010).
The Rc gene has six known alleles, including the wild type Rc, domestication alleles Rc and Rc-s found in Asian cultivated rice, revertants Rc-g and Rcr, and the unique Rc-g1 allele present exclusively in African rice cultivars (Li et al 2014).
Diversity of rice genetic resources in Vietnam
Vietnam is renowned for its rich biodiversity and is regarded as the origin of numerous essential food crops The country's varied natural conditions, climate, topography, and agricultural practices have led to the preservation of hundreds of rare plant varieties and species, significantly contributing to the development of its natural resources This includes a diverse array of plant species, particularly rice genetic resources.
Upland rice has been cultivated in Vietnam for over 6000 years, playing a significant role in the cultural life of various ethnic groups (Tran Van Dat, 2010) Today, it is primarily grown in the northern midland and mountainous provinces, as well as the North Central region and the Central Highlands However, the area dedicated to upland rice has drastically declined from 450,000 hectares in the late 1990s to just 130,000 hectares by 2009, representing a 72 percent reduction This decline is largely attributed to improved food security in the country and the implementation of various support policies for mountainous ethnic communities, such as Program 135 and initiatives aimed at promoting sustainable agricultural practices.
5 million hectares of forest, Program of Agricultural Extension etc
The genetic resources of indigenous rice varieties are at risk of significant loss without effective strategies for collection, storage, and conservation Rapid urbanization and the expansion of industry, tourism, and services have reduced agricultural land, contributing to the erosion of these genetic resources The Plant Resource Center has successfully collected and stored approximately 2,700 upland rice genetic resources from various ecological regions across the country Notably, the Northwest region contributes the largest share, representing 41% of the total resources in the Seed Gene Bank, primarily from provinces like Son La, Lai Chau, Hoa Binh, and Yen Bai The Northeast region follows with 25%, while the North Central Coast accounts for 22%, and the Red River Delta has the least representation at just 0.15%.
In short-term rice breeding, the collection and evaluation of genetic resources are crucial Vietnam boasts a diverse array of short-term genetic resources, including indigenous and imported varieties from countries like Japan, China, the USA, and the Philippines, encompassing both japonica and indica groups However, there is a lack of research analyzing the growth stages and periods to assess their impact on the total time from sowing to harvesting, which is essential for agricultural applications Crossbreeding efforts aim to develop rice varieties with shorter growth durations Additionally, evaluating the genetic diversity of short-term varieties is vital for breeding programs, as genetic diversity provides a foundation for utilizing a rich pool of genes and alleles in various crosses, ultimately leading to hybrid advantages This evaluation can be conducted through morphological, agronomic, and DNA molecular analyses.
Purple sticky rice, characterized by its black color, boasts high nutritional value, with protein content 6.8% higher and fat content 20% higher than other rice varieties It is rich in carotene, eight essential amino acids (including anthocyanin), and trace elements like iron and zinc, which are vital for the body (United Press International - UPI, 2010) This specialty rice has been cultivated for generations and plays a significant role in various cultural practices in Vietnam, being featured in nearly every festival and contributing to the nation's cultural identity Grown across diverse ecological regions, purple sticky rice exhibits a wide range of phenotypes Research into the genetic diversity of this rice group is crucial for preserving local varieties and selecting high-quality breeding materials in Vietnam (Ngo Thi Hong Tuoi, 2014).
Sticky rice is cultivated across various provinces in the country, thriving in both upland and field environments The genetic diversity of sticky rice is crucial for preserving unique rice varieties and enhancing the selection of high-quality strains Since 1977, efforts to collect and conserve rice genetic resources have led to the preservation of over 8,000 samples of both plain and sticky rice at the Plant Resource Center The classification of genetic diversity remains a key focus (Bui Huy Dap, 1980) As reported by Nghia et al (2011), the national plant gene bank houses more than 20,000 plant genetic resources, including approximately 8,000 rice varieties This highlights the significant role of local rice varieties in adapting to global climate change, with many being restored and maintained in agricultural production.
According to the study on "Evaluating genetic diversity of local sticky rice genetic resources based on phenotypes and fecal markers" by Doan Thanh
In 2016, Quynh conducted a study at the Institute of Crop Research and Development, Vietnam Academy of Agriculture, assessing the genetic diversity of 42 local sticky rice varieties from various origins using phenotypic traits and molecular markers The analysis revealed that these varieties could be classified into 11 genetically distinct groups, highlighting their significant diversity, which is essential for selecting high-yielding and quality sticky rice varieties Among the 38 SSR markers utilized, 33 showed polymorphic DNA bands across 33 loci, resulting in 106 different alleles, with an average of 3.03 alleles per locus This research is crucial for understanding the genetic relationships among sticky rice varieties, aiding in the management, conservation of genetic resources, and the development of new varieties.
We also found another study, which also provides important information for the study of quality and specialty rice breeding by molecular markers
The study "Analysis of Genetic Diversity of Rice Varieties with Markers" by Ngo Thi Hong Tuoi, published in the journal Science and Development in 2014, investigated the genetic diversity of 46 rice lines/varieties, including both glutinous and non-glutinous types, using SSR molecular markers A total of 35 SSR markers were employed, revealing 9 monomorphic and 26 polymorphic markers, which resulted in 68 polymorphic alleles and an average of 2.62 alleles per locus The genetic diversity coefficient (PIC) varied from 0.08 to 0.74, with a mean value of 0.46 The analysis categorized the rice varieties into two main groups and also assessed the anthocyanin content in the samples.
Previous research on glutinous rice has focused on the genetic diversity of specialty rice varieties, significantly aiding the development of high-quality rice Building on these findings, we propose a study titled "Sequencing and Analyzing Genetic Characteristics of the Rc Gene Regulating Anthocyanin Synthesis." For this research, we selected high-quality purple sticky rice samples from Yen Bai, provided by the Vietnam National University of Agriculture.
Studies on phylogeny of Vietnamese local varieties
In a study by Higgins, Santos et al (2021), researchers analyzed 672 rice genomes from Vietnam, including 616 newly sequenced samples representing various rice varieties across the country's diverse ecosystems They classified 48 samples (11%) as Indica and eight samples (4%) as Japonica, using Nipponbare as a reference variety.
(Temperate Japonica), Azucena (Tropical Japonica), and IR64 (Indica) were classified as J4, J1 and I1, respectively
The population structure and distribution of Indica and Japonica subpopulations in Vietnam were analyzed using STRUCTURE results For the Japonica subtypes, with a mean of 10 replicates at K = 4, 211 subtypes were evaluated, with a cutoff of 0.6 for subpopulation inclusion, resulting in a classification of 8 samples as admixed In contrast, the Indica subtypes were assessed at K = 5, involving 426 samples, where each color in the results represents a distinct subpopulation, and 48 samples were identified as admixed Additionally, the I5 subpopulation results were expanded to display individual samples The analysis also revealed the proportion of each population originating from 8 regions in Vietnam, based on a subset of 377 samples, with 54% of Indica and 85% of Japonica samples represented.
Figure 1.9 Population structure and location of the Indica and Japonica subpopulations within Vietnam (Higgins, Santos et al 2021)
Figure 1.10 Population structure and location of the Indica and Japonica subpopulations within Vietnam (Higgins, Santos et al 2021)
MATERIAL AND METHODS
Material
The NepCam YB pigmented rice variety, sourced from Yen Bai province, was utilized in this study and provided by the Department of Molecular Biology and Applied Biotechnology at the Vietnam National University of Agriculture.
Methods
Sample seeds were sown in the test field and the total DNA was extracted from young leaves using the previous procedures (Dellaporta 1983)
Extraction buffer: 1M Tris-HCl pH 8.0, 0.5M EDTA pH 8.0, 2.5N NaCl, 10% SDS
Isopropanol, absolute and 70% ethanol, TE 0.1X
Briefly, incubate extraction buffer was prepared (1M Tris-HCl pH 8.0,
0.5M EDTA pH 8.0, 2.5N NaCl, 10% SDS) at 65°C
Cut the vacuum dried leaves sample into 0.5-4 cm sections into1.5 mL tube, and then put in three iron beads
Crush the sample with a multi bead shocker (Yasui Kikai) at 1800 rpm for
60 seconds, rest 10 seconds, repeat twice
To each ground leaf sample, add 600μL extraction buffer, mix and incubate sample for 30 minutes at 65°C
Add 200μL 5M potassium acetate and mix gently
Incubate sample for 30 minutes in fridge
Centrifuge for 10 minutes at 4°C (9000 rpm)
Transfer the supernatant (roughly 400μL) to a new tube Add equal volume of Isopropanol, mix gently
Centrifuge for 30 minutes at 4°C (9000 rpm)
Remove the supernatant Wash the pellet with 1000μL 70% ethanol, drop gently
Centrifuge for 10 minutes at 4°C (9000 rpm), remove supernatant Dry the DNA at room temperature or 37°C until there is no smell of ethanol
Dissolve the pellet in 50 μL TE (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0), store in the fridge
DNA electrophoresis for 20 minute at 100V, using 1% agarose gel (with ethidium bromide) and TAE buffer0.1X
Hot start at 95 o C for 5 min
Annealing followed by 35 cycles with denaturation at 95 o C for 30 seconds Annealing at 45 o C – 62 o C for 30 seconds
Final elongation for 7 min at 72 o C
PCR products were electrophoresed on 2% agarose gel premixed with Ethidium bromide at a potential of 160V for 16-20 minutes
To sequence the complete Rc gene, approximately 6400 bp in length, we divided the gene into smaller fragments of less than 1000 bp and sequenced them using Sanger methods A total of 18 primers, as outlined by Li et al (2014), were utilized to ensure overlap along the Rc gene We first optimized the PCR conditions, specifically the annealing temperature for each primer pair, before amplifying the unique fragments for sequencing (see Table 2.2).
Table 2.1 Component of the PCR reaction
2 x PCR master mix Solution Primer
Table 2.2 List of 18 primer pairs for amplifying DNA fragments of Rc gene
No Primers Forward sequence Reverse sequence
2.2.3 Sequencing and identification of gene model
Eighteen amplified fragments were firstly qualified by agarose electrophoresis 2% and sent to 1 st Base Co., Singapore for purifying and sequencing by Sanger method
Sequences were assembled and aligned using Mega X software The assembled sequences were analyzed to identify exons and introns, and a gene model was constructed with the GSDS2.0 (Gene Structure Display Server) tool.
GSDS 2.0 is designed for the visualization of annotated features for genes, and the generation of high-quality figures for publication Besides the main features (i.e., coordinates of exons/ CDS), other annotated features such as conserved elements and binding sites can also be displayed GSDS 2.0 supports features in four types of formats, including BED, GTF/GFF3, GenBank Accession Number/GI, and Sequences in FASTA After inputting these features, GSDS would transform them into a uniform format for figure generation To facilitate evolutionary study, a phylogenetic tree can be uploaded and displayed on the side-panel of the figure After the first generation of figures, users can turn on/off the display of specified features, and modify the sizes, shapes and colors of displayed features Moreover, the generated figures can be sent to a built-in SVG-edit server for further refining Finally, users can export the figure as SVG, PNG, or PDF
Nucleotide polymorphisms were analyzed using BioEdit and DnaSP v6.0 (Librado and Rozas 2009), incorporating 8 reference sequences from the NCBI These sequences represent 4 subpopulations: indica, aus, tropical, and temperate japonica, which include both red and white grain color accessions.
Table 2.3 Rc gene sequences of 8 accessions from the NCBI
1 Bengkongang DQ885810.1 red Indica Indonesia
2 Kasalath DQ885812.1 red Aus India
3 Gangdodo DQ885805.1 red Temperate japonica Korea
4 Pae DayaIndolobye DQ885808.1 red Tropical japonica Indonesia
5 Dhala Shaitta Q885821.1 white Aus Banglade
6 Dee Geo Woo Gen DQ885818.1 white Indica Taiwan
7 Koshihikari DQ885803.1 white Temperate japonica Japan
8 Jefferson DQ885802.1 white Tropical japonica USA
Phylogentic analysis was constructed using the Maximum likelihood (ML) in MEGA – X(Tamura et al.2011).
RESULTS
Amplification and sequencing of Rc gene
To obtain the complete 6400 bp sequence of the Rc gene (Furukawa et al 2007, Sweeney et al 2007), we divided the gene into 18 segments and utilized 18 primer pairs for amplification, followed by purification and sequencing Each primer pair was optimized for annealing temperature to ensure the amplification of unique DNA bands The high-quality PCR products were then sent to 1st Base Co in Singapore for purification and sequencing The archived Rc sequences were assessed for quality using chromatograms analyzed in Chromas software, and only the sequences of good quality were used for assembly and alignment.
Figure 3.1 Electrophoresis photo of Rc gene fragments amplified by PCR
(DNA ladder was KAPPA universal ladder 100 bp)
The Rc gene sequence from the purple sticky YB variety was aligned with the reference sequence Oryza sativa (accession number KX549256), revealing a 97% similarity The analysis covered a total length of 7500 bp, encompassing the entire Rc gene.
Sequence Rc fragments
Our analysis and sequencing revealed 18 gene segments of varying lengths, with AF8 being the shortest at 432 bp and AF15 the longest at 986 bp We assessed the quality of all gene segments by examining pigments and nucleotide sequence variations using Chromas software.
Figure 3.2 Part of the gene sequencing results expressed through Chromas software
In our initial sequencing attempt, we discovered that AF4, AF7, AF17, and AF18 did not yield the expected sequences, while AF12 produced a poly AT sequence However, following a second sequencing round, we achieved favorable results, with sequence lengths ranging from 432 bp to 986 bp.
After successfully cloning and sequencing 18 gene fragments, we achieved sufficient quality for sequence assembly Initially, we eliminated noise "N" to prevent interference during the alignment with the DNA protein sequence Following the sequencing of the 18 gene segments, we proceeded to merge them into a complete Rc gene sequence of Nepcam.
YB is 7500 bp in length
Bài viết này trình bày một chuỗi DNA dài với nhiều đoạn mã hóa gen khác nhau Các đoạn này có thể chứa thông tin di truyền quan trọng, ảnh hưởng đến sự phát triển và chức năng của sinh vật Việc phân tích chuỗi DNA này có thể giúp hiểu rõ hơn về cấu trúc gen và các biến thể di truyền Ngoài ra, nghiên cứu về chuỗi DNA còn có thể hỗ trợ trong việc phát triển các phương pháp điều trị mới cho các bệnh di truyền Sự đa dạng trong cấu trúc DNA cũng cho thấy sự phong phú của sự sống trên Trái Đất.
Dưới đây là đoạn văn tóm tắt nội dung của bài viết:Bài viết chứa một chuỗi DNA dài với nhiều đoạn mã hóa gen khác nhau, thể hiện sự đa dạng di truyền và cấu trúc gen Các đoạn mã này có thể liên quan đến các chức năng sinh học quan trọng, như sự phát triển và chức năng của tế bào Sự phân tích chuỗi DNA này có thể cung cấp thông tin quý giá về các đặc điểm di truyền, cũng như các bệnh lý tiềm ẩn Việc hiểu rõ về cấu trúc và chức năng của các gen này là rất cần thiết trong nghiên cứu sinh học phân tử và y học.
TATGTTTTACCGGGCATCCGATTTTTAAAAAATTCAGAATGAAGAAAATTGAATCTTTTTTATGGATTTGA ATAAATCTTGATAAATTCGAAAAAATTTCCGAACTTTTGGCCAGAAGTGAATCCTACCCGTATCCACCGGT AATAAACCTAAATTTTTGGGAGTAATGAATTAATGTTATATATAATCCATGAATTATATAGTTCCAAACTA CTCCGTAACAAATTTTCAGGAGTAGTGAAATTAATATTATTACAATCTCAGAAAAAAATGGCAGAAACAA TTAATCTGTTTTCAATTATTAATTAATTTGTTTTTGTGTCCAGATGGACAAGGCGTCGATACTAGGCGACA CGATCGAGTACGTGAAGCAGCTAAGGAACCGCATACAAGAGCTCGAGTCGTCGTCGTCGTCGTCACGAGC AGCCGCCCGGGCGCCATCGGCGGCGGCCGCCGGGAGGCGGAGGAAGAGATCCGCCGCCGCCGCCACTGC CACGGCGGCGGAAGGGATGAGCAGCAGCAATGGCCGCAATGGCGGCGAGGCGGCGGAGGTGGTGCAGG TGTCCATCATCGAGAGCGACGCGCTGCTGGAGCTCCGGTGCGGTTGCGGCGGCGGCGGCGGCGGTGTGGT GCTGCTCCGGGTGATGCAGGCGATGCAGGAGCTCCAGCTGGAGGTCACCGCCGTCCAGGCCTCGTGCGCC GGTGGCGAGCTGCTCGCCGAGCTGCGCGCCAAGGTCGTCGTTATGATCCTGATCTGCATGAAAATGCAGA TGCAGATGCAAATGCAGAATTAAGCTTTCATTCTTGCTCCTCTGAATTCTGAATTTATATATTCACCCTTCT TTCGATCTGCTCGTACGTTCGTTTCGCCTAAATTATGTACAAATTAACTGAATCTTTGAACTGAAAATAAC TGAATCTTTTTTGTGTGTTTTTGTGTGGGTGAATTGGTTGGCGCAGGTGAAGGGGAGGAGGAGGAGCAGC ATCGCTCAGGTGAAGAGGCCCATCCTTTCCCTTTTTTCTTTGCTCATGGGGATTTTTTTCCAACGGTTATAT ATATAGAAAGTTCACATATATAAATCATGATAATTACCTTTTAGAAAATTCAAACAAAGAATCGATATAT ATAGTTCTATCTATGCATCATTTTCCTAAATCACGATAATTACCTCAAGCTTGATAAAATGGCATTACATT ACTATTATTATTACTAGAGTTTTTTCACTTCCCTGATCTAACCAATTTGCCACGATGGTTAGATATATAGAT GGTTCTAATTAATCTAATGTTAATGTACTCCACATATATGATATATGTACATACATATCTAGTTTTAACATT TGCAAATGAATTGCAATCAGAATATATATATGCATGCAGCACAGGTGCTTGATTGCAATTATATGATTATT ACTCTCTCACATATTGATATGGTAAATTTGTATATATTTGTGGCATGCATTCATGCACGAAGCTAGATTAA TTATTAATTAATTCAGGGTGATATCGATGAGGAGGAGAGGACGAGATGGATGGCCCTCTTCACCTGAGCG ATGCTGCTCCTCCTCCTCCCCTTCACCTGCGCCAACCAATTCACCCACACAAAAACACACAAAAAAGATTC AGTTATTTTCAGTTCAAAGATTCAGTTAATTGTACATATTGGAACGG
Figure 3.3 Full sequence of NepCam YB
Blue color: upstream and downstream; Yellow color: exons; White color: introns
Red color: AF2 (forward, reverse); Grey color: AF15 (reverse)
Green color: AF3 (forward, reverse); Pink color: AF17 (forward, reverse) Blue color: AF3 (forward, reverse); Purple color: AF18 (reverse)
Structural analysis of Rc gene
The Rc gene model, identified in the Nipponbare GRASP5.0 database, features a scaled representation with 8 introns (black lines) and 9 exons (yellow boxes) derived from the purple sticky YB rice variety Additionally, two regions upstream and downstream of the Rc gene are indexed (blue boxes).
Figure 3.4 Exon intron structure of Nepcam YB
The Rc gene, which encodes for the bHLH protein, has been sequenced in various rice varieties, revealing a model of intron-exon structure Initial sequencing by Furukawa et al (2007) identified 8 exons and 7 introns, while subsequent studies by Li et al (2014) and Meng et al (2021) confirmed these findings In contrast, research by Sweeney et al (2007) reported a different structure with 9 exons and 8 introns These variations in the Rc gene structure across rice varieties suggest a divergence that may significantly influence the domestication process of these crops.
The Rc allele Rc-s, identified in various white pericarp rice genotypes, shows a base transition from C to A in the seventh exon, contrasting with the absence of a 14 bp fragment observed in red pericarp rice, as noted in the study by Sweeney et al (2007).
The Rc gene of Nepcam YB exhibits a 14-base deletion in the fifth intron, specifically from nucleotide positions 5061 to 5075 Co-linearity analysis of the two alleles for Rc indicates that Nepcam YB possesses the Rc genotype, which is associated with red pericarp traits and represents domestication alleles identified in Asian cultivated rice (Li et al., 2014).
Figure 3.5 Collinear analysis of the fifth intron's partial sequence of Rc
This study compares the complete 7500 bp gene sequence of Nepcam YB with various global rice varieties, utilizing BioEdit software to analyze eight distinct rice types The research aims to explore the genetic diversity associated with a 14-bp deletion in the Rc gene, building upon previous findings in the field.
In a study by Sweeney et al (2007), eight magnetic sequences were analyzed, consisting of four white rice varieties and four red rice varieties The red varieties represent populations from India, Australia, tropical regions, and temperate japonica, along with complete Nepcam YB gene sequences A summary of the DNA polymorphisms in the Rc gene is illustrated in Figure 10.
14bp deletion previously described leading to white rice bran disease in rice was 95.5% (21/22)
A second mutation causing white rice bran disease was identified in 4.5% of the rice varieties studied, specifically on a japonica background This mutation, represented by a C → A switch in exon 6 of the Rc gene, results in an early stop codon that truncates the bHLH protein, leading to the white rice bran husk phenotype Research indicates that loss-of-function mutations in the Rc gene, which is crucial for proanthocyanin synthesis, inhibit the development of the rice bran coat.
Based on articles (Sweeney, et al., 2006) (Furukawa, et al., 2007)
Genetic analysis revealed that Nepcam YB has a mutation resulting in the loss of a 14-base segment, which is characteristic of the rc gene This mutation leads to a reduction in the synthesis of proanthocyanidins and the associated pigment in white rice bran husk.
The YB variety is characterized by its purple color In the future, I plan to investigate the role of the Rc gene in anthocyanin synthesis and examine how environmental factors affect anthocyanin content during the cultivation of Nepcam YB.
3.3.3 Sequence polymorphism of Rc gene
Sequence polymorphism of Rc gene for comparison of the sequence of Rc
The NepCam YB gene was analyzed alongside eight reference sequences from the Oryza sativa indica, japonica, and aus groups, resulting in the identification of 54 SNPs Among these, 20 SNPs were found in exon regions and 21 SNPs in intron regions of the RC gene in NepCam YB.
Figure 3.6 The SNPs in RC gene of NepCam YB compared with 8 representative rice accessions
Further analysis of amino acid sequences and protein functions is essential to comprehend the impact of mutations and SNPs on pro-anthocyanin accumulation in Nepcam YB.
Figure 3.7 G-C content, A-T content chart of each variety
The Guanine plus Cytosine (GC) content in genomes ranges from 20% to 80%, a variation primarily caused by mutation biases during replication and repair processes This GC content significantly impacts the hydrophobicity of proteins, which is a key factor in determining their folding stability.
The high-GC gene exhibited significantly increased rates of both mitotic and meiotic recombination GC content plays a crucial role in genome evolution, as it influences the stability of genes During evolution, higher G-C content leads to greater stability, making genes less susceptible to change through point mutations.
G-C content was identified in the Bioedit (Figure 6.) The average GC- content in 8 varieties genomes ranges from 37.24% to 37.59% G-C content of NepCam YB the shortest is 35.55%, thereby showing that Rc gene of NepCamYB is unstable.
Phylogenetic analysis
To investigate the origin and genetic relationships of Nepcam YB with other rice varieties, a phylogenetic tree was created using eight representative Rc gene sequences The analysis revealed that Nepcam YB is closely related to the japonica Jefferson cultivar from the USA However, the genetic distance illustrated in the tree did not distinctly separate red and white pericarp rice varieties.
Jeffersion from USA, have white grain, subpopulation is Tropical japonica
Japonica rice (Oryza sativa subsp japonica) is a key variety of Asian rice, primarily cultivated and consumed in East Asia Originating from Central China, it was first domesticated around 9,500 to 6,000 years ago along the Yangtze River basin In contrast, India rice dominates in most other regions.
Figure 3.8 Phylogenetic tree of Nepcam YB with 8 rice varieties.
Protein features
Proteins are the final products of a transcription process initiated by the information in a cell's DNA They serve as the structural and motor components within cells and function as catalysts for nearly all biochemical reactions in living organisms This remarkable variety of functions arises from a straightforward code that dictates a highly diverse range of structures.
Each gene in a cell's DNA encodes the instructions for a specific protein structure These proteins are formed from unique sequences of amino acids, which are connected by various bonds and folded into distinct three-dimensional shapes The final conformation of a protein is directly influenced by its linear amino acid sequence.
A comparison of the physicochemical parameters of the protein from NepCam YB with eight control varieties revealed that the number of amino acids varied between 445 and 636, while the molecular weight ranged from 49,615.95 to 69,697.34 The theoretical isoelectric point was found to be between 4.91 and 5.55 The instability index, which indicates protein stability, was greater than 40, ranging from 58.19 to 64.29, suggesting that both NepCam YB and the eight varieties are unstable Additionally, the aliphatic index varied from 62.45 to 77.52, and the grand average of hydropathicity (GRAVY) ranged from -0.590 to -0.431 A hydrophobicity score below 0 indicates a higher likelihood of being globular, classifying these proteins as hydrophilic.
Table 3.1 Physical and chemical parameters of protein of NepCam YB with 8 reference varieties
Sub cellular localization and 3Dmodel
The subcellular localization of the Rc gene was identified using TargetP, revealing that the Rc gene of the eight reference varieties remains unknown Notably, the Rc gene of NepCam YB was found to be located in the mitochondria, with a mitochondrial targeting peptide (mTP) value of 0.535, in contrast to the chloroplast transit peptide (cTP) value of 0.197 and the secretory pathway signal peptide (SP) value of 0.017.
The 3D model of Rc protein of Nepcam YB Was constructed with similarity with models of other 8 varieties (Figure 3.9)
Figure 3.9.Model structure Rc of each variety.
CONCLUSION AND SUGGESTION
The PCR conditions were optimized successfully for amplification of 18 fragments using 18 primer pairs
Using 18 primer pairs, we successfully assembled and aligned the full-length sequence of the Rc gene from the NepCam YB variety, achieving a length of 7500 bp with BioEdit software.
NepCam YB variety contains 8 introns and 9 exons
Analysis of sequence polymorphism in the Rc gene of the NepCam YB rice variety revealed a 14 bp deletion in the fifth intron and identified 54 SNPs when compared to eight representative rice cultivars The physicochemical characterization of the NepCam YB protein indicated that it is hydrophilic and unstable Additionally, the Rc gene of NepCam YB was found to be located in the mitochondria.
A phylogenetic tree was created using the full-length sequence to explore the origin and relationships of Nepcam YB with other rice groups globally This preliminary finding serves as a foundation for further research on the domestication of Vietnamese local rice.
I am still continuing to sequence more varieties of sticky rice in other localities to somewhat understand the domestication and evolution of Vietnamese rice.
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DNA SEQUENCE POLYMORPHISM OF Rc GENE IN 'YEN BAI' LOCAL
Bui Thi Thanh Hien 1 , Nguyen Thi Yen Chi 1 , Chu Duc Ha 3 , Bui Van Hung 4 , Ngo Thi Hong Tuoi 2 , Nguyen Quoc Trung 1 , Tran Van Quang 2
1 Faculty of Biotechnology, Vietnam National University of Agriculture
2 Faculty of Agronomy, Vietnam National University of Agriculture
3 Faculty of Agricultural Technology, University of Engineering and Technology, Vietnam National University of Hanoi
4 Agricultural Science Institute of Northern Central Vietnam
The Rc gene plays a crucial role in the accumulation of proanthocyanidins in the rice pericarp, making it a significant regulatory gene This study focuses on the mutation and sequence polymorphism of the Rc gene to enhance our understanding of rice cultivar evolution We aimed to obtain a complete genomic DNA sequence and analyze the polymorphism of the Rc gene in the NepCam YB rice, a local pigmented variety from Yen Bai province Utilizing 18 primer pairs, we successfully assembled and aligned a full-length sequence of the Rc gene, including its upstream and downstream regions, totaling 7500 bp, using BioEdit software.
The YB variety features 8 introns and 9 exons, with sequence polymorphism analysis identifying a 14 bp deletion in the fifth intron and 54 SNPs in the Rc gene when compared to 8 representative rice cultivars Phylogenetic analysis indicates that Nepcam YB is closely related to the Japonica Jefferson cultivar This study successfully archived the full-length sequence of the Rc gene in the red pericarp Nepcam YB, providing preliminary data for understanding the domestication of Vietnamese local rice.
Keywords: Proanthocyanidin, pigmented rice, gene model, SNPs, phylogenetic tree, Rc gene
Rice (Oryza sativa) is a vital cereal crop with a rich history of cultivation in Asia, serving as a staple food for over 100 countries and providing the primary carbohydrate source for more than half of the global population Numerous rice varieties exist worldwide, differing in grain characteristics such as shape, amylose content, and pericarp color The color of the bran shell is influenced by the accumulation of pigments, including flavonoids, carotenoids, and betalains Notably, flavonoid compounds like anthocyanins, flavonols, and proanthocyanidins contribute to the red, purple (black), and brown hues observed in rice.
The Rc gene is a crucial regulatory element in proanthocyanidin accumulation within the rice pericarp, located on chromosome 7 and encoding a basic helix-loop-helix (bHLH) protein The predominant domestication allele, found in over 97% of non-pigmented rice cultivars, features a 14-bp deletion in the seventh exon, resulting in a non-functional gene and the characteristic white pericarp An alternative allele, Rc-s, occurs in less than 3% of white pericarp rice and is marked by a C to A base change in the same exon Additionally, the red pericarp rice cultivar Wells from the USA has a G-base deletion upstream of the 14-bp deletion, restoring proanthocyanidin accumulation Similarly, the Italian red pericarp variety PerlaRosso has a G-base deletion at a different site, designated as Rcr In Africa, a transversion mutation (A to T) in the seventh exon alters the pericarp color from red to white, associated with the rc-g1 allele In total, six Rc gene alleles have been identified, including Rc (wild type), rc, Rc-s (domestication alleles), Rc-g, Rcr (revertants), and rc-g1 (specific to African cultivars).
The aim of this study was to analyze the sequence polymorphism in Rc gene of Nepcam pigmented YB- rice a local cultivar in Yen Bai province, Vietnam
The purple sticky YB (pigmented) rice variety utilized in this study was sourced from the Department of Molecular Biology and Applied Biotechnology at the Vietnam National University of Agriculture in Yen Bai province.
Figure 1 Rice grain of NepCam YB
Sample seeds were sown in the test field, and total DNA was extracted from young leaves using established procedures The extraction buffer was prepared with 1M Tris-HCl (pH 8.0), 0.5M EDTA (pH 8.0), 2.5N NaCl, and 10% SDS, then incubated at 65°C Dried leaf samples were cut into 0.5-4 cm sections, placed in Eppendorf tubes with iron marbles, and crushed using a multi bread shocker at 1800 rpm for 60 seconds, with rest intervals To the ground leaf samples, 600μL of extraction buffer was added, mixed, and incubated for 30 minutes at 65°C After adding 200μL of 5M potassium acetate and gently mixing, the samples were incubated in the fridge for 30 minutes and then centrifuged at 4°C for 10 minutes at 9000 rpm The supernatant was transferred to a new Eppendorf tube, mixed with an equal volume of isopropanol, and centrifuged again at 4°C for 30 minutes The pellet was washed with 1000μL of 70% ethanol, centrifuged, and dried until the ethanol smell dissipated The DNA was dissolved in 50μL of TE buffer and stored in the fridge Finally, DNA electrophoresis was performed for 20 minutes at 100V using 1% agarose gel with ethidium bromide and TAE buffer (0.1X).
The PCR protocol involves an initial hot start at 95 °C for 5 minutes, followed by 35 cycles consisting of denaturation at 95 °C for 30 seconds, annealing at temperatures ranging from 45 °C to 62 °C for 30 seconds, and elongation at 72 °C for 1 minute The process concludes with a final elongation step at 72 °C for 7 minutes.
To sequence the full Rc gene, approximately 6400 bp in length, we divided the gene into several fragments, each less than 1000 bp, and employed Sanger sequencing methods A total of 18 primers were utilized, as referenced in [1], ensuring overlap along the Rc gene The PCR conditions were optimized to determine the appropriate annealing temperature for each primer pair, which facilitated the amplification of the 18 fragments for sequencing (see Table 1).
Table 1 List of 18 primer pairs for amplifying DNA fragments of Rc gene
No Primers Forward sequence Reverse sequence Ta ( o C)
Sequencing and identification of gene model
Eighteen amplified fragments were initially qualified through 2% agarose electrophoresis and subsequently sent to 1st Base Co in Singapore for purification and Sanger sequencing Sequence assembly and alignment were performed using Mega X, resulting in a total length of 7500 bp The obtained sequences allowed for the identification of exons and introns, leading to the construction of a gene model using GSDS2.0 (Gene Structure Display Server) software.
Analyses nucleotide polymorphisms were calculated using BioEdit, DnaSP v6.0 [8] Analyses were performed on
The Rc gene sequences of eight accessions from the NCBI, representing indica, aus, tropical, and temperate japonica varieties with both red and white grain colors, were analyzed.
Table 2 Rc gene sequences of 8 accessionsfrom the NCBI
No Name Gene code in NCBI
1 Bengkongang DQ885810.1 red Indica Indonesia
2 Kasalath DQ885812.1 red Aus India
3 Gangdodo DQ885805.1 red temperate japonica Korea
4 Pae Daya Indolobye DQ885808.1 red tropical japonica Indonesia
5 Dhala Shaitta Q885821.1 white aus Bangladesh
6 Dee Geo Woo Gen DQ885818.1 white indica Taiwan
7 Koshihikari DQ885803.1 white temperate japonica Japan
8 Jefferson DQ885802.1 white tropical japonica USA
Phylogenetic analysis was constructed using the Maximum likelihood (ML) in MEGA – X[9]
Amplification and sequencing of Rc gene
To achieve the complete sequence of the Rc gene, measuring 6400 bp, we divided the gene into 18 segments and utilized 18 primer pairs for amplification, followed by purification and sequencing Each primer pair was optimized for annealing temperature to ensure the amplification of unique DNA bands The high-quality PCR products were sent to 1st Base Co in Singapore for purification and sequencing The archived sequences were assessed for quality using chromatograms viewed in Chromas software, and only the sequences of good quality were used for assembly and alignment.
The Rc gene sequence from the purple sticky YB variety was aligned with the reference sequence Oryza sativa (accession number KX549256), revealing a 97% similarity The analysis covered a complete length of 7500 bp of the Rc gene.
Figure 2 Full sequence of NepCam YB- Blue color: upstream and downstream; Yellow color: exons; White color: introns
Red color: AF2 (forward, reverse) Grey color: AF15 (reverse)
Green color: AF3 (forward, reverse) Pink color: AF17 (forward, reverse)
Blue color: AF3 (forward, reverse) Purple color: AF18 (reverse)
Figure 3 Electrophoresis photo of Rc gene fragments amplified by PCR (DNA ladder is KAPPA universal ladder 1000 bp) Intron-exon structures of Rc gene