Validation of molecular markers associated with amylose content of F8 and F9 lines resulting from a crossing of Black Rice and Mentik Wangi

The development of black rice with a fluffier texture at Universitas Jenderal Soedirman, which began in 2004, was carried out by crossing local black rice with the Mentik Wangi variety. At present, six black rice advanced breeding lines are ready for multi-location yield trials. Meanwhile, developing molecular marker-based plant breeding methods can facilitate a more efficient selection process. An important stage in developing molecular markers is marker validation, which aims to test the effectiveness in determining phenotypes in various genotypic and in new populations that differ from others based on the link between the marker and the expected character identified. This research aimed to determine molecular markers that can differentiate the level of amylose content in populations of F8 and F9 lines. The research was carried out on 10 (ten) genotypes consisting of the control variety Logawa, IRPM 112-19- 56, black rice, Mentik Wangi, and six lines. The amplification of the RM190, Wx and SSIIa markers showed polymorphic band. Based on the simple linear regression analysis, in F9, the SSIIa marker had the highest coefficient of determination compared to other markers. Hence, it had the greatest possibility of being a marker for selecting rice based on amylose content.


Introduction
The increasing level of public awareness of healthy lifestyles after the Covid-19 pandemic and the prevalence of degenerative diseases such as Diabetes Mellitus (DM) and cancer in Indonesia have made the need to consume pigmented rice as a functional food increasingly important [1].As a degenerative disease that can be a comorbid or accompanying disease of Covid-19, the number of Diabetes Mellitus cases continually increases annually.Based on data released by Basic Health Research in 2018, the prevalence rate of Diabetes Mellitus in Indonesia was 8.5%, an increase compared to 2013 (6.9%).The prevalence rate of cancer in Indonesia's population of all ages in 2018 was 1.79‰, with Central Java Province ranking in the top 10 (2.1‰).According to the Ministry of Health of the Republic of Indonesia [2], Diabetes Mellitus is the third deadliest disease in Indonesia after stroke and heart disease, so it is a significant problem that needs attention from the Indonesian people.
Consuming functional foods such as pigmented rice can effectively prevent degenerative diseases and improve the immune system.Black rice is a functional food ingredient with five time higher antioxidant content than other pigmented rice [3].Anthocyanins help boost the immune system, prevent liver cell 1297 (2024) 012046 IOP Publishing doi:10.1088/1755-1315/1297/1/012046 2 cell damage to kidney function and cancer/tumors, as antioxidants and anti-diabetes [4] [5].
The amylose content plays an essential role in determining rice's taste quality and fluffiness.Even though black rice has the potential to be a functional food, this rice has a high amylose content, making cooked rice dry, so people's preference for this rice as a staple food is still low [6].Rice with moderate amylose content (20-24%) is in great demand by farmers and the public because of its high selling value in local and international markets.Breeders must develop rice breeding methods with appropriate amylose content to meet consumer demand and improve farmer welfare [7].
Selection is essential in plant breeding to obtain lines/mutants with desired characteristics.The use of Marker Assisted Selection (MAS) in selection is more profitable compared to conventional selection.With the help of molecular markers, selection is carried out on plant genetic characteristics, which are not influenced by environmental factors, making the selection process faster, more precise, and relatively more cost and time-effective [8] [9].The selection process, with the help of molecular markers, requires information on markers associated with the desired trait, so there is a need for a validation/testing process for the ability of these markers to identify related traits.
This research was conducted to determine molecular markers that can differentiate the level of amylose content in F8 and F9 lines resulting from the crossing of black rice and Mentik Wangi.The previous lines had been examined through several studies [10] [11].Validation was carried out by comparing data from amylose content and polymorphic bands of DNA using regression analysis [12].Thus, the molecular markers examined can be used to differentiate the level of rice amylose content in specific cross populations, thereby increasing the selection efficiency in plant breeding programs based on certain desired traits.

Sample preparation for DNA extraction
Rice seed samples were soaked in clear plastic measuring 15 x 20 cm 2 to break dormancy for one night.The seeds were then planted in polybags measuring 40 x 40 cm2 [16].Rice leaves were harvested in the 2-3rd week, about 1.5 grams or four leaves.The rice leaves were then put in sterile plastic and stored in an ice box [17].The rice leaves were crushed using a mortar, pestle, and liquid nitrogen until a smooth leaf sample.Liquid nitrogen was added to facilitate the grinding and keep the DNA from being damaged.The finely crushed leaves were put into 1.5 µl microtubes and stored in the freezer at -20 o C [18].

Genomic DNA extraction
DNA extraction was carried out using the Cetyl Trimethyl Ammonium Bromide (CTAB) method [19], which was modified by adding 2% PVP (Polyvinylpyrrolidone) and β-mercaptoethanol.The addition of PVP is carried out to reduce polyphenolic compounds that can bind to DNA, while the compound βmercaptoethanol was added to the extraction buffer to degrade proteins in the sample so that pure and high DNA results will be obtained [20].

Visualization and quantification of DNA
Visualization of DNA was carried out by mixing 2 µl of 5x loading dye with 5 µl of extracted DNA solution.The homogenized DNA solution and DNA Ladder were injected into the well of a 1% agarose gel (0.5 grams of agarose, 50 ml of 1x TBE buffer, and 5 µl of fluorosafe), which was submerged in 1x TBE buffer solution and ran at 50 volts for one hour.After the electrophoresis process was complete, the agarose gel was transilluminated under a UV transilluminator [20].The purity of the DNA solution was determined if there was a single genomic DNA band and there was no smear on the agarose gel resulting from electrophoresis [17].
Quantification of DNA was carried out using a NanoDrop UV spectrophotometer at wavelengths of 260 nm and 280 nm.The DNA quantity was quantified based on the tool manual issued by Implen.Before using the tool to read the extracted DNA sample, the tool was calibrated by dropping 3 µl of TE buffer (blank solution) onto the cuvette and then pressing the blank button.After the indicator screen shows the number 0, 3 µl of DNA sample was dropped into the cuvette, and the sample button was pressed until the reading results appeared.Every time the solution was changed, the cuvette was wiped using a tissue with the aim of minimizing contamination from the previous solution.A DNA molecule was said to be pure if the λ260/λ280 ratio ranges between 1.8-2.0.If the comparison result was ≤ 1.6, then there was contamination from phenolic compounds, proteins, and other contaminants [21].

Amplification of molecular markers using Polymerase Chain Reaction (PCR)
The PCR method was based on the MyTaq™ HS Red Mix protocol issued by Meridian.PCR solution preparation was carried out in a 0.2 ml PCR tube by mixing several ingredients as follows: The initial concentration and volume could be adjusted to the final concentration required, but it would be better to optimize to get the right concentration.The DNA template and primer solutions were diluted to obtain a uniform concentration and make the mixing process more manageable.The PCR amplification process consisted of initial denaturation, denaturation, annealing, extension, and final extension.The annealing temperature for each primer was different, so optimization needs to be done [22].The following PCR program was based on the MyTaq™ HS Red Mix standard protocol:

Determination of amylose content
The amylose content could be determined using the spectrophotometric method at visible wavelengths.Isolation of amylose and amylopectin in starch was carried out based on their solubility level in water.Amylose dissolved in hot water and added to iodine solution produces a blue color that can be measured using a UV/Vis spectrophotometer at a wavelength of 625 nm.The amylose content could be determined by comparing the absorbance reading of the sample with the amylose standard curve series [23].Analysis of amylose content in rice using spectrophotometry (SNI 6128:2015), through two stages, namely making a standard curve and determining samples.

Data analysis
The variables observed in the study were the presence of DNA bands, the number of DNA bands, the size of the DNA bands, and the amylose content.Molecular data analysis was carried out by observing the relationship between the DNA band patterns formed from each sample and its amylose content.The scoring of the DNA bands formed was used as a value in the analysis to determine the relationship between the markers and the genes related to the physiological characters that appear in the parents of the crossing.Visualized DNA band scoring was differentiated based on (1) if the band was present and (0) if the DNA band was not present or did not match.Analysis of the relationship between molecular markers and amylose content was calculated using simple linear regression analysis and one-way ANOVA with the IBM SPSS Statistics version 25 program.The linear regression method could be used to determine the relationship between independent variables (molecular markers) and dependent variables (amylose content) by looking at the values regression coefficient, while oneway ANOVA could be used to validate that the three primers can differentiate amylose content levels [24] [25].

Analysis of molecular markers associated with amylose traits in F8 lines
The results of marker amplification showed polymorphic band products.The size of the product band resulting from RM190 amplification was in accordance with the Gramene library, where the DNA band ranged from 104-124 bp.The PCR product visualization showed that high and medium amylose content tends to have a larger base size (118-124 bp) compared to low amylose samples (104-115 bp).Samples PHMW 482-1-14 (D) and PHMW 482-17-18 (G) showed different results from the comparison samples, where sample PHMW 482-1-14 had a high band size (124 bp) while PHMW 482-17 -18 has a lower band size (106 bp) (Figure 1).The results of the Wx marker amplification showed the variation of DNA band sizes with values of 199 -213 bp.However, the DNA band size obtained did not match the literature (198/217 bp).Control samples IRPM 112-19-56 (-), Mentik Wangi (B), PHMW 482-17-7 (E), PHMW 482-1-4 (F), PHMW 482-17-18 (G) were assumed to have variations the A allele is due to the DNA band product produced during the amplification process being higher (207 -213 bp), while the Logawa (+), black rice (A), PHMW 482-1-14 (D), PHMW 482-9-134 (H), PHMW 487-24-8 (I) carries the C allele due to the smaller base size [14].In the Wx primer amplification results, the size of the A allele product was higher because it has 19 M13-tailed base pairs at the end of the Wx forward primer (5'-CACGACGTTGTAAAACGAC -forward primer -3').The presence of M13-tailed would increase the size and specificity of the amplified DNA band product [14].
The results of the SSIIa primer amplification showed that no DNA bands were formed in the parents (black rice and Mentik Wangi).The results of amplification of the resulting DNA bands were polymorphic, with DNA band sizes ranging from 240 -270 bp.The Logawa variety as a positive control had a low base size, namely 240 bp, while IRPM 112-19-56 had a higher base size, namely 264 bp.
The results of simple linear regression analysis of the three markers (RM190, Wx, and SSIIa) showed various similarities and coefficients of determination.The primary determination coefficient of RM190 had a value of 0.243 or 24.3%, which means that the RM190 marker caused 24.3% of the variation in amylose content.In comparison, 75.7% were influenced by other factors outside the marker.Apart from the coefficient of determination value, the results of the one-way ANOVA analysis showed a significance value (p-value) of 0.148, where the marker had a value of >0.05.Hence, the marker is still unable to differentiate the average level of amylose content.The coefficient of determination of the Wx marker showed a low value of only 0.0565 or 5%, so 95% of the variation in amylose content was not associated with the Wx marker.Apart from the low coefficient of determination value, the probability value from the ANOVA analysis between the two variables from this marker was high, namely 0.509.The results of the regression analysis on the SSIIa marker showed a low coefficient of determination, namely 0.1107, where only 11% of the SSIIa gene influenced the variation in amylose content in the genotype.In comparison, 89% were influenced by other factors.The probability value (p-value) shown by the SSIIa primer from the analysis results was also high, namely 0.421.Based on the results of the one-way ANOVA analysis, the three primers used in this study had a high p-value where the null hypothesis was accepted (h0 = there was no significant difference between the three types of amylose content of tested rice) so that the three primers could not differentiate the level of amylose content, on the tested genotypes.The RM190 marker had a higher ability to identify amylose content compared to the Wx marker, but its ability to differentiate each genotype based on amylose content still very low.The amylose trait expressed in rice plants is influenced by several allelic variations on the same chromosome (Chromosome 6) or is a haplotype [26].One of them is that the microsatellite repeats of C and T bases in intron one influence low, medium, and high amylose content, where the base repeats (CT)10-11 tend to be carried by rice that carries high amylose content (Wxa), (CT)17-20 has amylose content.Low (Wxb) and (CT)18-22 are owned by rice, which has medium amylose content (Wxin) [27].
Apart from variations in microsatellite repeats, the presence of 1 base polymorphism at several loci also influences the amylose content/Single Nucleotide Polymorphism (SNP).At the intron one cutting site, differences in the G and T base arrangement can influence Wxb allele variations (affecting low to medium amylose content).Apart from that, in exon 6, there were variations in A and C bases, which can determine whether the amylose content is medium-low or high.So, there is a need for further tests regarding the RM190 and Wx markers with other amylose-carrying genes in rice genotypes [28].

Analysis of molecular markers associated with amylose traits in F9 lines
Marker amplification with the 3 SSR primers showed a polymorphic band.Polymorphism or allelic diversity resulting from a molecular marker is an illustration of the diversity or variation that occurs in each gene [29].All markers had a product length of around 100 -250 bp.The Wx marker has a band length of around 104 -124 bp.Meanwhile, the SSIIa marker had a quite significant band length of 149 -258 bp.The band size of RM190 was 124 -156 bp.Based on the linear regression analysis that was carried out on 3 (three) polymorphic markers, a coefficient of determination value in a range of 0.2147 to 0.4043.The SSIIa marker had the largest coefficient of determination compared to other markers, namely 0.4043 or 40.43%.The RM190 had a coefficient of determination of 0.2147 or 21.47%.The Wx marker had a coefficient of determination of 0.2423 or 24.23%.Based on the interpretation value of the coefficient of determination, the markers Wx, SSIIa, and RM190 had a coefficient of determination value in the medium category so that they had the possibility of being used in genotype selection based on amylose content.The Wx and RM190 markers are specific markers related to the waxy gene.Amylose synthesis in the endosperm was controlled by a single dominant gene, waxy (Wx), which codes for the Granule-Bound Starch Synthase I (GBSSI) enzyme.The GBSS enzyme plays a role in catalyzing the formation of amylose.DNA analysis of the waxy gene allele using the RM190 marker showed that the gene allele is polymorphic [30].Likewise, the Wx marker was closely related to the waxy gene, which controls the expression of amylose traits.The Wx gene is located on chromosome 6 [31].Polymorphism information based on molecular marker analysis using the Wx marker was proven to support the differentiation of rice subspecies based on amylose content [32].
The SSIIa marker was the marker that has the highest coefficient of determination value, so it was the marker most likely to be used for genotype selection based on amylose content.This is in line with research by Slamet et al. [12], which stated that the SSIIa marker can be used to analyze amylose content using the molecular marker analysis method.Lestari et al. [33] also stated that SSIIa is able to distribute alleles of starch synthesis genes polymorphically in rice plants, illustrating that this marker is able to differentiate rice varieties based on amylose content.

Conclusion
Based on the simple linear regression analysis on binary electrophoregram data of marker amplification and amylose content, the value of the coefficient of determination of the RM190 primer was 0.243 or 24.3% with a p-value of 0.148, the coefficient of determination of the Wx marker had a very low value of only 0.0565 or 5% with a p-value of 0.509 and the SSIIa marker determination coefficient of 0.110 or 11% with a p-value of 0.421.However, in F9, the SSIIa marker has the highest coefficient of determination (40.43%) compared to other markers, so it has the greatest possibility of being a marker used for selection in plant breeding based on amylose content.

Acknowledgement
The author would like to thank the Research and Community Service Institutions (Universitas Jenderal Soedirman) for the Grant of the Applied Research scheme in 2021-2022.

Table 3 .
PCR Visualization of PCR productsPCR visualization was carried out by mixing 2 µl of 5x loading dye with 5 µl of PCR product.The homogenized PCR product and DNA ladder were inserted into a 2% agarose gel well (1 gram of agarose, 50 ml of 1x TBE buffer, and 5 µl of fluorosafe) submerged in an electrophoresis tank containing 1x TBE buffer solution and run at 50 volts for one hour.Visualization of the results of agarose gel electrophoresis could be seen under the UV transilluminator.