
Seed quality could be identified from its physical, physiological, and genetic aspects. In hybrid seeds, parental-inherited characteristics play a pivotal role in detecting seed purity. Seeds that do not share similar characteristics as their parents may lead to contamination during crossing (Hazmy
Hence, the need to perform genetic purity tests in genotypes of F1, F2, and BC1 generations of the upland and lowland rice parents is expected to be used as one of the guidelines for the development of the next rice variety (Ardiarini
Research activities were carried out both in the experimental field and the Plant Biotechnology Laboratory of
Seedling trays, nameplates, manure, NPK fertilizer, and polybags were used for planting the samples. Next, the young rice leaves produced were isolated using the DNeasy Plant Mini Kit procedure. When the results of the DNA isolation were obtained, a qualitative test of rice quality was carried out. The next stage involved DNA amplification by PCR technique using the DreamTaq Green PCR Master Mix procedure. The results of the PCR were electrophoresed using agarose gel and EtBr for analysis to obtain the data in the form of DNA banding patterns.
Data analysis was performed based on visible DNA bands on the gel doc. Then, the value of Polymorphism Information Content (PIC) was calculated. The data that had been obtained were analyzed using the Power Marker software to create binary data and the NTSYS 2.1 software with the UPGMA method to create genetic similarity matrixes and genetic distance dendrograms.
The use of molecular markers in this research is to measure the genetic purity of seeds from the crossing of upland rice with that from lowland rice. The genetic purity of seeds can be determined by the genetic similarity and genetic distance shown by amplified DNA banding patterns. In this study, six drought-specific and high-yielding primers were used, namely RM5, RM211, RM232, RM249, RM255, and RM258.
Electrophoresis was performed after isolating the rice plant DNA. The success of the electrophoresis process is affected by the use of KIT and the purity of the DNA solution (Adriansyah
In the amplification of the RM5 and RM211 primers, sample number 7 on the RM5 primer and sample number 12 on the RM211 primer did not form bands indicating that the used primers did not match with the target DNA and thus inhibited the doubling or amplification process during electrophoresis (Hazmy
Meanwhile, based on the analysis of the PIC value, the average PIC value of the six primers that were used is 0.3775. In this research, primer RM255 has a PIC value > 0.5 and it is therefore categorized as having a high PIC value. Four primers (RM5, RM211, RM232, and RM249) have moderate PIC values of 0.25 < PIC < 0.50 while one primer has a low PIC value of < 0.25 (Table 1).
Table 1 . Number of alleles and
Primer | Number of alleles | PIC value |
---|---|---|
RM5 | 11 | 0.3557 |
RM211 | 11 | 0.3557 |
RM232 | 12 | 0.3047 |
RM249 | 12 | 0.4598 |
RM255 | 19 | 0.5499 |
RM258 | 12 | 0.2392 |
Average | 12.8 | 0.3775 |
However, the obtained PIC values are different from those in the previous research. In Rokhmah
The results of scoring on the DNA banding patterns in this study through the genetic similarity values and the genetic distance dendrogram were obtained, in which the former refers to the inverse of the formed latter (Bonato
Observed from the genetic distance and similarity coefficient, it is known that the F1 generation tends to resemble the lowland variety as the male parent as opposed to upland rice as the female parent. The F1 generation is the result of crossing the varieties of
Based on genetic similarities (Table 2), F1 SBCH and F1 SBCB belonging to F1 generation genotypes possess genetic similarities with the same value of 1.00 despite having originated from different male parents. Nugroho
Table 2 . Similarity and genetic purity of each rice geno-type to its parents.
Genotype | Genetic similarity | Genetic purity | |
---|---|---|---|
Male parent | Female parent | ||
F1 SBCH | 0.81 | 0.19 | Pure |
F1 SBCB | 0.88 | 0.19 | Pure |
F1 TWCH | 0.85 | 0.62 | Pure |
F2 SBCH | 0.69 | 0.19 | Pure |
F2 SBCB | 0.63 | 0.25 | Pure |
F2 TWCB | 0.75 | 0.44 | Pure |
BC1 SBCH | 0.38 | 0.63 | The female parent is more dominant or there is contamination |
BC1 TWCB | 0.64 | 0.56 | Pure |
For the F2 generation, the resulting band pattern has a lower similarity coefficient with the two parents compared to the F1 generation. This is contrary to Mendel’s first law for monohybrid crosses, in which the F2 generation will show different characteristics from the F1 generation but are more similar to their parents because of random segregation. F1 generated plants were later re-crossed with the upland rice parent as the restorer or recipient. Based on distance and genetic similarities, it can be seen that BC1 SBCH has a quite distant genetic similarity from its restorer parent,
From the results of genetic purity testing based on genetic similarity, the F1 and F2 seeds of all cross sets (SBCH, SBCB, and BC1 TWCB) could be regarded as pure seeds and did not show a mixture of other genetic traits. The BC1 SBCH seeds were detected to have a mixture of other genetic traits because of the very low genetic similarity of the male parent (
The F1 SBCH and F1 SBCB generations had a genetic similarity of 1.00 because of having been produced by the same female parent (Situ Bagendit). In the F2 generation, all genotypes had lower genetic similarities compared to the F1 generation; thus, the resulting genetic distance was also further apart. The results of backcrossing F1 SBCH and F1 TWCB generations with restorer parents showed differences in genetic inheritance. Meanwhile, BC1 SBCH had a large genetic distance compared to BC1 TWCB, which is in the same cluster as its restorer parent.
The authors are grateful to DIKTI and LPPM UB of Universitas Brawijaya for providing great support and assistance in the research project. Gratitude is due to Elfita Rahma Aulia for her contribution to the research.
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