Segregation of molecular markers associated with Bph3 gene in BC5F2 population derived from Ciherang rice variety as the recipient parent

Brown planthopper (BPH), an important insect pest of rice, can cause severe damage and significant yield loss. An efficient and environmentally friendly method to control this pest is by planting resistant varieties. The brown planthopper resistance gene Bph3 from Rathu Heenati variety is closely linked to the Waxy gene that regulates amylose synthesis, the primary determinant of rice eating and cooking quality. The purposes of this study were to analyze the segregation ratio of SSR markers associated with Bph3 gene and to identify individual plants carrying homozygous allele for Bph3 gene in a BC5F2 population derived from a backcross that used Ciherang as the recurrent recipient parent. Analyses using RM589, RM586, and RM588 markers linked to Bph3 gene and RM190 marker linked to the Waxy gene indicated that the ratio of genotype segregation for each marker related to Bph3 gene did not deviate from the expected ratio with the χ 2 value was 0.956, 0.587, and 1.467 for RM589, RM586, and RM588, respectively (χ2 table = 5.991, α=0,05). All individual plants had a homozygous allele from Ciherang for Waxy gene based on the RM190 marker. As many as 25 individual plants which have homozygous Bph3 allele from Rathu Heenati and homozygous Waxy allele from Ciherang can be further developed as promising BPH-resistant rice varieties with eating and cooking quality similar to Ciherang.


Introduction
One of the important pests of rice plants in rice-producing areas is Brown planthopper (BPH), Nilaparvata lugens Stål, which can cause severe damage and significant yield loss.This insect pest directly damages rice plants by sucking plant cell fluids and can also act as a carrier of viruses that cause ragged stunt and grassy stunt diseases [1].In the planting season of 2021 BPH attacked 14,159.88hectares of rice field in Indonesia [2].An efficient and environmentally friendly method to control BPH is by planting resistant rice varieties.Therefore, the availability of genes resistant to BPH is a main requirement to develop new rice varieties that are resistant to this insect pest.
Rathu Heenati, a Sri Lankan traditional rice variety [3,4], was identified as resistant to a BPH population gathered from East Java Province, Indonesia [5].This variety has two BPH resistance genes, i.e.Bph3 [6] and Bph17 [7].The Bph3 gene showed a broad-spectrum resistance against BPH population in Thailand [8,9] and was durable against BPH population in the Philippines.In addition, Jena and Kim [11] showed that Bph3 gene from Rathu Heenati was resistant to four BPH biotypes The Bph3 gene is located between the flanking markers RM588 and RM589, which also contain RM586 marker, on the short arm of rice chromosome 6 [8].These markers can be employed in a marker-assisted selection (MAS) method to monitor the introgression of the Bph3 gene, which can improve the effectiveness and accuracy of selection stages in developing new rice varieties resistant to BPH.Jairin et al. [9] and Sansanoh et al. [12] have successfully incorporated Bph3 from Rathu Heenati into Thai rice varieties through MAS strategy.The location of Bph3 gene on chromosome 6 is close to Waxy locus [9], which is closely linked to RM190 marker [13].Waxy gene in rice seeds is involved in the formation of amylose which is an important component that affects the quality of taste and cooking of rice [14].Rathu Heenati has 26.5% of amylose content [15] which is considered high [16] and affects its taste qualities.
Ciherang, a mega rice variety in Indonesia [17], have been used as the recurrent recipient parent in our rice breeding program to develop a rice variety resistant to BPH.Bph3 gene from Rathu Heenathi has been introgressed into Ciherang genome through MAS strategy and resulted in a BC5F2 population.The BC5F1 plant that generated the BC5F2 population was homozygous for Waxy allele from Ciherang based on RM190 molecular marker [18].Ciherang has 23.2% amylose content and good taste and cooking quality [19].Thus, there is a high probability to obtain BC5F2 plants containing homozygous Bph3 allele from Rathu Heenati and Waxy allele from Ciherang that can be further developed as a new rice variety that is both resistant to BPH and has the good taste and cooking quality of Ciherang.Those markers can be used to assist the selection of plants carrying the preferred alleles in BC5F2 population.
Segregation distortion, defined as deviation from the expected Mendelian ratio, could naturally occur in either interspecific or intraspecific crosses of rice [20][21][22][23] between divergent parents [24].Molecular markers are suitable tools to be used for analysis of segregation distortion [22].Accordingly, the occurrence of segregation distortion of Bph3 gene in the BC5F2 population can be detected using molecular markers linked to this gene.Based on the genotyping analysis of our BC5F2 population utilizing molecular markers linked to the Bph3 gene, we observed that the proportion of homozygous allele of Rathu Heenati was consistently higher than that of Ciherang.Besides, the genotype proportion of heterozygous allele was always higher than that value of the expected Mendelian ratio.Therefore, we were interested in analyzing whether segregation distortion significantly occurred in our backcross population.
This study aimed to analyze the segregation ratio of Bph3 allele using the associated molecular markers and identify individual plants carrying homozygous Bph3 gene allele from Rathu Heenati with homozygous Waxy allele of Ciherang in BC5F2 population derived from Ciherang variety as the recurrent recipient parent.

Plant materials
Ninety-two BC5F2 plants descended from Ciherang as the recurrent recipient parent of Bph3 allele originated from Rathu Heenati were used in this study.Both varieties were included as the reference sizes of Bph3 and Waxy alleles in the following molecular marker analysis.Seeds of BC5F2 population, Ciherang and Rathu Heenati were germinated in petri dishes lined with wet tissue paper.After seven days of incubation at room temperature, the seedlings were planted in plastic trays containing mud soil where they were let to grow for seven days prior to transplanting to pots with mud soil.Up to the time of seed harvest, the plants were regularly irrigated and fertilized.Young leaf from each plant were sampled for DNA extraction.

Plant genotyping using SSR markers
The genomic DNA was isolated from leaf samples of individual BC5F2 plant, Ciherang and Rathu Heenathi using the plant DNA minipreparation method [25].The SSR markers RM589, RM586, and 3 RM588 were used to identify Bph3 allele genotype [8] while RM190 marker was used for detection of Waxy allele genotype [13,26,27].PCR amplification of SSR markers was conducted in a total volume of 15 µL containing of 7.5 µl of MyTaq TM HS Red Mix 2x (Bioline, A Meridian Life Science Company), 1.5 µl of 5 µM primer, 2 µl of 10 ng/µl DNA and 4 µl of ultrapure water.PCR amplification was performed using TM100 Thermal Cycler (Bio Rad Laboratories, Inc.) with the cycle profile: 3 minutes at 94 o C for initial denaturation, followed by 35 cycles consisting of 15 seconds at 94 o C for denaturation, 15 seconds at 55 o C for primer annealing, and 20 second at 72 o C for primer extension, and finalized with 5 minutes at 72 o C for final elongation.The products of PCR were separated by electrophoresis on 8% polyacrylamide gels in 1x TBE buffer solution at 80 volts for 90 minutes.The gel was then soaked in 5 µg/ml Ethidium Bromide solution and agitated on a rotary shaker for 10 minutes.Next, the gel was immersed in water and shaken for 10 minutes.DNA bands were visualized and documented using a UV transilluminator (GelDoc, Bio Rad).The DNA banding pattern was scored as homozygous allele similar to the gene recipient or homozygous allele similar to the gene donor for a single band and heterozygous for double bands, respectively, based on the comparison with the DNA fragments from Ciherang and Rathu Heenati varieties.

Segregation analysis of Bph3 gene in BC5F2 populations
The segregation ratio of RM589, RM586, and RM588 marker genotypes was analyzed using the Chisquare test with the following formula [28]: where: p : number of classes ni : observed number of units falling into class i Ei : number of units expected to fall into class i The null hypothesis of the test was that the markers segregate in a 1:2:1 ratio.The observed segregation ratio was declared not deviating from the expected ratio if the value of calculated was less than the value of table at α = 0.05.

Genotype and segregation analyses
The SSR markers flanking Bph3 gene -RM589, RM586, and RM588showed segregation patterns that show homozygous or heterozygous for alleles from Ciherang or Rathu Heenati alleles were all present in BC5F2 population (figure 1a-c).Meanwhile, all BC5F2 plants had homozygous alleles similar to Ciherang for RM190, the SSR marker linked to Waxy gene (figure 1d), and thus it can be inferred that the preceding BC5F1 plant had homozygous allele similar to Ciherang for this SSR marker [18].The value of observed ratio of each SSR marker for Bph3 gene did not seem to exactly follow the expected 1:2:1 Mendelian segregation ratio, i.e. the number of homozygous Rathu Heenati allele was always higher than that of Ciherang (table 1).However, the results of Chi-square analysis showed that the calculated values for the segregation ratio were 0.956, 0.587, and 1.467 for RM589, RM586, and RM588, respectively, which are smaller than the value threshold of 5.991 at α level 0.05.Consequently, it can be concluded that the ratio of segregated genotypes for each molecular marker does not deviate from the predicted Mendelian ratio.The absence of marker segregation distortion in this BC5F2 population might be due to that the population is already an advanced backcross population with high level genetic background of Ciherang as the recipient parent or the genetic distance between Ciherang and Rathu Heenati is likely close.A previous study also reported no segregation distortion found in the BC4F2 rice population produced by an intraspecific cross between YK3CSSL-6.1 and Kirara397 [29].Segregation distortion is commonly found in crosses among divergent genotypes [30].In addition, the chromosome segment containing Bph3 gene might not be related to the location where several loci related to segregation distortion have been mapped in rice [31][32][33].[12].In addition, it was successfully developed BC3F6 rice lines which had a combination of Bph3 allele from Rathu Heenati and Waxy allele from jasmine rice [9].Our promising results should support the establishment of new rice varieties that are both BPH resistant and have good taste and cooking quality.As an advanced backcross population, these selected plants are expected to have genetic background similar to Ciherang, the recurrent parent.Besides having good taste and cooking quality [19], Ciherang also has an excellent grain quality [34].Further research would be still needed to prove the resistance levels of these selected plants against BPH and the content of amylose in grains.Subsequently, plants showing useful resistance to BPH biotypes or prevailing population can be developed as promising rice varieties with good grain quality including taste and cooking characteristics similar to Ciherang variety.

Conclusion
BC5F2 population segregated normally for Bph3 gene based on the SSR markers linked to Bph3 gene (RM589, RM586, and RM588 markers).Twenty five BC5F2 plants had homozygous alleles for Bph3 gene from Rathu Heenati and all 92 BC5F2 plants showed homozygous alleles for Waxy gene from Ciherang based on linked SSR marker RM190.The homozygous lines for both genes can be further developed as BPH resistant promising rice varieties with high grain quality, good taste and cooking characteristics similar to that of Ciherang.The rice lines proved resistant to BPH and having good grain quality could be proposed to be released as new rice varieties according to the applied regulatory for releasing plant crop varieties.At the end, the released rice varieties are expected to be adopted by farmers and support the rice production and its stability.
Biotypes 1,2,3,and 4).Therefore, Bph3 gene can be used as a promising candidate gene for developing new rice varieties resistant to BPH populations in Indonesia. 2(

Table 1 .
Genotype proportion of BC5F2 population based on rice brown planthopper resistance Bph3 and Waxy gene-linked markers.The SSR markers RM589, RM586 and RM588 are associated with Bph3 gene, a BPH resistance gene in rice, whereas RM190 is associated with Waxy gene, a gene that determines amylose content in rice seed b C: homozygous Ciherang allele, H: Heterozygous, and R: homozygous Rathu Heenathi allele 3.2.Identification of plants carrying beneficial homozygous alleles for Bph3 and Waxy genes Twenty five of 92 BC5 F2 plants were identified as carriers of homozygous Rathu Heenati allele for Bph3 gene based on RM589, RM586, and RM588 markers, as well as all 92 BC5 F2 plants showed homozygous alleles for the Waxy gene from Ciherang using RM190 marker (table 1).This means that Bph3 allele and Waxy allele of Rathu Heenati have been unlinked and replaced with Waxy allele from Ciherang.A successful attempt to break the linkage between Bph3 gene from Waxy gene was previously reported in the introgression of Bph3 allele from Rathu Heenati into Pathumthani 1, which is susceptible to BPH and has low amylose content, and were able to produce F4 rice lines that were resistant to BPH with low amylose content a