Screening of Rice Varieties for Bacterial Leaf Blight Resistance by Using SSR Markers

Genetic variations of seventeen rice germplasms were studied using two SSR markers (RM122 and RM390) linked to the rice bacterial leaf blight (BLB) resistance genes.The average number of allele per locus was 10, with a range of 7 (RM122) to as many as 13 (RM390). The highest level of gene diversity value (0.9135) was observed in loci RM390 and the lowest level of gene diversity value (0.7059) was observed in loci RM122 with a mean diversity of 0.8097. The PIC values ranged from a low of 0.6715 (RM122) to a high of 0.9069 (RM390) and averaged of 0.7892. A dendrogram based on genetic distance using UPGMA, indicates segregation of the 17 rice varieties in six major clusters, BR10 and Binadhan 5, which were single variety formed cluster-1 and cluster-2 respectively. BRRIdhan 38, Malshira, Binadhan 7, BRRIdhan 29, BRRIdhan 31 were grouped in cluster-3, in which sub cluster-I include BRRIdhan 38, Malshira and sub cluster-II includes Binadhan 7, BRRIdhan 29, BRRIdhan 31. Cluster-4 was the largest cluster comprised of seven rice varieties. These are Iratom 24, BRRIdhan 34, Binasail, Binadhan 4, BR 26, BR 16 and Binadhan 6. Only BRRIdhan 33 was formed cluster-5. BR 14 and BRRIdhan 32 formed cluster-6. Thus, using the same linked marker RM122, BR 14 and BR 10 might be resistant to BLB diseases. SSR markers offer a potential, simple, rapid and reliable method in marker assisted breeding.


I. Introduction
Rice (Oryza sativa L.) (2n = 24) belonging to the family Poaceae is the staple food for one third of the world's population that occupies almost one-fifth of the total land area covered under cereals (Chakravarthi and Naravaneni, 2006).Over half of the world's population depends on rice as staple food.More than 90% of the world's rice is grown and consumed in Asia (Khush, 2005).In Bangladesh, rice is the principal crop sharing over 94% of the total food grain production of 26.7 million metric tons.Rice provides 75% of the calories and 55% of the proteins in the average daily diet of the people of Bangladesh (Bhuiyan, 2005).By the year 2025, 21% increase in production over that year 2000 of will be needed (Bhuiyan et al., 2002).To achieve a substantial improvement in grain yield in a limited period, a 'second green revolution' based on advanced plant biotechnology and plant genomics is needed (Conway and Toenniessen, 2000).
Bacterial leaf blight (BLB) caused by the rod-shaped bacterium, Xanthomonas oryzae pv.Oryzae (Xoo) is one of the most devastating diseases in rice (Oryza sativa L.).Outbreaks of BLB usually occur in irrigated and rain-fed lowland ecologies throughout Asia.Leaf blight symptom can occur at all stages of rice plants.BLB disease can cause yield loss typically ranging from 20-30% but in severe cases, it can cause as high as 80% yield reduction.However, it depends on rice growth stages, geographic locations or seasonal conditions (Singh et al., 1977;Ou, 1985).Since the bacterial races vary continually influenced by the artificial and natural selection of genes resistance to bacterial blight, it is critical to explore and identify the new resistant resources to control the changeful races (Xia et al., 2012).Since the chemical control is not effective, the utilization of resistant varieties carrying resistance genes have been considered to be the most effective way to control the disease (Nino-Lui et al., 2006).As biotechnological development progresses identification, cloning, and functional analysis of a gene can be performed relatively rapidly.In the case of BLB resistance, 35 BLB resistance genes have been identified in cultivated rice and the wild relatives (Nino-Lui et al., 2006;Singh et al., 2007;Wang et al., 2009).
Unlike protein markers DNA segregate as single gene and they are affected by the environment.Molecular markers based on the DNA sequence are more varied and reliable.Several molecular markers viz.RFLP, RAPD, SSRs, ISSRs, AFLP and SNPs are presently available to assess the variability and diversity at molecular level (Joshi et al., 2000).With the development of a wide range of molecular techniques, marker assisted breeding is now used to enhance traditional breeding programs to improve crops (Frey et al., 2004).Among various PCR based markers, SSR markers are more popular in rice because they are highly informative, mostly mono locus, co-dominant, easily analyzed and cost effective (Gracia et al., 2004).Simple Sequence Repeats (SSRs) or microsatellites are most suited to routine application in breeding programs.SSRs or microsatellite markers are proved to be ideal for making genetic maps (Islam, 2004;Niones, 2004), assisting selection (Bhuiyan, 2005) and studying genetic diversity in germplasm.Microsatellite marker analysis is promising to identify major gene locus for BLB resistance that can be helpful for plant breeders to develop new cultivar.Bangladeshi rice varieties have been developed traditionally by selection, hybridization and back crossing with locally adapted high-yielding lines.The conventional methods of plant selection for BLB resistance are not easy because of the large effects of the environment and the low narrow sense heritability of BLB resistance.This hinders the development of an accurate, rapid and reliable screening technique.Individual with target gene in a segregating population can be identified with the assistance of DNA markers.Recent progress and technical advances in DNA marker technology permit the rapid and accurate identification of individuals that contain gene (s) for BLB resistance.In the present study, 17 rice genotypes were fingerprinted using 2 Simple Sequence Repeats (SSRs) primers in view to identify and tag the BLB resistance/susceptible Bangladeshi rice varieties using SSR markers linked to xa5 gene.

II. Materials and Methods
Seventeen germplasm of rice viz.BR 10,BR 14,BR 16,BR 26,BRRIdhan 29,BRRIdhan 31,BRRIdhan 32,BRRIdhan 33,BRRIdhan 34,BRRIdhan 38,Malshira (Local Farmer: Rangpur), Iratom 24, Binasail, Binadhan 4, Binadhan 5, Binadhan 6 and Binadhan 7 were used in the study.These varieties were different in their parent and also in breeding strategies.Seeds were collected from the Bangladesh Rice Research Institute (BRRI), Bangladesh Institute of Nuclear Agriculture (BINA) and different districts of Bangladesh.Seed germination was performed and germinated seeds were sown in the pot at Biotechnology field.Fresh and young leaf samples were collected from 18 day old seedlings and used as the source of genomic DNA.In order to carry out SSR analysis, youngest leaves from each of the seventeen germplasm were collected randomly from each population as the source of genomic DNA.

Collection of sample and isolation of Rice genomic DNA:
Young and actively growing fresh leaf tissues were collected for the isolation of genomic DNA.Total genomic DNA was isolated from rice leaves following Phenol: Chloroform: Isoamyl alcohol purification and ethanol precipitation method.Finally DNA samples were stored at -20 0 C.The extracted genomic DNAsamples were evaluated both qualitatively and quantitatively using agarose gel electrophoresis (0.8%, 200 ml) and spectrophotometer respectively.After electrophoresis, DNA samples were documented through Image Documentation System.
Quantification of DNA Concentration: DNA was quantified through spectrophotometer (260 nm) and absorbance readings for the other samples were taken in the same way and recorded according to the following formula: The concentration of DNA in the extracted leaf samples as quantified through absorbance reading using spectrophotometer is given in Table 1.Absorbance reading and DNA concentration is varies from 0.005-0.015and 250-750µl respectively.
Preparation of Working Solution of DNA Samples: Before PCR, Original stock solution concentration of each DNA sample was adjusted to a unique concentration (50 ng/μl) using the following formula: Where, V1= Final volume of DNA solution (µl) , S 1 = Final DNA concentration (ng/µl) , V 2 = Initial volume of DNA solution (µl)and S 2 = Initial DNA concentration (ng/µl)

PCR Analysis for Microsatellite Markers
Primer selection: Polymorphism survey of 17 rice varieties was carried out using two microsatellite markers.These two primers showed clear polymorphism which were used in genotyping the varieties.RM 122 and RM 390 were used for this.The details of the primers are given in Table 02.

Preparation of PCR Cocktail:
The following components were used to prepare PCR cocktail (Table 03).The total volume of PCR cocktail for this study was 8.37 µl per sample.Analysis of Microsatellite Data: Allele frequencies were calculated directly from the observed genotypes.Allelic variations and fit to Hardy-Weinberg proportions were estimated by the software POPGENE (version 1.31) by a chi-square (χ 2 ) test (Yeh et al.,1999) with 1000 simulated samples.Molecular weight for each amplified allele was measured in base pair using Alpha Ease FC 5.0 software.The allele frequency data from Power Marker Version 3.25 (Liu and Muse, 2005) was used to export the data in binary format (allele presence = 1 and allele absence = 0) for analysis with NTSYS-PC Version 2.2 (Rohlf, 2002).Polymorphisms information content (PIC) value of a marker was calculated according to a simplified version after Andersons et al. (1993).
Where, Xijis the frequency of the jth allele for the ith marker.
The summary statistics including the number of alleles per locus, major allele frequency, gene diversity, Polymorphism Information Content (PIC) values were determined using Power Marker Version 3.25 (Liu and Muse, 2005).Expected (H e ) and observed heterozygosity (Ho) were also calculated after (Nei, 1972) using the following formula and with the help of POPGENE (version 1.31) (Yeh et al., 1999) computer package program: Where, he ~ is the expected heterozygosity of each locus, n is the number of sampled individuals, x i is the frequency of i-th allele at each locus and N is the number of loci examined.Thus, the average heterozygosity (H e ) was calculated as, Where, r is the number of loci examined (Nei and Roychoudhury, 1973).Nei's (1972) genetic distance value was computed using the formula as described in the POPGENE (Version 1.31) software user manual (Yeh et al., 1999).Genetic distance values (D) (Nei, 1972) were calculated as Where, J x =∑ X i 2 / r in population X,J y = ∑Y i 2 / r in population Y, J xy =∑X i Y i , X i and Y i are the frequency of the i-th allele of a given locus in the two populations compared andr = the number of allele frequencies for all possible pairs of populations.
For the unrooted phylogenic tree, genetic distance was calculated using the 'C.S. Churd 1967' distance (Cavalli-Sforza and Edwards, 1967) followed by phylogeny reconstruction using neighborjoining as implemented in Power Marker with tree viewed using the Tree view.The allele frequency dated from Power marker was used to export the data in binary format (allele presence = 1 and allele absence = 0) for analysis with NTSYS-PC Version 2.2 (Rohlf, 2002).A similarity matrix was calculated with Simqual Subprogram using the Dice coefficient, followed by cluster analysis with the SAHN Subprogram using the UPGMA clustering methods implemented in NTSYS-PC was used to construct a dendrogram showing relationship among the genotypes.The similarity matrix was also used for principal coordinate analysis (PCoA) with Dcenter, Eigen, Output and MX Plat subprograms in computer program Numerical Taxonomy and Multivariate analysis system (NTSYS-PC).The software GDA (Lewis and Zaykin, 2001) was used for estimating genetic distance.The unweighted pair-group method with arithmetic mean (UPGMA) dendrogram was drawn by using the software TREE VIEW (NTSYS-PC Version 2.2) (Rohlf, 2002).

Allelic and loci variation within the genotypes
The microsatellite enriched libraries were constructed using the standard procedure with some modifications.In this study 17 varieties of rice were analyzed using two primer pairs (RM122 and RM390).Amplified microsatellite loci were analyzed for polymorphism using Polyacrylamide Gel Electrophoresis (PAGE) (Figure 01-02) and the result revealed that all the primer pairs detected polymorphism among the rice genotypes analyzed.The microsatellite loci were also multi-allelic (seven to thirteen allele per locus with a mean of 10/locus in the present study) and the alleles were co-dominant suggesting their relative superiority in detecting DNA polymorphism over some other markers.

Number of alleles per locus
Using two SSR markers, a total of 20 alleles were detected among the 17 rice varieties.The average number of allele per locus was 10, with a range of 7 (RM122) to as many as 13 (RM390) (Table 04).

Rare alleles
Rare alleles were observed at all of the SSR loci in one or more of the 17 accessions with an average of 7 rare alleles per locus and a total of 14 across all the loci (Table 04).Among 17 rice varieties marker RM390 detected the greatest number of alleles (13), which also detected higher number of eISSN 2312-7945 Khan, et. al. 2015  Published with open access at http://www.journalbinet.com/rare alleles (9) and marker RM122 detected the lower number of alleles ( 7), which also detected lowest number of rare alleles (5).Rare alleles are highly informative in fingerprinting of the varieties.

Gene diversity
According to Nei's;(1972), the highest level of gene diversity value (0.9135) was observed in loci RM390 and the lowest level of gene diversity value (0.7059) was observed in loci RM122 with a mean diversity of 0.8097 (Table 04).The maximum number of repeats within the SSRs was also positively correlated with the genetic diversity.

Allele size range
The size variation between the smallest and the largest allele at a given SSR locus was correlated with the number of alleles per locus.Thus, RM390 presented the smallest allele size range (76-128bp) and had 13 alleles in a locus, while RM122 had the largest allele size range (229-244 bp) and a total of 7 alleles (Table 04).

PIC values
Asa measure of the in formativeness of microsatellites, the PIC values ranged from a low of 0.6715 (RM122) to a high of 0.9069 (RM390) and averaged of 0.7892 (Table 04).PIC values also showed a significant, positive correlation with the number of alleles and allele size range for microsatellites evaluated in this study.The allele size range and the number of alleles were themselves also highly correlated.Deviation from Hardy-Weinberg proportion:There are no significant deviations from Hardy-Weinberg Equilibrium (HWE) for all microsatellite loci (Table 08).

Table 08. Deviation from Hardy-Weinberg expectations in 17 rice varieties according to locus
Locus name Degrees of Freedom Chi-square ( χ 2 ) G-square RM122 21 184.59.9 RM390 66 324 96.9 Gene diversity and gene flow in 17 rice varieties according to locus:According to Nei's (1987) the highest level of Shannon's information index (2.4255) was observed in loci RM390 and the lowest level of Shannon's information index (1.5285) was observed in loci RM122 (Table 09).The highest effective number of allele (10.7037) was observed in loci RM 390 and lowest effective number of allele (7.0519) was observed in loci RM122 (Table 09).Average gene flow for all microsatellite loci in 17 rice varietiesis 0.00 (Table 10).

Detection of BLB resistance and susceptible rice varieties
Amplification of xa5 linked DNA fragment was carried out using tightly linked PCR based markers RM 122 (Chen et al., 1997).The bands of xa5 gene were standardized by the amplified DNAs of IRBB-5 and IR-24 used as control.IRBB-5 showed the amplification DNA fragment having a gene of about 240 bp was considered as resistance line and IR-24 showing the DNA band of about 230 bp was considered as susceptible line (Naveed et al., 2010).The PCR product was measured as polymorphic bands pattern (Table 13).

Genetic distance:
The values of pair-wise comparisons of Nei's (1972) genetic distance (GD) between varieties were computed from combined data for the 2 primers, ranged from 0.000 to 0.500 (Table 14).The average genetic distance among the 17 rice varieties was quantified as 0.139 (Figure 03).The means of genetic distances between varieties were used to evaluate the genetic diversity of different rice varieties.From the difference between the highest and the lowest genetic distance value it was revealed that there were wide variability's among 17 rice varieties and genotypes.High genetic variability within varieties and significant difference between varieties indicate rich genetic material of a species.This study indicated that varieties those showed the lowest genetic variation can be used as parental source for breeding line to improve rice varieties.

IV. Conclusion
BR 14 and BR 10 were showed the DNA band of 244bp and 241bp using the same marker RM122.BR 14 and BR 10 are complete resistant or partial resistant to BLB diseases.Binadhan 5 was showed the DNA band of 239bp

Figure 03 .
Figure 03.UPGMA dendrogram based on Nei's (1972) genetic distance summarizing on differentiation the data among 17 rice varieties according to SSR analysis

Table 03 . Components of PCR cocktail (For 17 reactions)
PCR Amplification Profile: The PCR tubes were set on the wells of the thermo cycler plate.Then the machine was run according to the following setup: Initial denaturation at 94 o C for 3 min, Denaturation at 94 o C for 1 min, Annealing at 55 o C for 1 min, Polymerization at 72 o C for 2 min, Cycle to step 2 for 34 more time and Incubation at 72 o C for 7 min.PCR Products Conformation was done by Agarose (1.5%) Gel Electrophoresis.Then, Polyacrylamide (8%) Gel Electrophoresis was done for Microsatellite Analysis.Polyacrylamide Gel Electrophoresis of PCR Products was stained with silver nitrate following (Ethidium Bromide Staining) Pro-mega silver staining protocols with some modification.
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Table 05 . Size, Frequency, Variance and Standard Deviation of alleles at two SSR loci of 17 rice germplasm
Data on sample size, number of observation, major alleles and polymorphism information content (PIC) found among 17 rice genotypes for 2 SSR markers were observed in table 06.
SL.No.Locus Allele size (bp) Frequency This work is licensed under a Creative Commons license.Published with open access at http://www.journalbinet.com/Major allele

Table 07 . Heterozygosity for allmicrosatellite loci of 17 rice varieties
(N=Number of alleles, H o =heterozygosity observed, H e =heterozygosity expected)

Table 10 . F-Statistics and gene flow for all microsatellite loci of 17 rice varieties
Cluster-4 was the largest cluster comprised of seven rice varieties.These are Iratom 24, BRRIdhan 34, Binasail, Binadhan 4, BR 26, BR 16 and Binadhan 6.Only BRRIdhan 33 was formed cluster-5.BR 14 and BRRIdhan 32 were formed cluster-6.From this study, the dendrogram revealed that the genotypes that derivatives of genetically similar type clustered together.
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Table 11 . Allele frequency of two SSR markers in all 17 rice varieties
. Binadhan 5 may be partial resistant to BLB diseases.Malshira, Iratom 24, Binasail, Binadhan 4, Binadhan 6, BR16, BR 26, BRRIdhan 34 were showed the DNA band of 236bp which was considered at the middle between 240bp of IRBB-5 and 230bp of IR-24.Malshira, Iratom 24, Binasail, Binadhan 4, Binadhan 6, BR16, BR 26, BRRIdhan 34 may be partial resistant and partial susceptible to bacterial blight diseases.BRRIdhan 38, Binadhan 7, BRRIdhan29, BRRIdhan 31 were showed the DNA band of 234bp which is partially same with the band of 230bp of IR-24.BRRIdhan 38, Binadhan 7, BRRIdhan29, BRRIdhan 31 may be partial or complete susceptible to BLB diseases.BRRIdhan 32 and BRRIdhan 33 were showed the DNA band of 231bp and 229bp respectively.BRRIdhan 32 and BRRIdhan 33 are complete susceptible to bacterial blight diseases.The results may be utilized as the local source of xa5 gene for elite molecular breeding program and used as a baseline for improvement of rice varieties in Bangladesh.
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