Genetic diversity of mutative line group arising from Tam Thom rice variety

81 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2017-0058 Chemical and Biological Science 2017, Vol. 62, Issue 10, pp. 81-90 This paper is available online at GENETIC DIVERSITY OF MUTATIVE LINE GROUP ARISING FROM TAM THOM RICE VARIETY Nguyen Minh Anh Tuan 1 , Nguyen Minh Cong 2 and Nguyen Xuan Viet 2 1 Tuyen Quang Education and Training of Department 2 Hanoi National University of Education Abstract. The 45 indicators on morphological, agro-biological, productive and qualitative characteristics were used to determine phenotypic diversity of the 20 mutative lines arising from Tam Thom variety, when they were planted in the Spring crop and Autumn crop. The 31 SSR markers were used to determine the allelic diversity in 31 loci distributed on 12 chromosomes and genetic relationship among 20 mutative lines and original variety. A total of 602 bands appeared by using 31 SSR primers in lines/varieties studied. Out of 31 primers, 4 primers showed polymorphism (2 alleles per locus). The number of alleles per locus ranged from 1 to 2 with an average of 1.13. The PIC ranged from 0.00 to 0.32 with an average of 0.03. Using software NTSYS 2.1 pc to build a tree diagram of the genetic relationship between the mutation of lines and with original species, we have identified: The genetic diversity based on 45 morphology, agro-biology and rice quality indicators showed higher expression based on 31 SSR primers. The genetic diversity of the mutant lines planted in the spring crop expressed significantly higher than in the autumn crop. The T6, T9, T13 and T19 lines are promising to improve in yield or to create new varieties. Keywords: Genetic diversity, molecular markers, morphological indicators, Tam Thom. 1. Introduction Currently, there are many studies on the genetic diversity at the molecular level (DNA) of rice varieties: Thomson et. al. (2007) [1] analyzed 309 Indonesian rice varieties using 30 SSR markers and reported that a total of 394 alleles were detected at the 30 SSR loci, with an average of 13 alleles per locus, and an average PIC value of 0.66; Meti et. al. (2013) [2] studied the genetic diversity of 48 indigenous aromatic Indian varieties using 12 SSR markers, resulting in a PIC ranging from 0.00 to 0.74, with an average value of 0.58. In Vietnam, there have been a number of genetic diversity studies at the agonist- morphological and molecular level of some rice varieties. For example, Ngo Thi Hong Tuoi et al. (2014) [3] used 35 SSR markers to analyze 46 black lines/varieties genetic Received May 3, 2017. Revised July 12, 2017. Accepted July 19, 2017. Contact Nguyen Minh Anh Tuan, e-mail address: nmatuan@tuyenquang.edu.vn Nguyen Minh Anh Tuan, Nguyen Minh Cong and Nguyen Xuan Viet 82 YM X H  % diversity and achieved a total of 68 alleles, 9 of which were monotypic and 26 polymorphic alleles, with an average of 2.62 alleles per locus; the coefficient of genetic diversity ranged from 0.08 to 0.74, with an average value of 0.46. Nguyen Tien Thang (2011) [4] studied the genetic diversity of 24 hibrid lines between two mutant lines produced from two special rice varieties in the North (Tam Xuan Dai and Hai Hau) for an average PIC of 0.22. However, until now, studies have not identified the genetic diversity of mutantive lines derived from a variety, especially the specialty rice variety that has a characteristic aroma, strong photoperiodic sensitivity based on agronomic, and quality characteristics of rice when cultivated in different seasons and is based on SSR markers. Our research aims at identifying the relationship between variation in DNA molecular structure and the variety of morphological, agronomic, and grain quality variations of mutant lines arising from Tam aromatic rice seeds by gamma irradiation (Co 60 ) into seed germination; and at the same time, selecting the elite lines to help improve the old varieties and create new varieties of rice. 2. Content 2.1. Materials Tam Thom, which is a specialty rice variety for aromatic, sticky and delicious characteristics, is very popular in the North. However, Tam Thom has strong photoperiodic sensitivity, weak and lodging trees, and low productivity. Therefore it does not meet the requirements of production. 20 mutant lines in the 12 th generation (M12) in the research are the products of the basic State-level research project managed by the Hanoi National University of Education, code 4.5.10 (1996-1997) and 6.5.10 (1998 - 2000), "Determination of the rule of mutagenesis of some Asian rice cultivars (O. Sativa L) in the treatment of mutations". 31 SSR primers supplied by Operon company to determine the presence of alleles, which are located on the 12 chromosomes of the rice genome. 2.2. Methods Morphological and agrinomic characteristics are assessed according to "Standard Evaluation System for Rice", IRRI (2013) [5]; "National Technical Regulation on test of Cultivated value and use for Rice Varieties" (2011) [6] and "National Technical Regulations on test of stability, diversity and uniformity for Rice Varieties" [7], the Ministry of Agriculture and Rural Development (2011). To determine the genetic relationship among mutant lines (in molecular respect), we use 31 SSR primers. The results obtained from PCR electrophoresis are determined by the presence or absence of DNA bands amplified by PCR. PIC (Polymorphic Information Content): The PIC was estimated as follows: PIC = 1- ∑Pi 2 , where Pi is the frequency of the i th allele. The rate of heterosis (H%) of each rice variety is given by the formula: Genetic diversity of mutative line group arising from Tam thom rice variety 83 where: X is a total marker appeared 2 alleles per 1 locus SSR; M is the total number of primers used in research; Y: the total number of SSR primers does not appearing DNA. The rate of defect is given by the formula: where: Z is the total number of primers that do not appear DNA band; M is the total number of SSR primers used in study. The experiments were conducted at the Vietnam Agricultural Academy of Sciences (Vinh Quynh commune, Thanh Tri district, Hanoi) in the Spring and Autumn crops of 2014; Laboratory of Molecular Genetics, Department of Genetics, Department of Biology, Hanoi National University of Education. Data analysis and processing: Data on morphological and agronomic indicators (*) were processed, analyzed using Excel version 5.0 software and NTSYS pc 2.1 software [2] as well as in the case of electroenergetic processing PCR products for establishing tree diagrams: Two genetic diversity diagrams on 45 morphological, agronomic and quality indicators in Spring and Autumn crop, a plot of genetic diversity at the molecular level (DNA). (45 morphology, agronomic and quality indicators were investigated including growth time, plant height, tillering, number of effective panicles/hill, panicle length, panicle neck length, number of full grains/panicle, 1000 grains weight, individual productivity, thickness of stem, stem length, root stem diameter, plant stiffness, stem angle, flag leaf length, flag leaf width, flag leaf angle, use leaf length, use leaf width, use leaf angle, leaf colour, leaf aroma, panicle stalk, secondary branch, rice grain length, rice grain width, ratio of length/width of rice, ratio of empty grain, grain kernel length, grain kernel width, ratio of length/width of rice kernel, photoperiodic sensitivity, seedling length, seedling vitality, ratio of wholegrain, ratio of rice kernel, chalkiness, endosperm color, aroma on seeds, amylose content, protein content). 2.3. Results and discussion Genetic relationships between mutant lines and original variety were established based on 45 morphological, agronomic and qualitative indicators identified; of which 10 indicators are the most prominent manifestations presented in Table 1. 2.3.1. The results of genetic diversity analysis at the morphological level Table 1. Some morphological, agronomic and quality characteristics of the 20 mutative lines deriving from the Tam Thom variety showed distinct differences when cultivated in the spring and the autumn crops Line /var Crop GT (days) PH (cm) NP/H NG/ P REG (%) P1000 (gram) IP (gram/hill) GKL (mm) EC AC (%) CT Aut 162 152.75 6.95 105.38 21.07 19.8 14.5 5.73 TT 21.46 T1 Spr 140 133.23 7.36 115.34 23.23 20.1 17.1 5.41 TT 20.15 Aut 127 135.02 7.45 134.43 18.65 20.1 20.1 5.44 TT 19.24 T2 Spr 142 132.11 6.73 118.42 30.24 19.8 15.8 5.78 TT 20.23 M Z M % Nguyen Minh Anh Tuan, Nguyen Minh Cong and Nguyen Xuan Viet 84 Aut 120 133.28 6.87 127.17 26.41 19.9 17.4 5.77 TT 19.43 T3 Spr 142 131.25 6.77 134.07 27.45 17.7 16.1 6.67 TT 20.76 Aut 121 132.56 6.74 136.35 25.57 19.8 18.2 6.73 TT 19.32 T4 Spr 140 133.17 7.21 115.32 27.34 19.5 16.2 5.28 TT 20.38 Aut 124 133.45 7.42 127.48 20.43 19.9 18.8 5.38 TT 19.75 T5 Spr 138 129.39 8.79 107.56 26.73 19.7 18.6 6.39 TT 20.67 Aut 118 130.32 8.83 113.21 20.01 19.9 19.9 6.41 TT 19.45 T6 Spr 141 117.28 9.03 101.44 25.62 20.0 18.3 5.61 TT 20.83 Aut 123 116.35 9.04 111.34 19.02 20.1 20.2 5.67 TT 19.44 T7 Spr 136 130.45 6.57 114.53 26.43 19.6 14.7 5.54 TT 20.52 Aut 119 132.43 6.75 126.53 21.31 19.8 16.9 5.52 TT 19.54 T8 Spr 134 136.29 7.45 137.34 26.58 19.6 20.1 5.39 TT 20.45 Aut 124 135.72 7.52 145.13 21.34 19.8 21.6 5.36 TT 19.34 T9 Spr 136 131.83 7.21 147.34 27.43 20.0 21.2 5.61 TT 20.47 Aut 118 132.04 7.23 156.18 22.19 20.1 22.7 5.61 TT 19.54 T10 Spr 142 127.04 7.34 127.41 35.29 19.9 18.6 6.55 TT 20.35 Aut 123 128.33 7.45 143.44 20.83 20.1 21.5 6.56 TT 19.35 T11 Spr 144 120.92 6.95 134.37 26.41 19.2 17.9 5.67 TT 20.37 Aut 125 119.62 7.04 157.3 19.35 19.5 21.6 5.63 TT 19.73 T12 Spr 136 137.41 6.98 120.43 23.72 20.0 16.8 6.36 TT 20.50 Aut 119 135.34 7.06 133.14 17.24 20.2 19.0 6.45 TT 19.46 T13 Spr 138 139.27 8.79 111.12 28.72 20.1 19.6 5.78 TT 20.23 Aut 122 140.34 8.87 122.53 20.03 20.2 22.0 5.82 TT 19.34 T14 Spr 141 133.61 7.19 141.04 26.83 19.7 20.0 6.84 TT 20.25 Aut 121 135.34 7.21 147.15 25.09 19.8 21.0 6.89 TT 19.36 T15 Spr 140 139.19 7.13 127.54 27.44 20.1 18.3 5.32 TT 20.47 Aut 128 140.37 7.17 130.29 20.47 20.2 18.9 5.35 TT 19.38 T16 Spr 143 132.49 6.78 116.45 25.86 20.2 15.9 5.18 TT 20.36 Aut 132 133.43 6.89 128.35 20.35 20.3 18.0 5.24 TT 19.74 T17 Spr 137 140.31 7.09 122.92 26.52 19.9 17.3 6.31 TT 20.71 Aut 123 140.38 7.11 129.24 20.15 20.0 18.4 6.34 TT 19.35 T18 Spr 138 137.67 6.75 120.71 25.84 19.2 15.6 7.37 TT 20.65 Aut 117 139.45 6.78 127.55 18.35 19.5 16.9 7.34 TT 19.76 Genetic diversity of mutative line group arising from Tam thom rice variety 85 T19 Spr 137 121.68 7.34 120.43 24.59 20.1 17.8 5.25 TT 20.58 Aut 115 120.42 7.43 132.48 21.52 20.3 20.0 5.32 TT 19.56 T20 Spr 138 138.65 6.23 124.74 25.71 19.9 15.5 5.28 TT 20.45 Aut 119 137.34 6.45 134.67 22.47 20.0 17.4 5.30 TT 19.47 Note. Var: variety. CT: control. Spr: Spring. Aut: Autumn. GT: growth time. PH: plant height. NP/H: number of effective panicles/hill. NG/P: number of full grains/panicle.REG: ratio of empty grain. P1000: 1000 grains weight. IP: individual productivity. GKL: grain kernel length. EC: endosperm color. AC: amylose content. T1, T2,... T20: mutative lines. * Genetic diversity of 20 mutant lines and original variety in the autumn crop At the genetic similarity coefficient of 0.29, 21 lines/variety in this study were grouped into 3 major clusters. Cluster 1 contained only Tam Thom variety (original variety- control); cluster 2 contained 16 lines (T1, T2, T3, T5, T6, T8, T9, T10, T12, T13, T15, T16, T17, T18, T19 and T20); cluster 3 contained 4 lines (T4, T7, T11, and T14). At the genetic similarity coefficient of 0.34, the 21 mutant lines and the original seed were grouped into 10 cluster s (Figure 1). Figure 1. Genetic relationships of 20 mutant lines and original variety (in autumn crop) based on 45 morphological, agronomic and rice quality indicators Note. d/c: Control. T1, T2,T20: mutative lines * Genetic diversity of 20 mutant lines and original variety in spring At the genetic similarity coefficient of 0.29, 20 mutant lines were grouped into 6 major clusters. Cluster 1 included 8 lines (T1, T4, T6, T9, T10, T12, T15 and T19); cluster 2 consisted of 2 lines (T14 and T17); cluster 3 consisted of 4 lines (T3, T11, T16 and T18), and cluster 4 included 1 line (T7); cluster 5 contained 4 lines (T2, T8, T13 and T20); cluster 6 contained 1 line (T5). At the genetic similarity coefficient of 0.34, the 20 mutant lines were grouped into 13 clusters (Figure 2). Nguyen Minh Anh Tuan, Nguyen Minh Cong and Nguyen Xuan Viet 86 The data in Table 1 and Figures 1 and 2 showed that the diversity of the 20 mutant lines in the spring crop is more pronounced than that of the autumn one. At the genetic similarity coefficient of 0.29, in the autumn crop, 20 mutant lines and original variety were grouped into 3 major clusters; while in the spring crop, they were grouped into 6 major clusters. At the genetic similarity coefficient of 0.29, the lines/varieties in this study were grouped into 10 clusters in the autumn crop and 13 clusters in the spring crop. In our opinion, this difference is due to the fact that the mutated lines in this study were derived from Tam aromatic rice variety, (this variety is strong photoperiodic sensitivity, good growth in the Autumn crop, but non-flowering in the spring crop - at the wrong time). Thus, mutant lines have lost their photoperiodic sensitivity but genes and genomes that control other traits have not fully adapted to spring conditions. Each line had a different response to spring conditions, which led to differentiated expression at different rates compared to the autumn crop. Breeders need to exploit a phenotype that is distinctly different from the original but only in the spring to produce unique characteristics unfound in the original variety. Figure 2. Genetic relationships of 20 mutant lines and original variety (in spring crop) based on 45 morphological, agronomic and rice quality indicators Note. T1, T2,T20: mutative lines 2.3.2. Genetic diversity at the molecular level of 20 mutant lines and original variety * PIC coefficients, number of alleles and total number of DNA bands are shown on each pair of primers Genetic diversity of mutative line group arising from Tam thom rice variety 87 Analysis of 31 SSR primers (Table 2) with 20 mutant lines and original variety, we yielded a total of 602 DNA bands of 35 different alleles. Of these, 4 pairs of primers (RM341, RM13, RM223 and RM316) gave the polymorphic locus (2 alleles/locus), 27 primers for single locus. Thus, the lines/varieties in this study were less diverse in allelic compositions at the SSR loci used in the study. The PIC values of the 31 primers ranged from 0.00 to 0.32 with an average of 0.03, was significantly lower than that of Ngo Thi Hong Tuoi et al. (2014)[3] on genetic diversity of black rice varieties with the PIC ranging from 0.08 to 0.74 with an average of 0.46, and Nguyen Tien Thang (2011) [4], genetic diversity study of 24 hidrid lines between two mutant lines were generated from two different rice varieties (Tam Xuan Dai and Du Hai Hau) for PIC of 0.22. In our opinion, this difference may be due to the use of different objects in the studies. Table 2. Number of alleles expressed and PIC coefficients of 31 SSR primers Order Name of primes No. of chro Number of alleles PIC order Name of primes N o of chro Number of alleles PIC 1 RM104 1 1 0.00 17 Arol 1 8 1 0.00 2 RM128 1 1 0.00 18 RM28 8 1 0.00 3 RM166 2 1 0.00 19 RM223 8 2 0.18 4 RM174 2 1 0.00 20 RM264 8 1 0.00 5 RM211 2 1 0.00 21 RM284 8 1 0.00 6 RM318 2 1 0.00 22 RM337 8 1 0.00 7 RM341 2 2 0.26 23 RM515 8 1 0.00 8 RM135 3 1 0.00 24 RM23120 8 1 0.00 9 RM143 3 1 0.00 25 RM245 9 1 0.00 10 RM156 3 1 0.00 26 RM296 9 1 0.00 11 RM241 4 1 0.00 27 RM316 9 2 0.32 12 RM13 5 2 0.18 28 RM333 10 1 0.00 13 RM30 6 1 0.00 29 RM224 11 1 0.00 14 RM314 6 1 0.00 30 RM229 11 1 0.00 15 RM345 6 1 0.00 31 RM322 11 1 0.00 16 RM346 7 1 0.00 Total 35 0.94 Average 1.13 0.03 Note. N o of chro: number of chromosome * Rate of heterozygosity (H%) and percentage of defects (M%) of the studied rice lines The percentage of defects (M%) and the heterogeneity (H%) of the 20 mutant lines and original variety based on the analysis of 31 SSR primers are presented in Table 3. Nguyen Minh Anh Tuan, Nguyen Minh Cong and Nguyen Xuan Viet 88 The data in Table 3 show that the mutative lines have very high genetic purity. Of the 21 lines/variety studied, 18 lines/seed had a heterologous rate of 0% which means that these lines were homozygous in all 31 SSR primers (only single allele/locus), 3 lines with heterologous ratio greater than 0% were T4, T10, and T14 (3,2%). Average heterologous rate in the studied lines/variety was 0.46%. The percentage of data defect (M%) of the mutant lines was very low; only 4 lines had data defect: T3, T7, T12 and T19 (3,2%). Average defect rate of 31 primers was 0.61. Thus, the data obtained from the 21 lines/variety in our study were highly reliable for statistical evaluation and genetic correlation determination. Table 3. Heterosis (H%) and percentage defect (M%) of the studied rice lines Order Lines/Variety M% H% Order Lines/Variety M % H % 1 T1 0.00 0.00 12 T12 3.20 0.00 2 T2 0.00 0.00 13 T13 0.00 0.00 3 T3 3.20 0.00 14 T14 0.00 3.20 4 T4 0.00 3.20 15 T15 0.00 0.00 5 T5 0.00 0.00 16 T16 0.00 0.00 6 T6 0.00 0.00 17 T17 0.00 0.00 7 T7 3.20 0.00 18 T18 0.00 0.00 8 T8 0.00 0.00 19 T19 3.20 0.00 9 T9 0.00 0.00 20 T20 0.00 0.00 10 T10 0.00 3.20 21 TT 0.00 0.00 11 T11 0.00 0.00 Average 0.61 0.46 Figure 3. Photograms of PCR products of 20 mutant lines and original variety with RM 316 marker Note. 1: Control. From 2 to 21: 20 mutant lines from 1 to 20, respectively Genetic diversity of mutative line group arising from Tam thom rice variety 89 2.3.3. Genetic relationships of 20 mutant lines and original variety The data obtained from the PCR product template with 31 SSR primers from 20 mutative lines and original variety were statistically analyzed by NTSYSpc version 2.1 software, which was used to set up a table of genetic similarity coefficient and construct a tree diagram of genetic relationships at the molecular level (DNA). The analytical results showed that: genetic diversity of 21 lines/variety was low. Genetic similarity coefficient ranged from 0.88 to 1.00. At the genetic similarity coefficient of 0.91, 21 lines/variety were grouped into 3 major clusters: Cluster 1 contained 5 mutative lines: T4, T7, T11, T13, T20 and original variety (TT- control); Cluster 2 contained 9 mutative lines: T1, T3, T5, T6, T9, T10, T12, T14 and T19; Cluster 3 contained 6 mutative lines: T2, T8, T15, T16, T17 and T18. Figure 4. Genetic relationships of 20 mutant lines and original variety based on analytical results using 31 SSR markers Note: TT: control. T1, T2,T20: mutative lines 2.3.4. The relationship between diversity at the morphological and molecular levels When using agronomic, qualitative and molecular indicators to study the genetic diversity of the lines/variety, we found that: the diversity of lines/variety based on morphological, agronomic and qualitative indicators were higher than those using 31 SSR primers and there was a similarity in grouping into major clusters of lines/variety in our study. However, when grouping into small clusters, there are still some differences such as when using the morphological indicators at the similarity coefficient of 35% the T4, T7, T11 and T14 lines belong to the same cluster, TT (control) belongs to a group and the other lines belong to another group. While using 31 molecular markers at the similarity coefficient of 0.91, the T4, T7 and T11 lines are in the same cluster as TT (control), T20 and T13; the T14 line belongs to another group. Especially, the T14, T1, T3, T5, T6, T10 and T12 lines had a similarity coefficient of 1.00. This shows that, when researched on a variety based on morphological indicators, there is a higher precision than molecular indicators. Coefficient 0.88 0.91 0.94 0.97 1.00 T10 TT T20 T4 T11 T13 T7 T1 T3 T5 T6 T10 T12 T14 T19 T9 T2 T8 T15 T17 T16 T18 Nguyen Minh Anh Tuan, Nguyen Minh Cong and Nguyen Xuan Viet 90 3. Conclusion Cultivating in the spring crop, 20 mutant lines arising from the special rice variety of the North Vietnam (Tam Thom variety) showed a clearer diversity compared to the cultivating in the Autumn crop (based on the results of the evaluation of 45 indicators on morphology, agronomy and quality rice); correspondingly, the average similarity coefficient of a mutant line compared to the original variety in the spring and autumn crops were 0.28 and 0.32, respectively. The genetic relationships of the mutant lines expressed through tree diagram, which was build based on the results of the above 45 indicators, are more complete and detailed than their genetic relationships as shown in the tree diagram, which was build based on based on analytical results using 31 pairs of SSR primers. The T8, T9, T13 and T14 lines are promising with many improvements compared to the original variety such as reduced plant height, shorter growth time, and greater productivity. 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