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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Specialization: Biotechnology Code: 62 42 20 01 NGUYEN THI MY DUYEN BREEDING OF SALINITY TOLERANT RICE WITH HIGH IRON AND LOW AMYLOSE CONTENT Can Tho, 2019 THIS STUDY WAS COMPLETED AT CAN THO UNIVERSITY Scientific supervisor: Assoc Prof Doctor TRAN THI CUC HOA NGUYEN THI LANG The dissertation defended at the university examination committee At The Room of Thesis Defend (Room 3), The Second Floor, Operation Building, Area 2, Can Tho University At … hour …, on date … month … year … Referee 1: Prof.Dotor.Nguyen Hoang Loc Referee 2: Doctor Truong Thi Bich Van Confirmation of the Chairman of the board Prof.Dotor.Le Van Hoa The dissertation is available at Libraries: Central library of Can Tho University National library of Vietnam PUBLISHED PAPERS Nguyen Thi My Duyen, Vu Anh Phap and Tran Thi Cuc Hoa, 2018 Evaluation of genetic material for salt tolerance rice with high iron content and low amylose Science and Technology Journal of Agiculture and Rural Development, 1:24-28 (in Vietnamese) Nguyen Thi My Duyen, Vu Anh Phap and Tran Thi Cuc Hoa, 2018 Breeding salt – tolerant rice lines by backcrossing to the salt – tolerant rice variety Pokkali and using molecular marker selection Science and Technology Journal of Agiculture and Rural Development, 11:11-16 (in Vietnamese) Nguyen Thi My Duyen, Vu Anh Phap, Tran Thi Bich Xuan and Tran Thi Cuc Hoa, 2018 Improved quality of OM5451/Pokkali by backcross breeding Can Tho University Journal of Science 45(7B): 6-12 (in Vietnamese) Nguyen Thi My Duyen, Vu Anh Phap and Tran Thi Cuc Hoa Hydridization and selection salinity tolerant rice lines from the hybrid and backcross OM238/Pokkali Can Tho University Journal of Science (Accepted, in Vietnamese) Chapter 1: INTRODUCTION Necessity of the dissertation Rice (Oryza satiava L.) is the staple food of more than half of the world's population, especially countries in Asia, Africa and Latin America countries In Vietnam, rice is the main agricultural crop In 2015, Vietnam ranked the third in the world in terms of rice exports after India and Thailand In particular, the CuuLong River Delta (CLRD) is the largest rice export region According to statistics of the General Statistics Office, the total area of 2017 rice cultivation in Vietnam was estimated at 7.72 million hectares (down by 26.1 thousand hectares compared to 2016), the estimated yield was 5.55 tons/ha (down by 0.02 ton/ha compared to 2016), the production was estimated at 42.8 million tons (down by 318.3 thousand tons compared to 2016) (Vietnam Business Monitor, 2017) Agricultural production has been facing climate change, in which salinity is the second most important factor after drought About a fifth of the world's irrigated arable land has been affected and over 800 million hectares have been damaged by salinity In CLRD region, in the period of 2015-2016, the extent of the influence of the "4 g/l" saltwater boundary (salinity cannot be drained or produced) affected 10/13 provinces of this region, with a total area rice damage was nearly 139,000 Among them, 86,000 of rice damaged over 70% and 43,000 of rice damaged 3070% of productivity (vtv.vn, 2016) According to the General Department of Irrigation, the Ministry of Agriculture and Rural Development (MARD), the flow of the Mekong River to CLRD had been changing complicatedly, sharply decreasing compared to the beginning of the dry season, the possibility of increasing invasion Saline intrusion was higher than the same period in 2017 (Bich Hong, 2018) Thus, with the influence of global climate change, the area of rice cultivation in the CLRD has been increasing, leading to a serious reduction in Vietnam's rice production Coping with this problem, the selection of salinity-tolerant rice varieties has been studied by scientists This was also one of the solutions given in MARD's proposal in 2015 (TTO, 2015) In addition to some salinity tolerant varieties, it has been selected to create and cultivate effectively in some countries around the world Many sources of local rice varieties such as Nona Bokra, Bura Rata were well tolerated with saline conditions, equivalent to the Pokkali varieties that have been identified In the late 20th century, breeders used genetic changes to create rice varieties with high yield potential, good rice quality, resistance to some major pests, and tolerance to the disadvantage conditions such as drought, flooding, salinity In CLRD, rice varieties grown in saline areas have limitations such as high-yielding rice varieties that are low or medium tolerant to salinity, or low quality rice Local rice varieties are saline tolerant such as Tep Hanh and Mot Do Do, but with long duration, low yield and hard grain Therefore, it is necessary to breed tolerant rice varieties at the average salinity threshold (EC dS/m) and high salinity tolerance (EC above dS/m) or more, and other interested characteristics such as short duration, high yield and good resistance to pests and diseases Currently, consumer’s demands are getting higher and higher The quality of rice with low in amylose content and rich in micronutrients is being paid attention The creation of rice varieties with high iron content in grain to contribute to reducing the rate of diseases related to iron deficiency, especially poor farmers in remote areas is very necessary Therefore, breeding of rice varieties is both able to adapt well to saline conditions and high yield, good quality (low amylose content) and rich nutrition Moreover, nowaday, the rapid development of biotechnology, especially molecular genetics, has effectively supported the new plant breeding, helping to select the main hybrid more accurate, shorten the time for breeding In particular, the SSR (Simple sequence repeats) was chosen by many scientists because of its homogeneous nature (codominan), a high degree of reliability (Nguyen Thi Lang and Bui Chi Buu, 2011) In 2011, CuuLong Delta Rice Institute released the iron rich rice variety OM6976 with medium salinity tolerance, but high amylose content, and OM5451 variety with low amylose content (18%) but low salinity tolerance Currently, there is no rice variety that is not only saline tolerant but also high iron and low amylose content in grain Therefore, the thesis "Breeding Of Salinity Tolerant Rice With High Iron And Low Amylose Content" was carried out through backcrossing method and detection of salinity tolerance gene by molecular markers SSR, thereby, rice varieties with both high salinity tolerance and high quality grain (low amylose and iron-rich content) to meet regional production and market demand for rice Objectives 2.1 General objective: Breed and select rice lines that adapt and thrive in saline ecological regions, and have high iron content and low amylose content (≤ 20%) in grain by the combination of traditional backcrossing and molecular markers 2.2 Specific objective: One to two rice lines were bred and selected by traditional methods combined with molecular markers These lines were capable of adapting in saline ecological areas (~ ‰) and good in quality (amylose content ≤ 20% and high iron content (iron in brown grain > 14 mg/kg and iron in milled grain ≥ mg/kg) Research subjects and scope of the study Improved rice varieties from Genebank of Department of Biotechnology, CuuLong Delta Rice Institute Location and duration The experiments were conducted at the CuuLong Delta Rice Institute, Can Tho; Kien Giang (Minh Luong at Summer-Fall crop and An Bien at Winter-Spring); and Soc Trang (Tran De) Duration: from January 2014 to February 2018 New contributions of the dissertation Genetic resources were effectively exploited through the selection of materials for Saltol genes (well-known salt-tolerant rice varieties was Pokkali) Pokkali was affected by the photoperiod, the height of plant was over 150 cm, the grain quality was very bad, and the grain yield was not high However, effective application of SSR molecular markers in selection in rice backcross populations, the elite rice lines that were both salininity tolerant and high iron and low amylose (≤ 20%) in grain were selected In addition, these selected rice lines not only achieved the thesis objectives but also attached other interested traits, such as strong plant, long grain, less chalkiness, wide adaptation, high yield and insect and disease resistance Outline of the dissertation The thesis was 146 pages in length, including parts, introduction, overview, materials and methods, results and discussions, conclusions and recommendations, references and appendix The thesis consited of 74 tables, 44 figures and 142 references Chapter 2: MATERIALS AND METHODS 2.1 Materials Rice varieties: 36 of improved rice lines/varieties and controls (Pokkali, IR29, IR68144, KDML105, IR64, OM6976, OM5199) from the genebank of the Department of Biotechnology - CuuLong Delta Rice Institute Molecular markers: for saline gene Saltol on chromosome (RM1287, RM10694, RM3412b, RM7075, RM490, RM140, RM310); for genes that regulate amylose content Waxy on chromosome (Wx-In1) 2.2 Experimental methods 2.2.1 Content 1: Evaluation of parent materials used in the rice breeding for salinity tolerance and high quality 2.2.1.1 Evaluation of salinity tolerance by screening in saline condition Table 2.1 Standard evaluation system for rice (SES) for salinity tolerance at seedling stage SES DESCRIPTION TOLERANCE Normal growth, only the old leaves show white tips while no symptoms on young leaves Near normal growth, but only leaf tips burn, few older leaves become whitish partially Growth severely retarded; most old leaves severely injured, few young leaves elongating Complete cessation of growth; most leaves dried; only few young leaves still green Almost all plants dead or dying Highly tolerant Tolerant Moderately tolerant Susceptible Highly susceptible (Source: IRRI, 1997) The artificial salinity screening in the tray with the solution of Yoshida (1976) plus salt at seedling stage according to the method of IRRI (1997) 2.2.1.2 Analysis of amylose content, gel consistency and gelatinization temperature by biochemical method Analysis of amylose content based on the method of Graham (2002) Analysis of gel consistency and gelatinization temperature belonged to the method of IRRI (1996) 2.2.1.3 Analysis of iron content in brown and milled rice grain Analysis of iron content in rice grain were done by the method of Hossain and Virk (IRRI, 2006) 2.3.1.4 Application of SSR markers related to Saltol gene to identify donors from the material set DNA extraction by CTAB, PCR and electrophoresis based on Kumar et al (2007) 2.2.2 Content 2: Creation of the backcrossing populations with desirable traits * Objectives: Breeding offspring populations with both salinity tolerance and good quaility * Content: Combination of high salinity tolerance from donor parent (Pokkali) and high iron and low amylose content from recurrent parent was in order to creat the F1 offspring populations 2.2.3 Content 3: Breeding and selecting of BC populations by salinity screening and identifying gene related to salinity tolerance through SSR markers * Ojectives: Aim to shorten the time and increase the effect of selection of salinity tolerant lines, after each generation, separated lines would be selected by molecular markers * Content: Breeding to creat the offspring populations to BC3 generation (Hình 2.1) Individual selection in all generations based on: - Salinity tolerance at seedling stage through screening in saline condition - individuals carried genes associated with salinity tolerance (Saltol) - Evaluation of agronomic traits and yield - Analysis of iron content and amylose content in grain Figure 2.1 Breeding process of backcross population with salinity tolerance and high quality (high iron and low amylose content) (P1: Recurrent Parent; P2: Donor Parent) 2.2.4 Content and 5: Field trial of elite rice lines tolerant to salinity, high iron content and low amylose content at different ecological regions at the 2017 Summer-Fall crop and the 2018 Winter-Spring crop * Objectives: The elite rice lines of BC3F4 and BC3F5 were grown in ecological regions (normal – CuuLong Delta Rice Chapter 3: RESULTS AND DISCUSSIONS 3.1 Content 1: EVALUATION OF INITIAL MATERIALS FOR BREEDING OF SALINITY TOLERANT AND HIGH QUALITY RICE VARIETIES 3.1.1 Evaluation of salinity tolerance in parental materials by screening in saline environment The results of screening of 36 rice varieties/lines showed that varieties/lines were salinity tolerance in group I (score 1.0 - 2.9) and similar with Pokkali, such as OM107, OM6976, OM109, OM9577, SH1317, SH333 -91, OM108-200 Besides, there were 19 varieties/lines in group II (score 3.0 - 4.9) for salinity tolerance consisted of SH62, OM11, SH200, SH1-12, OM5451, OM241-5, OM40, OM326, OM121, OM10636, OM232, OM9584, SH514, OM241-7, OM240, SH713, SH891, OM239, OM9581 The rest varieties/lines belong to group III (above score 5.0), in the same group with IR29 including OM259, OM238, OM231, OM193, SH893-4, OM236, SH921, OM178, OM238-2, OM238-3 3.1.2 Analysis of amylose content, gel consistency and gelatinization temperature by biochemical method Results of amylose content’s analysis recorded 15/36 varieties/lines with low content (≤ 20%) including OM238, OM231, OM5451, OM241-5, OM326, OM121, OM193, OM232, SH514, OM241-7, OM240, SH713, SH333-91, OM238-2 and OM238-3 All of these lines/cultivars had a gel strength of 72.7 100.0 mm, belonging to the low group (soft rice), in the same group as KDML105 Meanwhile, varieties/lines with high gelatinization temperature (score 1-3) were OM238, OM231, OM326, OM121, OM193, OM232, OM241-7, OM240 and SH33391 Five (5) varieties/lines with average gelatinization temperature (score 4-5) were OM5451, OM241-5, SH514, SH713 and OM238- 11 Only one line was OM238-3 with low gelatinization temperature (score 6) 3.1.3 Analysis of iron content in brown rice and milled rice Iron content in milled rice of 36 rice varieties/lines varied from 3.04 to 6.74 mg/kg OM121 had the highest iron content (6.74 mg/kg) Other varieties/lines rich in iron in milled rice (≥ 6.5 mg/kg) included OM11, OM5451, OM121, OM6976 and SH514 Among of 36 varieties/ lines, 12 varieties/lines had high iron content in milled rice (6-8 mg/kg), in the same group as IR68144, accounting for 33% Thus, based on salinity tolerance, Pokkali has been selected as the father (donor) for the Saltol gene The varieties OM238, OM231, OM5451 and OM121 were selected as mothers (recurrents) because of their low amylose content and high iron content 3.1.4 Application of SSR molecular markers associated with saline tolerance genes to detect genetic material that carried the desired gene Evaluation of polymorphism in parents with Saltol gene with molecular markers is RM140, RM310, RM1287, RM3412b, RM7075, RM490 and RM10694 In particular, two molecular markers RM1287 (150 - 175 bp) and RM10694 (250 - 350 bp) were selected as indicators for gene Saltol in selection of salinity tolerant rice lines 3.2 BACKCROSSING TO CREAT NEW RICE LINES WITH DESIRABLE TRAITS In F1 generation, the OM231/Pokkali popualtion collected 52 seeds, the OM 238/Pokkali population was 60 seeds, the OM5451/Pokkali population was 80 seeds and the OM121/Pokkali population was 48 seeds 12 3.3 BREEDING AND SELECTING OF BC POPULATIONS BY SALINITY SCREENING AND IDENTIFYING GENE RELATED TO SALINITY TOLERANCE THROUGH SSR MARKERS 3.3.1 The generation of F1, BC1-3F1 3.3.1.1 Breeding results in the OM231/Pokkali//OM231 population Figure 3.1 Scheme of breeding in the OM231/Pokkali//OM231 population (P1: Recurrent Parent; P2: Donor Parent) 13 In F1 generation, individuals, such as F1-1, F1-2, F1-3, F1-4, F1-5, F1-6, F1-7, F1-9, F1-12 and F1-13, were heterozygous at Saltol gene In BC1F1 generation, through screening in the nethouse and genotyping with markers RM1287 and RM10694, individuas were selected because of carring heterozygous Saltol gene including BC1F1-2, BC1F1-3, BC1F1-10, BC1F1-11 and BC1F1-15 In BC2F1 generation, 10 rice lines, BC2F1-1, BC2F1-6, BC2F1-8, BC2F1-13, BC2F1-14, BC2F1-15, BC2F1-16, BC2F1-19, BC2F1-22 and BC2F1-24 were selected In BC3F1 generation, elite rice lines, BC3F1-1, BC3F1-3, BC3F1-10, BC3F1-11, BC3F1-17, BC3F1-18, BC3F1-26, BC3F1-36 and BC3F1-39 were good at salinity tolerance and carried Saltol gene Combination of phenotype and genotype evaluations in the OM231/Pokkali//OM231 population showed that BC3F1 individuals were relatively homogenous about genetic of salinity tolerance Their shape were similar to the mother, OM231 (high yielding variety), and salinity tolerance was similar to the father, Pokkali Therefore, these individuals selected in this generation would be self-pollinated, multiplicated and selected pure lines in the field 3.3.1.2 Breeding results in the OM238/Pokkali//OM238 population Table 3.1 Number of individuals from F1 generation to BC3F1 generation in the OM238/Pokkali//OM238 population Generation F1 BC1F1 BC2F1 BC3F1 Total number of individuals Number of screened individuals Number of genotyped individuals 60 140 250 300 45 90 180 180 15 12 29 35 Number of Saltolcarried individuals 13 14 Number of selected individuals 9 Finally, elite individuals selected as BC3F1-1, BC3F1-2, BC3F1-3, BC3F1-8, BC3F1-11 and BC3F1-15 These lines had some 14 desirable traits Since then, these lines serve as a source of materials for the selection of pure rice lines in the field 3.3.1.3 Breeding results in the OM5451/Pokkali//OM5451 population Table 3.2 Number of individuals from F1 generation to BC3F1 generation in the OM5451/Pokkali//OM5451 population Generation F1 BC1F1 BC2F1 BC3F1 Total number of individuals Number of screened individuals Number of genotyped individuals 80 120 700 900 45 90 180 180 17 23 28 40 Number of Saltolcarried individuals 12 14 18 Number of selected individuals 7 13 In the OM5451/Pokkali//OM5451 population, 13 elite lines, BC3F1-1, BC3F1-2, BC3F1-3, BC3F1-6, BC3F1-8, BC3F1-11, BC3F115, BC3F1-17, BC3F1-21, BC3F1-24, BC3F1-36, BC3F1-37 and BC3F1-39, carried Saltol gene and salinity tolerant similar to Pokkali, but, their shape was similar to OM5451 These individuals were evaluated as genetically relatively pure according to the study objective, so, they were candidated to select pure lines in the field 3.3.1.4 Breeding results in the OM121/Pokkali//OM121 population This population was bred and continuously selected to the BC1F1 generation However, it was noted that these plants in this population were infected with yellow leaf disease, so this population was removed 3.3.2 Breeding results of rice populations in the BC3F2 generation 3.3.2.1 Results in the OM231/Pokkali//OM231 population In 2016 Summer-Fall crop, 26 selfed rice lines in the BC3F2 generation were grown in the field of CuuLong Delta Rice Research Institute a Screening of salinity tolerance at seedling stage 15 Nine rice lines BC3F2-3, BC3F2-12 BC3F2-14, BC3F2-16, BC3F2-17, BC3F2-20, BC3F2-21, BC3F2-23, BC3F2-24 were tolerant to salinity at score 3-5 (moderatey tolerant to tolerant) b Identification of Saltol gene at rice lines by SSR molecular markers Genotyping of Saltol gene with markers, RM1287 and RM10694, 15 lines were selected, including BC3F2-5, BC3F2-14, BC3F2-16, BC3F2-17, BC3F2-20 and BC3F2-23 (homozygous Saltol) and BC3F2-3, BC3F2-4, BC3F2-12, BC3F2-13, BC3F2-18, BC3F2-19, BC3F2-21, BC3F2-24 and BC3F2-26 (heterozygous Saltol) c Analysis of agronomic traits Evaluate agronomic characteristics help to monitor the dissociation of the progenies compared with the original parents The results of agronomic evaluation showed that it was significantly improved compared to the father Pokkali, most of them were similar to mothers d Analysis of grain quality Grain length of most progenies significantly improved compared to Pokkali Four lines had low amylose content, below 20%, the iron content in grain of rice lines was medium to high in both brown and milled rice, the grain length was over mm Therefore, lines (including red rice lines (BC3F2-3, BC3F2-16) and white rice lines (BC3F2-17, BC3F2-26) were selected for further study 3.3.2.2 Results in the OM238/Pokkali//OM238 population Similar to the OM231/Pokkali//OM231 population, through evaluation of combination of phenotype and genotype for salinity tolerance of 30 lines of the OM238/Pokkali//OM238 population in BC3F2 generation, 15 lines were tolerant to salinity and carried Saltol gene Evaluation of agronomic characteristics and grain quality, elite rice lines with low amylose content (≤ 20%) and high iron content were BC3F2-3, BC3F2-4, BC3F2-6, BC3F2-20 and BC3F2-24 These lines would continue to be selected for BC3F3 16 generation 3.3.2.3 population Results in the OM5451/Pokkali//OM5451 Similarly, through the evaluation of salinity tolerance of 39 rice lines of the OM5451/Pokkali//OM5451 combination in BC3F2 generation, 18 rice lines with Saltol gene tolerant to salinity were found Based on results from evaluation of agronomic characteristics and grain quality, elite lines with low amylose content (≤ 20%) and high iron content in grain were BC3F2-1, BC3F2-5, BC3F2-8, BC3F2-9, BC3F2-12, BC3F2-17 These lines continued to be selected for BC3F3 generation 3.3.3 Breeding results of rice populations in the BC3F3 generation 3.3.3.1 Results in the OM231/Pokkali//OM231 population Similar to the BC2F3 generation, through the experiments of phenotyping and genotyping, among of 17 rice lines of the OM231/ Pokkali//OM231 population in the BC3F3 generation, elite lines were selected Then, combine with the evaluation of agronomic and grain quality characteristics, salinity tolerant rice lines, BC3F3-5 and BC3F3-11 were selected, with good agronomic characteristics and grain quality (low amylose content (≤ 20%), high iron content and less chalkiness) 3.3.3.2 Results in the OM238/Pokkali//OM238 population Three elite rice lines (BC3F3-22, BC3F3-24 and BC3F3-33) were selected as good agronomic characteristics and grain quality (low amylose content (≤ 20%), high iron content and less chalkiness) 3.3.3.3 Results in the OM5451/Pokkali//OM5451 population Two elite rice lines (BC3F3-42 and BC3F3-53) were selected as good agronomic characteristics and grain quality (low amylose content (≤ 20%), high iron content and less chalkiness) 17 3.4 FIELD TRIAL OF THE ELITE RICE LINES TOLERANT TO SALINITY AND HIGH RICE GRAIN QUALITY AT ECOLOGICAL REGIONS IN THE 2017 SUMMER-FALL CROP In the BC3F4 generation, 28 rice lines of backcrossing populations were used for experiments 3.4.1 Evaluation of salinity tolerance of the BC3F4 rice lines Figure 3.2 PCR products of Saltol gene in BC3F4 rice lines of backcrossing populations with RM1287 (a) and RM10694 (b) markers on the 2.5% agarose gel Notes: L1-ladder 25 bp; L2-ladder 100 bp; P1-Pokkali; P2-IR29; P3-OM231; P4-OM238; P5-OM5451; 1-28: BC3F4 offsprings Through genotyping of Saltol gene in 28 of BC3F4 progenies of backcross populations, OM231/Pokkali//OM231, OM238/Pokkali//OM238 and OM5451/Pokkali//OM5451 showed that all of individuals presented Saltol gene and were tolerant to salinity (4‰) 18 3.4.2 Evaluation of agronomic traits and grain yield In alluvial soils, not affected by salinity as in the CuuLong Delta Rice Institute, the offspring had a superior yield compared to their parents The yield of rice lines ranged from 6-7 tons/ha In Kien Giang, in terms of actual yield, in 24 of crossbred lines, lines were not high at grain yield and in the same group with Pokkali, 11 lines got high yield and similar to their mothers and local variety “OM576” In Soc Trang, the actual yield of rice lines was lower than in Kien Giang Most of offspring lines are more than Pokkali at grain yield, in which, lines got high yield and were in the same group as the local control OM576 Thus, in terms of yield in the 2017 Summer-Autumn crop, the rice lines with high yield (over 5.5 tons/ha) include: BC3F4-5-1, BC3F4-5-2, BC3F4-22-1, BC3F4-22-3, BC3F4-24-3, BC3F4-33-1, BC3F4-42-2, BC3F4-42-5 and BC3F4-53-3 3.4.3 Evaluation of grain quality of rice lines at the 2017 Summer-Fall crop For amylose content, in of the backcrossing populations, 13/24 lines were with low amylose content in grain Fiugure 3.3 PCR products of Waxy gene in BC3F4 rice lines of BC populations with marker Wx-In1 on the 2.5% agarose gel 19 Notes: L-ladder 50 bp; A: Pokkali; B: OM231; C: OM238; D: OM5451; E: KDML105; 1-24: BC3F4 offsprings Results of identification of Waxy gene in BC3F4 offspring lines showed that lines carried the homozygous Waxy, lines caried the heterozygous Waxy, and lines were without Waxy gene These lines carried Waxy gene would be selected Among 13 rice lines had Waxy gene and low amylose content (≤ 20%), lines were recorded high iron content in grain These lines included BC3F4-5-1, BC3F4-5-3 (OM231/Pokkali//OM231); BC3F4-22-1, BC3F4-24-3 (OM238/Pokkali//OM238); BC3F4-42-5, BC3F4-53-1 (OM5451/Pokkali//OM5451) These six lines were also fair at grain-milling quality On the whole, through phenotyping with many traits and genotyping with Saltol and Waxy genes, elite rice lines were chosen These lines carried Saltol gene, salinity tolerance, high yield (5 - tons/ha in the Summer-Fall crop), low amylose content (≤ 20%), high iron content in grain, and fair milling quality 3.5 FIELD TRIAL OF THE ELITE RICE LINES TOLERANT TO SALINITY AND HIGH RICE GRAIN QUALITY AT ECOLOGICAL REGIONS IN THE 2017-2018 WINTER-SPRING CROP In BC3F5 generation, 14 lines were evaluated and selected 4.5.1 Evaluation of salinity tolerance of the BC3F5 rice lines The BC3F5 rice lines were tolerant to salinity from score 3.00 to score 5.67, in which, lines were high tolerant (score 3.00 3.67), not significantly different from Pokkali Results of evaluation of gene related to salinity tolerance, all lines carried the Saltol gene 20 Figure 3.4 PCR products of Saltol gene in BC3F5 rice lines of backcrossing populations with RM1287 (a) and RM10694 (b) markers on the 2.5% agarose gel Notes: L1-ladder 25 bp; L2-ladder 100 bp; A-Pokkali; B-IR29; C-OM231; D-OM238; E-OM5451; 1-14: cá thể lai BC3F5 offsprings Combination of phenotypic and genotypic data for salinity tolerance, 10 BC3F5 lines were selected because they had the highest salinity tolerance These lines included BC3F5-5-1-1, BC3F5-5-3-1, BC3F5-5-3-2, BC3F5-22-1-1, BC3F5-22-1-2, BC3F522-1-3, BC3F5-24-3-1, BC3F5-24-3-2, BC3F5-24-3-3 and BC3F5-425-1 3.5.2 Evaluation of agronomic traits and grain yield Most BC3F5 lines were fair to good at agronomic characteristics 21 Table 3.3 Adaptability and stability index in offprings at the 20172018 Winter-Spring crop No Name of lines BC3F5-5-1-1 BC3F5-5-3-1 BC3F5-5-3-2 BC3F5-22-1-1 BC3F5-22-1-2 BC3F5-22-1-3 BC3F5-24-3-1 BC3F5-24-3-2 BC3F5-24-3-3 10 BC3F5-42-5-1 Pokkali (đ/c) OM231 (đ/c) OM238 (đ/c) OM5451 (đ/c) OM576 (đ/c) FL478 (đ/c) Average yield (tons/ha) 6.34 a 5.08 ab 5.54 ab 5.72 a 5.78 a 6.07 a 5.23 ab 5.24 ab 5.13 ab 5.83 a 3.64 c 5.43 ab 5.82 a 5.94 a 6.16 a 4.24 bc Stability index (Sdi 2) Adaptability index (bi) 0.034 1.387 -0.098 -0.037 0.244 0.042 0.964 0.919 -0.085 0.170 0.071 0.338 -0.099 0.191 0.264 -0.093 0.990 1.007 1.068 1.020 1.139 1.066 1.005 0.919 1.244 1.167 0.687 0.659 1.060 1.083 0.813 1.073 Notes: đ/c: control; deviation from regression (Sdi 2); the slope value (bi) For average yield, among experimental sites, rice lines, BC3F5-5-1-1 and BC3F5-22-1-3 were the highest yield (over tons/ha) Other lines got yield from 5.08 – 5.83 tons/ha For adaptability and stability of offsprings, Sdi value goes to zero, varietiy is more stable Through evaluating, almost of offsprings were stable Rice lines BC3F5-5-1-1, BC3F5-5-3-2, BC3F5-22-1-1, BC3F5-22-1-3 were wide adaptation (bi~1); lines BC3F5-22-1-2, BC3F5-24-3-3, BC3F5-42-5-1 were advantage adaptation (bi>1) 3.5.4 Analysis of grain quality 3.5.4.1 Amylose and iron content in grain Amylose content of the BC3F5 offsprings was recorded 22 similar to less than 20% in all of three populations Ten rice lines selected were genotyped for Waxy gene with marker Wx-In1 7/10 lines were considered carrying the target gene, including BC3F5-5-1-1, BC3F5-22-1-1, BC3F5-22-1-2, BC3F522-1-3, BC3F5-24-3-2, BC3F5-24-3-3 and BC3F5-42-5-1 The rest lines also carried the heterozygous Waxy Figure 3.5 PCR products of Waxy gene in BC3F5 rice lines of BC populations with marker Wx-In1 on the 2.5% agarose gel Notes: L-ladder; A: Pokkali; B: KDML105; C: OM238; D: OM5451; E: OM231; 1-10: BC3F5 offsprings In the above lines with low amylose content, there lines were high at iron content in grain, such as BC3F5-5-1-1 (Febrow = 16.15 mg/kg, Femilled = 6.09 mg/kg), BC3F5-22-1-1 (Febrow = 15.85 mg/kg, Femilled = 6.02 mg/kg) and BC3F5-22-1-3 (Febrow = 14.70 mg/kg, Femilled = 5.39 mg/kg) 3.5.4.2 Analysis of milling quality Three lines, BC3F5-5-1-1, BC3F5-22-1-1 and BC3F5-22-1-3, had grain length at over mm This length was significantly improved compared to the father Pokkali Seed color was white Seeds were no awn Lines had high head rice grain (over 50%) and less chalkiness (2 - 4%) Thus, all of three lines (BC3F5-5-1-1, BC3F5-22-1-1 and BC3F5-22-1-3) were selected and developed 23 Chapter 4: CONCLUSIONS AND RECOMMENDATIONS The study created salinity-tolerant rice varieties with high iron and low amylose content in grain with the following results: Through evaluation of 36 rice varieties/lines from the Department of Biotechnology at CuuLong Delta Rice Institute, varieties were selected as mother plant (♀, recurrent individual) because they had high iron and low amylose content, including OM238, OM5451, OM121 and OM231, and Pokkali was father plant (♂, donor individual) because of high salinity tolerance a Evaluation results of offsprings from F1 to BC3F3 generations: The OM231/Pokkali//OM231 population: Two lines, BC3F35 and BC3F3-11, were good at agronomic traits and grain quality, amylose content was low (
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