Research and application of molecule markers in breeding salt-Tolerant Bacthom 7 rice variety

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES DONG THI KIM CUC “Research and application of molecule markers in breeding salt-tolerant Bacthom 7 rice variety” Major: Genetics and Breeding Code: 62.62.01.11 DOCTORAL THESIS SUMMARY OF AGRICULTURE HaNoi - 2014 The Doctoral thesis was completed in: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Supervisos: 1. Assoc.Prof. Le Huy ham 2. Dr. Le Hung Linh Objection1: Objection2: Objection3: The Doctoral thesis is defelded at Institute Committee of PhD Dissertation Examination: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES At……day…….month …………..2014 PhD thesis can be found at: - Nationa Library of VietNam. - Library of VietNam Academy of Agricultural Sciences 1 INTRODUCTION 1. Imperativeness of the thesis Rice production and yield are significantly losses due to diseases and pests infestation and the environmental impacts. Of these, the noticeable factor is salt-influenced to rice. The cultivated lands have been affected from the salinity by 380 million ha, accounted for 1/3 total worldwide cultivated areas. Salt – affected lands is the main factor which has curbed to develop productivity of rice, and also caused influence to food security in general. Hence, to reduce the salinity affect to the rice plant has paid much attention to research [124]. To meet this demand, to generate the salinity tolerance rice variety is necessary work. It is needed to explore the natural plant resource against salinity tolerance by either directly selecting or by genetic selection, or marker assisted selection. Application of molecular markers may help to identify the present of salinity tolerance gene(s), which is very useful for the breeder to select the effective crossed combination. Hence, It would be accelerated to breed rice salinity tolerance, shortened the selection time, and expenses and labour. As aforementioned, the topic entitled “Application the molecular markers to improve salinity tolerance of Bac Thom 7” 2. Objectives 2.1. General objectives Study on evaluating and developing some salinity tolerance of rice which derived from the the IRRI and India, and some grown rice varieties in Vietnam were used in this thesis. Applying marker assisted backcrossing to improve salinity tolerance of rice which are adapted for the Red River Delta. 2.2. Specific Objectives Identifying the salinity tolerance and phenotype of the rice lines which carrying the Saltol locus (donor plant), imported from International Rice Research Institute, as well as selecting the polularly grown rice cultivar were used as the receipient plant Applying marker assisted backcrossing (MABC) to pyramid salinity tolerance Saltol locus into Bac Thom 7, in order to generate the high quality rice variety with salinity tolerance for growing the coastal areas in Red River Delta 3. Scientific and Practical Significance 3.1. Scientific Significance 2 Based on the successful archivements of application of marker assisted backcrossing to transfer salinity tolerance QTL into other rice varieties will be widely applied for rice breeding to cope with climate change in the foreseeable future. Application of molecular breeding to combine with the traditional breeding to accelerate and identify the salinity tolerance rice materials, to pyramid it into Bac Thom 7 which could help to overcome the constrains of traditional breeding, especially with the heterzygote salinity tolerance QTL, reducing the cost of experiments, shortening the time and rapidly applying in practice. 3.2. Practical Significance The success of Saltol transfering into Bac Thom 7 based on molecular breeding will be widely applied for rice molecular breeding. The improved Saltol salinity tolerance of Bac Thom 7 lines would be selected and grown in a larger scale, especially for the coastal areas in the north of Viet Nam, where the most adversed influence from the climate change are. The most other significance of the current thesis was to simultaneously develop the salinity tolerance line/variety with the highest genetic background of the Bac Thom 7 and carried the Saltol QTL. The newly improved lines would grow well in the salt areas. 4. Plant materials and the Scope study of thesis 4.1. Plant materials to study The inbred rice varieties carrying the salinity tolerance (Saltol) which were imported from the IRRI, and the inbred rice varieties are popularly grown in Vietnam, as well as using the related molecular markers in the current study. 4.2. Places and time to conduct experiments The experiments were carried out at the Molecular Biology Division, Agricultural Genetics Institute, (Tu Liem, Hanoi); and the Center of Technological Exchange and Extension (Thanh Tri, Hanoi), and Giao Thuy, Nam Dinh Province. Time period: From 2010 to 2013 5. Significant Contribution Application of molecular assisted backcrossing (MABC) is one of the initial research to improve Bac Thom 7 with salinity tolerance for growing in the coastal areas of Red River Delta. Applying MABC method which can be transferred the target gen/QTL in the other variety via 2-3 generations, while, traditional backcrossing has needed about 8 breeding generations 3 Application of molecular assisted backcrossing could pyramid the saltol into Bac thom 7 which also has carried the enough desire traits of Bac Thom 7, but can be grown in the salinity affected areas upto 6 ‰ 6. Structure of the Thesis The current thesis was presented by 159 pages, of which included 25 Tables and 31 Fingures, and separated into 4 chapters: Chapter I: An overview (50 pages), Chapter II: Materials and Methods (15 pages); Chapter III: Results and Discussion (88 pages); Chapter IV: Conclusion and Suggestions (2 pages). One hundred and ten literature references were used to cite for this thesis, in which there are 23 Vietnamese references and 89 English reference and 16 link webpage were also used. 4 CHAPTER I OVERVIEW AND SCIENTIFIC BACKGROUND 1.1. The adversed impacts from climate change to worldwide agricultural production and Vietnam 1.1.1. Adversed effects from climate change to worldwide agricultural production According to the report of FAO (2010), over 800 million ha of cultivated areas have been affected by salinity and 20%, approximately 45 million ha, have been also affected due to salinity penetration at different levels [38]. In Asia, if sea level rise will be at 1m, approximately 10.000 km2 cultivated and fishery areas will be influenced and become the salinity swarmp. 1.1.2. The effects of climate change to agricultural production in Vietnam Vietnam is among the most influenced by sea level rise. The scientists reported that when the sea level rise, some cultivated areas in Cuulong delta and Red River delta and some other coastal delta will be inundated by sea water, and the sea level rise will be more increase, the most effected areas will be Red River delta and Cuulong delta. 1.2. Salt affected land and salt affected areas in Vietnam 1.2.1. Salt affected land The land consists of 50-60% ratio of argillaceous. The land shows high tight level and poor absorbent level, tought patter, and chapped and difficult to do tillage. Because the salt land is composing of much Na+ under the NaCl dissovel, thus, the pressure of Na2SO4 endosmosis is so high that can be influenced to water and nutrition absobtion of the plant. Also, neutral and alkali in salt land are causing low activation of the microoganism 1.2.2. General introduction of the rice field affected salinity in Vietnam Accoring to Hoang Kim and Bien and Howeler (2003), in Vietnam, there are two larger rice field affected salt is: Red River Delta included some sub-area such as Thai Binh, Hai Phong, Nam Dinh, Ninh Binh, while approximately 1.8 to 2.1 millio ha of land have been affected by salinity where are located in Ca Mau, Bac Lieu, Ben Tre, Kien Gian, Tien Giang, Tra Vinh and Soc Trang Provinces. Most of cultivated land areas have been affected salinity and alum and flooding [4]. 1.3. Genetic reseaches on salinity tolerance of rice 1.3.1. Mechanism of salinity tolerance of rice • Phenomenom of salt prevention • Phenomenom of leaf to leaf patition • Phenomenom of re-absorbance • Tolerance ability by the tissues 5 • Moving from root to bud •Dillution influence 1.3.2. Genetics of Salinity tolerance 1.3.2.1. Reseach on genetics of quantitative traits of salinity tolerance According to Mishra et al (1998), the trait of salinity tolerance in plant is polygenic trait, negligible causing effect from the parental plant (recipient plant) because these genes are not located in the cytoplasm [72]. During development stage of rice, the plant height, and rice production under salt affection are controlled by the additative genes (Mishra et al 1990)[73]. 1.3.2.2. Research on salinity tolerance at molecular level Based on the QTL mapping, salinity tolerance is controlled by multi-genes. Some markers such as AFLP and STS have been used, the major gene has been identified and located on the chromosome 1 and named as Saltol. The QTL (quantitative trait loci) mapping has been applied in case of the target gen has been controlled by many genes (example as salinity tolerance trait) 1.3.3. Expression of salinity tolerance of gene Based on the morphological, physiological and biochemical characteristics of the experiment, we have observed the present of Saltol-rice varieties and the sensitive rice varieties (without Saltol) 1.4. Molecular markers and their application 1.4.1. Molecular marker Molecular markers (or DNA markers) are polymorphic markers. They are included the molecular flow of DNA or the sequence information which are available and transiting in the database or internet (for example, sequence of primers SSR, STS, RAPD, AFLP...). 1.4.2. Some popular markers-use  RFLP marker ((Restriction Fragment Length Polymorphism)  RAPD, AFLP, STS marker  SSR (Simple sequenced repeat) 1.4.3. Some applications of molecular markers 1.4.3.1. Study on genetic diversity Research on genetic diversity is very important to help to evaluate the plant germplasm, animals and use them more efficiency. Especially, it is possible to estimate the hererosis between the parents (the pairs of parents carrying higher the genetic distance which could obtain more the heterosis properly. 1.4.3.2. Studying on genetic mapping Application of molecular markers in QTL linkage mapping, has been applied the statistical analysis which could identify the linkage between the markers and gen loci (Quantitative trait loci). QTL mapping included 6 the architecture of genome mapping which could be useful to search for relationship between the traits and the polymorphic markers, and provide the close distance between QTL and markers, respectively. 1.4.3.3. Studying on plant breeding Along with the advanced development of molecular breeding technologies, the breeders have paid more attention to the issue as Marker assisted selection (MAS), has implied to use the markers that have linkage with the interested QTL/genes in plant breeding programe 1.4.4. Application of Marker assisted backcrossing (MABC) MABCis an a practical and efficient technique in transferring the interested QTL/gene into the elite rice variety to generate the improved rice variety in a short time carrying the desire QTL/gene and attain approximately 100% genetic background of the elite rice variety: The breeding programe may only implement at the BC3 or even thought in BC2 generation, respectively. 1.5. Some archievements in improving rice salinity tolerance 1.5.1. Some results and archivements in research on rice salinity tolerance in the world During the year 1977 to 1980, International Rice Research Institute (IRRI), was successfully selected the good rice salinity tolerance such as IR42, IR4432-28-5, IR4595-4-1, IR463-22-2, and IR9884-54-3 with the yield at 3,6 tones/ha. Gregorio et al (2002)[45] developed TCCP226-2-49-B-B- 3 rice cultivar with high salinity tolerance ability. Some local rice varieties which were derived in the East Asia have often been high salinity tolerance such as Nona Bokra (India), Pokkali (Sri Lanka), Getu (India), SR26B, Damodar, Cheriviruppu, Pat and Solla (India), Ketumbar (Indonesia), Khao Seetha (Thailand). Some rice varieties were in the template (subtropical countries) such as Harra (Spanish), Agami (Egypt), and Daeyabyeo (Korea). Several Japonica rice varieties such as Moroberekan have high salinity tolerance, which were origined in the affected salt areas. This variety has been researched and used as the donor plant (salinity tolerance) and population mapping (Kim et al, 2009)[55]. The rice varieties were Oryza glaberrima, which are mostly grown in the West African show lower salinity tolerance ability to compare with the rice varieties (Oryza sativa) (Awala và cs, 2010)[2]. Recently, in 2013, researchers in IRRI have successfully developed the high-super salt tolerance that could be very useful for the farmers to grow this rice cultivar in the affected salt areas such as the coastal areas. 1.5.2. Application of molecular markers in improving salinity tolerance of rice The fine mapping of Saltol QTL was made on the chromosome 1 by the researcher groups (Gregorio 1997; Bonille et al 2002; and Niones 2004) which explained about 40-65% salinity 7 tolerance in rice [44][28][85]. Mohammadi – Nejad et al (2010) used 33 SSR polymorphic markers on the chromosome 1 “Saltol QTL” in order to identify the linkage and the utinity of the markers for rice breeding [76]. 1.5.3. Some results and archivements of research on salinity tolerance of rice in Vietnam 1.5.3.1. Use of the SSR markers which have tightly linked to Saltol QTL in rice breeding Application of molecular markers and anther culture to improve salinity tolerance of rice were conducted, total 72 rice lines were generated by anther culture (Lang et al, 2008). Also, Buu et al (2000) used 30 SSR markers to map the salinity tolerance trait in the F3 generation that including 257 segregation population from the crossed between IR28/Đoc Phung. 1.5.3.2. Improving the rice salinity tolerance Đo Huu At (2005) made mutation by Coban (Co 60) to generate CM1, CM5, ... [1]. Also, Dang Minh Tam et al (2003), reported that 10 rice line were developed from the local and high yield rice cultivars, shown a medium salt tolerance (3-5 point). Moreover, they showed high regenerative percentage in the NaCl culture at 1,0 and 1,5% [35]. Ngo Dinh Thuc (2006) applied the anther culture technique to create 8 soma variation liné from the OM576, IR64, Basmati and VD20 which could withstand salinity tolerace at 5 level at the test of rice seeding with EC = 12 dS/m [19]. 1.5.3.3. Screening salinity tolerance of rice From 1992 – 1995, Institute of Southeast Agriculture and Science reported that 14 potential rice cultivars involving salinity tolerance were selected as the following: Nep ao Gia; Trang Diep; Mong Chim; Mong Chim Roi; and Nep Bo Rieng [8]. Also, Cuulong Rice Research Institute has reported to attain 30 rice lines with promising in salinity tolerance as from 2000 to present. The Field Crops Research documented that M6 is a salinity tolerance which obtained from the crossed Bau Hai Phong/1548 during the year of 2001-2005. 8 CHAPTER III MATERIALS, CONTENTS AND METHODS 2.1. Materials The rice materials included:  Total 14 rice lines/varieties carrying Saltol QTL salinity tolerance were imported from IRRI and some popularly grown rice varieties in Red River Delta  Chemical argents and research facilities: SSR markers used: 447 markers Research instruments: Experimental tools of Agricultural Genetics Institute 2.2. Areas to conduct experiments The Laboratory of Molecular Biology Division-Agricultural Genetics Insititute. Tuliem-Hanoi The Net house and paddy fields conducted for experiment at the Center of Technology Exchange and Extension, Vinh Quynh, Thanh Tri, Hanoi Experiments for evaluation of growth and development of the imported rice lines/varieties were conducted in two provinces: Nam Dinh and Hanoi Perious to implement: From 2010 to 2013 2.3. Contents 2.3.1. Content 1: Research, evaluation of the salinity tolerance and agronomical traits, rice yield and some sub-traits involving in rice yield of the Salton-lines/varieties imported from IRRI and some popularly grown in the Red River Delta. It has been an important for further research on rice salinity tolerance for the coastal areas in North Vietnam 2.3.2. Content 2: Application of marker assisted backcrossing to transfer the Saltol QTL into Bacthom 7, an elite rice cultivar 2.3.3. Content 3: Evaluating some main agronomical traits and several components involving in rice yield traits, salinity tolerance level, rice quality of the lines carrying Saltol QTL in the net house and the paddy field test 2.4. Methods * MABC (Marker Assisted Backcrossing) to improve salinity tolerance of rice 2.4.1. Methods to conduct field test 2.4.2. Methods for evaluation of salinity tolerance of rice: Screening in the artificial conditions. 2.4.3. Methods to implement experiments in the laboratory 9 2.5. Field test of the improved rice varieties 2.6. Statistical Analyses Field experiements (observation and evaluation…) were analysised by IRRISTAT 5.0; Cropstat7.2; Statistic 8.2, Excel 2007. Technical of data analysis in laboratory was carried out following the Graphical genotypes 2 (GGT2.0) and the other stasistical programes. Evaluating the parental materials was followed IRRI methods IRRI and Suprihatno, 1980. All data were documented in Excel and analysised by Graphical Genotyper (Van Berloo, 2008). Each SSR and alen relationship were recorded as homozygous to the recipient plant is “A” and homozyous to donor plant is “B” and heterzyous is “H”, respectively. CHAPTER III RESULTS AND DISCUSSION 3.1. Evaluation of the initial rice plant materials for improving salinity tolerance in rice 3.1.1. Evaluating salinity tolerance level of the rice lines/varieties in the artificial condition The results of screening the rice cultivars with salinity tolerance. 6g/l NaCl was added in the Yoshida as shown in the Table 3.1. Table 3.1. Artificial screening for salinity tolerance of rice varieties after 2 weeks with 6g/l NaCl (EC=12dS/m) No Line/variety Effect after 2 weeks treated NaCl 6‰ Effect after 3 weeks treated NaCl 6‰ Rep 1 Rep 2 Rep 3 Aver Rep 1 Rep 2 Rep 3 Aver 1 IR72046-B-R-8-3-1-3 3 5 3 3.7 7 5 5 5.7 2 IR52713-2B-8-2B-1-2 3 3 3 3.0 7 5 7 6.3 3 IR77674-3B-8-2-2-AJY5 3 3 3 3.0 5 5 7 5.7 4 NSIC Rc 106 3 3 5 3.7 7 7 7 7.0 5 IR45427-2B-2-2B-1-1 3 5 3 3.7 7 7 5 6.3 6 IR55179-3B-11-3 3 3 3 3.0 7 5 7 6.3 7 IR65196-3B-5-2-2 5 5 3 4.3 7 7 7 7.0 8 IR74099-3R-3-3 3 3 3 3.0 7 5 5 5.7 9 IR 4630-22-2-5-1-3 3 5 3 3.7 5 7 7 6.3 10 FL478 1 1 3 1.7 3 3 3 3.0 11 Bac thom 7 5 7 7 6.3 9 7 9 8.3 12 Khang dan 18 7 7 7 7.0 9 9 9 9.0 13 Pokkali (salinity tolerance ) 1 1 3 1.7 3 1 3 2.3 10 14 IR29 (Sensitive) 7 9 7 7.7 9 9 9 9.0 3.1.2. Evaluation of the growth and development of the imported rice varieties in the natural condition 3.1.2.1. Results of evaluating the ability of growth and development of some imported rice varieties at Thanh Tri, Hanoi, 2010 Table 3.3. Agronomical traits and morphological of the rice varieties used in the study at Thanh Tri, Hanoi - 2010 No Line/variety Days to heading (day) Plant height (cm) Panicle length (cm) Spring Summer Spring Summer Spring Summer 1 IR72046-B-R-8-3-1-3 139 115 96.0 e 96.3 h 24.2 ab 23.0 cd 2 IR52713-2B-8-2B-1-2 127 115 109.0 c 110.0 c 24.5 a 25.0 a 3 IR77674-3B-8-2-2-AJY5 155 130 109.0 c 110.0 c 24.0 ab 24.0 b 4 NSIC Rc 106 136 105 92.3 g 92.3 j 22.7 bc 23.0 d 5 IR45427-2B-2-2B-1-1 150 120 92.5 g 94.0 i 23.5 bc 22.3 e 6 IR55179-3B-11-3 145 120 113.0 b 115.3 b 23.7 c 24.0 b 7 IR65196-3B-5-2-2 145 130 115.3 a 115.7 b 22.7 d 24.0 b 8 IR74099-3R-3-3 135 120 94.3 f 98.0 g 24.3 ab 23.3 cd 9 IR 4630-22-2-5-1-3 142 115 113.0 b 106.3 e 21.3 d 20.3 f 10 FL478 135 120 103.3 d 102.3 f 20.3 e 20.7 f 11 Pokkali - 135 - 182.7a - 23.7bc 12 Bac thom 7(control) 135 125 112.0 b 107.3 d 22.0 d 21.7 e CV (%) 0.47 0.46 2.04 1.63 LSD0.05 0.84 0.87 0.8 0.63 3.1.2.2. Results of evaluation of the growth and development of some imported rice varieties grown in Gia Thuy, Nam Dinh in 2010 Table 3.5. Several agronomical traits and morphology of the rice varities grown at Giao Thuy, Nam Dinh Province in 2010 No Line/variety Days to heading (day) Plant height (cm) Panicle length (cm) Spring Summer Spring Summer Spring Summer 1 IR72046-B-R-8-3-1-3 135 120 98.3 ef 96.0 efg 24.3 ab 23.3 bc 2 IR52713-2B-8-2B-1-2 128 110 111.0 cd 110.7 bc 24.3 ab 25.7 a 3 IR77674-3B-8-2-2-AJY5 160 134 110.7 cd 105.0 cde 23.3 abc 23.0 bcd 4 NSIC Rc 106 140 110 95.0 f 87.7 g 22.3 bcd 22.7 cd 5 IR45427-2B-2-2B-1-1 152 125 94.0 f 92.0 fg 23.3 abc 22.3 cd 11 6 IR55179-3B-11-3 142 130 113.3 bc 113.7 bc 24.0 abc 24.3 ab 7 IR65196-3B-5-2-2 142 125 119.0 b 115.0 b 23.3 abc 23.0 bcd 8 IR74099-3R-3-3 140 115 98.7 ef 92.3 fg 25.3 a 22.7 cd 9 IR 4630-22-2-5-1-3 140 112 116.3 bc 108.0 bcd 21.7 cd 19.8 f 10 FL478 132 115 105.0 de 99.0 def 20.7 d 20.7 ef 11 Pokkali - 140 - 188.7a - 25.3a 12 Bac thom 7(control) 135 120 111.0 cd 115.0 b 21.7 cd 22.0 cde CV (%) 4.17 5.14 6.08 3.67 LSD 0,05 7.88 9.54 2.38 1.41 3.2. Application of Marker assisted backcrossing to improve salinity tolerance of Bac Thom 7 3.2.1. Results of identification of parental plants to improve QTL Saltol rice line In order to improve salinity tolerance of rice varieties grown in the Red River delta, we have used Marker assisted backcrossing method to transfer QTL Saltol into the receipient plant, but attaining its agronomical traits such as quality of rice. Based on the obtained results, the Bac Thom 7 is the variety that need to be improve salinity trait and used as the receipient Saltol material. 3.2.2. Resukts of applying Marker assisted backcrossing to pyramid QTL saltol into BT7 3.2.2.1. Identification of the markers linked with Saltol and polymorphic markers between BT7 and FL478 In this study, total 30 markers at the target gene of Saltol were used to identify the linkage markers between the donor and receipient plants. Fifteen polymorphic markers between the parental plants at the target gen were AP3206, RM3412b, RM10748, RM493, RM140, RM10825. G1a, G6a, G11a, Salt 4a, SCK1b, SCK1d, SCK2, SCK10, and SCK10a. The information of the polymorphic markers have shown in the Figure 3.2 and Figure 3.5 Figure 3.2. Polymophic markers between BT7 and FL478 with 3 markers as RM493, RM3421b and RM140 Note:: P1: Bacthom 7; P2: FL478 12 Figure 3.4. Position of QTL/gen Saltol located on the chromosome 1 3.2.2.2. The results of identification of polymorphic markers that were out of QTL Saltol region between BT7 and FL478 on the 12 chromosomes To identify the polymorphic markers which were located out of region of Saltol on the 12 chromosomes for determining the genetic background of the selected individual plants from the crossed population. Total 447 SSR markers were used to screen to find out the polymorphic markers, 102 polymophic markers were identified (accounted for 21,38%) between Bacthom 7 and FL478 Figure 3.7. Results of the polymorphic markers between BT7 and FL478 Note:: P1: Bac Thom 7; P2: FL478 13 Figure 3.8. The map of polymorphic markers between FL478 and BT 7 on the 12 chromosomes Note: The order of markers presents on the left of the chromosomes, the position of polymorphic markers were on the right of the chromoshomes. The black regions present the Saltol locus. The order and position of the markers were established on the Nipponbare map (TIGR v. 3 pseudomolecules available at www.gramene.org and atsliver.plbr.cornell.edu/SSR). 3.2.3. Results of improving salinity tolerance of BT7 by marker assisted backcrossing 3.2.3.1. Developing F1 from the crossed combination FL478/BT7 In this experiment, the polymorphic marker RM7643 to screen the individual plant F1. The result showed that 17/20 individual plants of F1 were heterozygote (H). Figure 3.10. Result of electrophoresis of RM7643 marker BT: Bac thom 7; FL: FL478; A: BT7; H: heterozygote, 1-20: the individual plants of F1 After selected 17 individual plants were heterozygote between FL478 and Bac thom 7, the backcross was conducted with BT7 to develop BC1F1. 3.2.3.2. Results of selecting the individual plants in the population of BC1F1 by molecular markers To identify the individual plants which were carrying the target gene Saltol in the crossed off- spring, it was identified 15 markers AP3206, RM3412b, RM10748, RM493, RM140, RM10825, G1a, G6a, G11a, Salt 4a, SCK1b, SCK1d, SCK2, SCK10, and SCK10a which were shown linked with Saltol and polymorphism between BT7 and FL478. Also, in this experiment, our results were 2 flanked markers RM493 and RM3412b which were closely linked with Saltol as the successful use of IRRI to select the individual plants carrying Saltol. Figure 3.10. Results of electrophothesis on 94 individual plants of BC1F1( RM493 markers). From 1-94 the individual plants BC1F1, BT7: Bac Thom 7, FL: FL478 A: Bac Thom 7, B:FL478, H: Heterozygote 14 Figure 3.11. Results of electrophothesis on 94 individual plants of BC1F1 (RM3412b markers). From 1-94 the individual plants BC1F1, BT7: Bac thom 7,FL: FL478 A: BT7, B:FL478, H: Heterozygote Combination of 2 markers namely RM493 and RM3412b, 14 individual plants have been screened to carry Saltol as the plant number: 5, 10, 11, 14, 19, 28, 29, 32, 36, 42, 45, 50, 71, 83. 3.2.3.3. Results of selecting the individual plants in the BC2F1 population by applying molecular markers -Results of selection of the individual plants carrying locus Saltol in the BC2F1 population In this experiment, the successfully crossed 141 individual plants in BC2F1. To identify the individual plants carrying Saltol in the population of BC2F1, two closely linked markers with Saltol as RM493 and RM3412b were consecutively used to select the individual plants that carrying the target gen. The results of selection of the individual plants carrying Saltol were shown in the Figure 3.13 and 3.14. Figure 3.13. Results of electrophoresis of 141 individual plants from BC2F1 (RM3412b) From 1-141, the individual plants BC2 F1- BT7:BT 7; FL: FL478; A: BT 7; B:FL478; H: Heterogyzote Figure 3.14. Results of electrophoresis of 141 individual plants from BC2F1 (RM493) From 1-141, the individual plants BC2 F1, BT7: BT 7,FL: FL478 A: BT 7, B:FL478, H:Heterogyzote The results of selection of the individual plants which have been carrying the target gen by applying markers RM3412 and RM493, we have selected 34 individual plants as the plant number: 1, 2, 7, 9, 11, 13, 15, 22, 23, 24, 30, 34, 36, 42, 45, 47, 51, 53, 57, 59, 60, 65, 74, 77, 81, 92, 93, 94, 96, 112, 114, 117, 136, 141. - Evaluation of the background of the individual plants carrying Saltol in the BC2F1 population Results of identifying the individual plants carrying Saltol and attained the maximum genetic 15 background of BT7 in the BC2F1, total 43 polymophic markers which have not linked in the regions of Saltol on the chromosomes to select background of the receipient plant. Figure 3.20. Statistical analysis of GGT2 for 10 individual plants carrying Saltol trong in the population of BC2F1 Figure 3.21. Statistical analysis of GGT2 for 10 individual plants on the 12 chromosomes As shown in Figure 3.20 and Figure 3.21, it was identified the plant number 8 (similar with the plant number 57 in the population BC2F1) that has the highest genetic background upto 80.7% Figure 3.22. Genetic map of the plant number 8 to analyse by GGT2 software 3.2.3.4. Results of selecting the individual plants in the population of BC3F1 by applying molecular markers To identify the individual plants carrying the targeted gene in the population of BC3F1, 2 previous markers RM493 and RM3412b have been used to select the individual plants carrying Saltol. 16 Figure 3.23. Results of electrophoresis for 369 individual plants of BC3F1(RM3412b) From 1-369 the individual plants BC3 F1, BT7: BT 7,FL: FL478 A: BT 7, B:FL478, H: Heterogyzote. Figure 3.24. Results of electrophoresis of 369 individual plants from BC3F1 (RM493) To identify the individual plants carrying Saltol in the population of BC3F1 by using 2 markers RM493 and RM3412, total 115 individual plants have obtained: 6, 7, 8, 10, 14, 16, 18, 22, 28, 29, 30, 32, 35, 36, 38, 41, 42, 45, 50, 54, 63, 64, 65, 70, 72, 73, 74, 75, 80, 82, 83, 84, 94, 101, 102, 109, 111, 112, 116, 122, 123, 135, 148, 157, 158, 166, 169, 174, 176, 178, 184, 188, 190, 192, 194, 197, 198, 200, 211, 215, 217, 218, 221, 234, 233, 237, 238, 246, 248, 254, 257, 259, 260, 263, 270, 273, 274, 275, 276, 277, 284, 289, 290, 293, 300, 302, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 317, 320, 324, 331, 332, 333, 335, 336, 344, 345, 351, 353, 357, 358, 359, 361, 366, 367. Among 115 individual plants carrying Saltol to identify by use of 2 markers RM493 and RM3412, 88 individual plants were to backcrossed as accounted from the individual plants number 8 (in the population of BC2F1). * Evaluation of the genetic background of the individual plants carrying Satol in the population of BC3F1 17 To identify the genetic background of the individual plants carrying Saltol in the population BC3F1, only 88 individual plants which were developed from the plant number 8 (genetic background 80,7% of BC2F1). Figure 3.29. Satistical analysis of genetic background of 88 individual plants from BC3F1 on 12 chromosomes by use of GGT1 As the result shown in Figures 3.30; 3.31, it was identified two individual plants number 30 and 32 which have had the highest genetic background 99,3% and 100% as the BT7. Figure 3.30. Genetic map of individual plant number 30 Figure 3.31. Genetic map of the individual plant number 32 Note that figures 3.30 and 3.31: The numberal chromosomes were expressed the below number, and the list of markers used to screen the genetic background was on the left side, equivalent with position of marker that was on the right of chromosome. Red region was indicated genetic 18 background of BT7 and marker position was established-based on the statistical analysis of GGT2.0 3.3. Evaluation of some main agronomical traits, component of yield and salinity tolerance of the improved rice lines carrying QTL/gen Saltol in the net house and paddy field 3.3.1. Results of evaluation of some agronomical traits and yield components of BT7-Saltol in the net house In the generation of BC3F1, 2 individual plants have been selected as the plant number IL-30 và IL-32 from the crossed combination of BC3F1 which attained the highest genetic background of BT7 as 99,3% and 100%, respectively. IL-32 was grown in the net house condition to develop BC2F2 for analysis and evaluation of its phenotype. Applying of MAS to select the individual plants to select 30 plants which have shown homogygote at locus markers RM1287, RM8094 which closely linked with QTL/gen Saltol (as the numbered from 1-30), each individual plant has been selfing to develop 30 different lines, to observe the growth and development, yield components to compare with BT7 as the control Table 3.17. Growth indicator and morphological characteristics of BT7- Saltol lines (BC3F2) in the Spring season crop 2012 at Thanh Tri, Hanoi No Name DTH (days) Plant height (cm) Panicle length (cm) Panicle exsertion (cm) Ear/panicle (ear) Color awn 1 IL32-1 135 112.9jk 20.6bcde 2.9ef 10.7hi Light yellow 2 IL32-2 135 111.2mn 20.7de 2.9bcdef 10.4i Light yellow 3 IL32-3 135 113.2hijk 20.5de 2.7f 10.7hi Light yellow 4 IL32-4 135 111.5lm 19.4f 2.6f 11.7cdefg Dark yellow 5 IL32-5 135 114.2efgh 20.6de 3.2a 12.1a Light yellow 6 IL32-6 135 109.5o 20.5de 3.5a 10.4hi Light yellow 7 IL32-7 135 117.9a 21.5bcd 2.8f 10.4hi Light yellow 8 IL32-8 135 113.2ghijk 20.4de 2.9cde 10.4hi Light yellow 9 IL32-9 135 116.9b 20.4de 3.3ab 10.7ghi Light yellow 10 IL32-10 135 111.5mn 29.7a 3.0abcde 11.7efghi Brown 11 IL32-11 135 113.2ijk 21.2ab 3.3ab 12.1ab Dark yellow 12 IL32-12 135 114.5ef 20.4de 3.3ab 11.1defghi Light yellow 13 IL32-13 135 115.2cd 20.4ef 3.0ab 10.7hi Light yellow 14 IL32-14 135 114.2ef 21.0bcde 3.0abc 11.1fghi Light yellow 15 IL32-15 135 113.5fghi 21.4bcde 3.3ab 12.1abc Dark yellow 16 IL32-16 135 111.9lm 21.1bcde 2.8cde 11.4abcdef Light yellow 17 IL32-17 135 108.9o 20.5de 2.6ef 12.1abc Light yellow 19 18 IL32-18 135 113.9hijk 20.5de 3.2abc 11.7bcdefg Light yellow 19 IL32-19 135 113.9ijk 20.7de 3.0ab 11.7abcde Light yellow 20 IL32-20 135 113.9hijk 21.9bc 3.1abcd 10.4hi Light yellow 21 IL32-21 135 113.2hijk 20.9cde 3.1abc 10.1hi Dark yellow 22 IL32-22 135 111.2n 21.9bcd 3.1ab 12.1ab Dark yellow 23 IL32-23 135 116.9ab 20.7de 3.4a 12.1abcd Light yellow 24 IL32-24 135 114.5de 20.9de 2.7f 11.1bcdefg Light yellow 25 IL32-25 135 117.2a 21.7bcd 3.4a 11.7defghi Brown 26 IL32-26 135 114.2efg 21.0bcd 3.2a 12.7abcd Brown 27 IL32-27 135 113.2hijk 20.2de 2.8def 11.7bcdefg Light yellow 28 IL32-28 135 112.9kl 21.5bcde 3.4a 12.1abcd Light yellow 29 IL32-29 135 116.2bc 19.8f 2.8ef 12.1abc Light yellow 30 IL32-30 135 113.2fghij 21.5bcde 2.8ef 10.4hi Brown BT7 (control) 135 116.2bc 21.2bcde 3.1abcde 11.6abcdef Light yellow CV (%) 0.48 2.3 9.82 4.13 LSD5% 0.89 0.79 0.49 0.77 3.3.2. Evaluation of salinity tolerance of BT7-Saltol line (BC3F3) in the artificial conditionKết Table 3.19. Evaluation of salinity tolerance (after 3 weeks) of some individual plants BT7 – Saltol (BC3 F3)- Summer crop in 2012 No Name No of plant survivor (plant) Percentage of plant suvivor sống (%) Point Evaluation 1 IL32-1 16.3 81.5 3-5 Tolerance 2 IL32-2 17.7 88.5 1-3 High tolerance 3 IL32-3 15.3 76.5 3-5 Tolerance 4 IL32-5 15.7 78.5 3-5 Tolerance 5 IL32-7 17.3 86.5 1-3 High tolerance 6 IL32-8 17.3 86.5 1-3 Tolerance 7 IL32-9 16.0 80.0 3-5 Tolerance 8 IL32-12 16.3 81.5 3 Tolerance 9 IL32-13 17.3 86.5 1-3 High tolerance 10 IL32-14 17.7 86.5 1-3 Tolerance 11 IL32-15 16.3 81.5 3 Tolerance 12 IL32-16 17.3 86.5 1-3 High tolerance 13 IL32-17 17.3 86.5 1-3 High tolerance 20 14 IL32-18 17.7 88.5 1-3 High tolerance 15 IL32-19 16.7 83.5 3 Tolerance 16 IL32-20 15.7 78.5 3-5 Tolerance 17 IL32-23 15.3 71.5 3-5 Tolerance 19 IL32-28 17.7 86.5 1-3 High tolerance 20 IL32-29 17.7 86.5 1-3 High tolerance BT7(control) 3.0 1.5 7-9 Tolerance FL478(cho gen) 17.0 85.0 1-3 High tolerance Pokkali (chuẩn kháng) 17.3 88.5 1-3 High tolerance Cv% 0.37 0.45 Thus, evaluation of the growth and development and salinity tolerance in the artificial condition of the individual plants of BT7 - Saltol showed that most of the carried QTL/gen Saltol that shown in the laboratory exhibited good growth and development to compare with the controlled variety. 3.3.3. Results of evaluation of some agronomical traits of the BT7- Saltol (BC3F3) under the paddy field. 21 Tabe 3.20. Indicators of some growth and development of some BT7–Saltol lines (BC3F3) in the summer crop in 2012 tại at Giao Thuy, Nam Dinh province No Name DTH (days) Leaf green level (point) Blade pubescence (điểm) Plant height (cm) Growth habit of plant (điểm) Panicle length (cm) Panicle exsertion (cm) Chiều dài lá đòng (cm) Awn color 1 IL32-1 112 5 5 109.2 3 22.5 5.4 33.5 Light yellow 2 IL32-2 112 5 3 111.8 3 21.0 7.7 29.1 Light yellow 3 IL32-3 112 3 3 107.9 3 20.8 5.3 27.8 Light yellow 4 IL32-5 112 5 3 106.7 5 22.1 5.3 32.0 Light yellow 5 IL32-7 112 5 5 105.3 3 20.9 6.8 28.6 Light yellow 6 IL32-8 112 3 5 106.0 5 22.3 9.3 33.6 Light yellow 7 IL32-9 112 3 1 110.4 3 21.3 14.0 27.8 Light yellow 8 IL32-12 112 7 3 104.3 3 21.0 6.0 28.0 Light yellow 9 IL32-13 112 5 3 110.3 5 22.0 6.3 28.0 Light yellow 10 IL32-14 112 5 5 104.4 1 22.1 7.6 30.0 Light yellow 11 IL32-15 112 5 5 98.7 3 21.2 6.0 30.1 Light yellow 12 IL32-16 112 7 3 107.9 3 22.8 6.8 31.4 Light yellow 13 IL32-17 112 5 3 105.6 1 22.1 8.0 33.8 Light yellow 14 IL32-18 112 3 3 101.0 1 21.0 4.8 25.8 Light yellow 15 IL32-19 112 7 1 101.9 3 23.0 5.4 31.4 Light yellow 16 IL32-20 112 5 5 104.2 3 23.2 6.0 28.6 Light yellow 17 IL32-23 112 5 3 100.1 1 23.5 4.4 26.4 Light yellow 19 IL32-28 112 3 3 97.2 3 19.8 5.2 23.5 Light yellow 20 IL32-29 112 5 5 99.9 3 20.6 5.0 25.9 Light yellow BT7 112 5 3 104.2 3 21.5 6.7 25.3 Light yellow Cv% 4.22 1.00 2.31 2.89 22 In terms of days to heading between the lines that shown the high similarity and were as the BT7 as the control variety is 112 days. The plant heigh was arranged from 97,2 to 111,8. The line number 2 showed the highest the height of plant, and the lowest was the line number 5 (97,2). The lines number 1, 7 and 9 have plant height that were higher than the control by 5-6 cm, the rest of those were similar with the BT7. However, if based on the variance with value of F-test > 0.05, it showed that the improved lines indicated no least significance at 95% as shown in Table 3.20. 3.4. Evaluation of some agronomical traits, yield components of the BT7- Saltol (BC3F4) in the field Amongst 20 lines of BT7- Saltol (BC3F3), after evaluating and checked salinity tolerance in the net house, and based on some agronomical traits, yield components, 8 promising lines were selected such as 1-3-4-9-10-12-13-15 which disclosed higher yield than the controlled BT7 but have similar agronomical traits of BT7 Table 3.22. Criteria of growth and morphological characteristics of BT7- Saltol (BC3F4) in Spring crop 2013, Giao Thuy, Nam Dinh province No Name DTH (days) Plant height (cm) Panicle length (cm) Panicle exsertion (cm) Flag leaf length (cm) BT7(control) 132 113.2 24.0a 3.9b 24.8d Line 1 IL32-1 132 112.8 22.3bc 6.0a 25.9b Line 3 IL32-3 132 113.0 21.9bc 4.6b 26.1a Line 4 IL32-4 132 113.5 21.5c 4.7b 23.9e Line 5 IL32-5 132 112.9 21.6bc 4.6b 25.4c Line 9 IL32-9 132 112.7 22.8b 5.9a 23.8e Line 10 IL32-10 132 114.2 21.7bc 4.7b 25.2c Line 12 IL32-12 132 114.5 24.1a 5.6a 22.4f Line 15 IL32-15 132 114.0 22.5b 5.7a 25.3c CV% 2.74 9.48 0.58 LSD0.05 1.06 0.43 0.25 As the results shown in Table 3.22, the potential BT7- Saltol lines showed days to heading that were similar with BT7 (132 days). The plant height of the lines were ranged about 112,7 cm (line number 9) to 114,5 cm (line number 12). Hence, there is no significant difference to compare with the BT7 as 113.2 cm. 23 3.5. Results of Rice quality of some BT7 – Saltol lines Table 3.24. Rice quality of BT 7 – Saltol lines Name Miling revovery (%) Head rice recovery (%) Translucent kernel (%) amyloza (%) Geltinization temperature Length grain (mm) Grain width (mm) (D/R) 1 line 69,02 86,87 57,65 15,72 Medium 5,57 2,04 2,73 5 line 69,00 89,22 55,85 17,52 Medium 5,70 1,98 2,88 12 line 68,74 87,28 57,03 17,00 Medium 5,52 1,94 2,84 BT 7 - (control) 68,96 87,54 56,22 16,02 Medium 5,61 1,97 2,85 Source: Department of Biochemical & Quality of agricultural farming –CLT&CTP, 2013. As the results shown in Table 3.24: 7- Saltol and BT 7 (control) revealed the similar good milling recovery (>65%), and head rice recovery >80%. Translucent kernel was measured by Kett machine after milling and devided into the following group: Good (>50%); medium (35-40%); rather good (40-50%). Therefore, both the above rice varieties showed good translucent kernel. Size and type of grain were rather lengthy (length/width 3,0). 24 CHAPTER IV CONCLUSIONS AND SUGGESTIONS 4.1. Conclusions -In the current study, some rice varieties with salinity tolerance and acceptable yield were evaluated (as FL478 and IR55179-3B-11-3 varieties). - FL478 was used as the donor (QTL/gen Saltol) which has some agronomical traits similar with some widely grown rice varieties in Red River Delta (50,2 ta/ha – 55,3 ta/ha), especially, its salinity tolerance showed at point 3, equivalent with Pokkali variety in the same experimented condition. - BT 7 was used as the recipient plant - 102 polymorphic markers were identified (accounting for 21,38 %) between BT7 and FL478 . - 94 polymorphic markers distributed on the 12 chromosomes were identified and use to select the carried Saltol gene that attained the highest genetic background of the recurrent parents in the backcrossed populations. - Applying marker assisted backcrossing to select the plants number IL30 and IL32 from the generation of BC3F1 had the maximum genetic background up to 99,3% and 100% of BT7 - BT7-Saltol (BC3F4) was developed by MAB which showed salinity tolerance at point 3, similar with salt tolerance ability of FL478. - Most of rice lines showed phenotypic characteristics similar with BT7 at 100% based on the criteria and carried QTL/gen Saltol with salinity tolerance at poit 3 as the Pokkali. - 8 potential BT7-Saltol (BC3F4) with acceptable yield and quality similar with BT7 were developed. 4.2. Suggestions The improved salinity tolerance lines should be developed as the commercial rice varieties. However, it is needed to screen them under the natural condition (field test) with different salt concentrations. Also, bio-physiological and bio-chemical of those rice lines should be further fully studie THE PUBLISHED ARTICLES IN RELATION TO THESIS 1. Le Hung Linh, Tran Dang Khanh, Nguyen Văn Luan, Dong Thi Kim Cuc, Le Duy Duc, Ta Hong Linh, Abdelbagi M Ismail, Le Huy Ham (2012); “Application of assisted backrossing to pyramid Salinity Tolerance (saltol) into rice cultiva BacThom7” (2012), VNU Journal of Sciences and Technology 28. P 87-99. 2. Le Hung Linh, Tran Dang Khanh, Nguyen Văn Luan, Dong Thi Kim Cuc, Ta Hong Linh, Abdelbagi M Ismail, Le Huy Ham (2012), “Application of Marker Assisted Backrossing (MABC) to improve Salinity and submergence tolerance in BacThom7” (2012), Sciences Med Vol3.No3. P251-258. 3. Le Hung Linh, Tran Dang Khanh, Nguyen Văn Luan, Dong Thi Kim Cuc, Ta Hong Linh, Abdelbagi M Ismail, Le Huy Ham (2012), “ Marker assisted backrossbreading to improve rice tolerance of salinity stress to cope with climate change in coastal areas of Vietnamese Deltas” (2012), 4. Đong Thi Kim Cuc, Le Hung Linh, Le Huy Ham (2013), “ Research and evaluation of plant materials to improve rice” (2013), Journal of agriculture and Rural Development. Vol 20.No 2 October. P9-18. 5. Đong Thi Kim Cuc, Le Hung Linh, Le Huy Ham, Nguyen Thi Hue (2013), “ Results of evaluating salinity tolerance of some BT7 lines by applying MABC” (2013), Journal of agriculture and Rural Development . Vol230. No 1 December. P3-13. 6. Ta Hong Linh, Le Hung Linh, Le Huy Ham, Dong Thi Kim Cuc, Tran Dang Khanh (2013), “Improving submergence tolerance of VietNamese rice cultivar by molecula breeding” (2013), Journal Plant Breeding and Genetics.01(03) 2013. 151- 162.