Source: LOUISIANA STATE UNIVERSITY submitted to NRP
PYRAMIDING GENES FROM MULTIPLE DONORS TO ENHANCE SALT TOLERANCE IN RICE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1015763
Grant No.
2018-67013-27618
Cumulative Award Amt.
$490,000.00
Proposal No.
2017-07788
Multistate No.
(N/A)
Project Start Date
Feb 15, 2018
Project End Date
Feb 14, 2023
Grant Year
2018
Program Code
[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production
Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
School of Plant,Env.&Soil Sci
Non Technical Summary
Salinity is a major climate-related risk for sustainable rice production. The major obstacle to design salt tolerant varieties is the narrow genetic base of US rice germplasm. Although we have successfully developed salt tolerant breeding lines, the level of salt tolerance in these lines needs further enhancement. Since multiple mechanisms contribute to salt tolerance and favorable alleles are distributed among various donors, pyramiding of genes from multiple donors is needed. The overall goal of this proposal is to exploit the natural genetic variation to develop rice cultivars with enhanced salt tolerance. Our specific objectives are: (a) pyramiding of favorable salt tolerance genes from selected donor germplasm, and (b) identification of superior salt tolerant alleles from multiple donors for rice improvement. Introgression lines developed from multiple donors will be utilized for developing superior salt tolerant lines through pyramiding. Evaluation of these lines for salt tolerance and agronomic performance in greenhouse and field experiments will be done to identify high yielding breeding lines with enhanced salt tolerance. Whole genome and transcriptome sequencing of pyramided lines and parents will be done to identify potential salt tolerant genes and their superior allelic variants. The project will generate advanced breeding lines with enhanced salt tolerance and genomic resources for the public sector breeding program to accelerate development of climate resilient rice varieties and elucidation of the molecular basis of complex salt tolerance mechanisms operating in rice.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011530108040%
2011530108160%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1530 - Rice;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
The overall goal of this proposal is to develop advanced breeding lines and rice varieties with enhanced salinity tolerance using classical breeding, modern genomics, and physiological tools. Our specific objectives are: (a) pyramiding of favorable QTLs/genes associated with salt tolerance traits from selected donor germplasm, and (b) identification of superior salt tolerant alleles from multiple donors.
Project Methods
For pyramiding favorable QTLs/genes associated with salt tolerance traits from salt tolerant donor germplasm, we will follow two approaches. The first approach will use the ILs of Pokkali and Nona Bokra already developed in the backgrounds of two medium and two long grain US cultivars. Two best performing ILs with non-overlapping introgressions and minimal donor genome will be selected from each population for generating BC1F2 populations which will be evaluated for salt tolerance to identify near-isogenic lines (NILs) with higher degree of salt tolerance. The second approach will use ILs developed from a complex cross involving two other donors (FL478 and Geumgangbyeo) in an US cultivar background. Two best performing ILs with desirable agronomic traits will be selected and backcrossed to the recurrent parent for one more generation followed by selfing to generate BC2F2 populations, which will be genotyped and phenotyped for salinity tolerance to identify NILs with minimal donor genome and improved salt tolerance. The NILs developed by both approaches will be evaluated in field experiments for potential release as variety in future.The salt tolerant NILs developed earlier along with recurrent parents will be resequenced to identify the donor segments and the donor specific SNPs relative to the corresponding recurrent parent of each cross. An RNA-Seq experiment will be performed using the NILs, donors, and recurrent parents to identify differentially expressed genes (DEGs) under salt stress, which will be validated through qRTPCR. The DEGs and their temporal and special expression patterns will be examined among NILs, donors, and recurrent parent.The whole genome sequence data will be integrated with transcriptomic data to determine the number of DEGs present in the introgressed regions in the NILs, the donor genes responding to salt stress compared to recurrent parents, and their superior alleles. The introgressed genes maintaining their expression pattern in NILs can be identified as useful targets for marker-assisted breeding. In case of multiparental NILs, the scanning of donor specific transcripts will allow identification of superior alleles, which can be useful for exploiting multiple donors for rice improvement.

Progress 02/15/18 to 02/14/23

Outputs
Target Audience:Rice producers, plant breeders, geneticists, pathologists and other researchers working with rice and abiotic stresses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunities for training and professional development of three graduate students, one visiting research scholar and one research associate. They were involved in molecular analysis, whole genome sequencing, RNA-sequencing, evaluation of advanced introgression lines for salinity tolerance traits, and identification of high yielding salt tolerant lines. How have the results been disseminated to communities of interest?Research findings from this project were presented at following national and international professional meetings and conferences: ASA, CSSA, and SSSA International Annual Meeting held at Salt Lake City, Utah, November 7-10, 2021; An International Symposium on Coastal Agriculture: Transforming Coastal Zone for Sustainable Food and Income Security, March 16-19, 2021; 38th Rice Technical Working Group Meeting held at Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020; National Association of Plant Breeders 2019 Annual meeting held at Callaway Garden, Pine Mountain, Georgia, Aug 25-29, 2019; 2018 meeting of the Southern Section of the American Society of Plant Biologists held at New Orleans, LA, 37th Rice Technical Working Group Meeting held at Long Beach, CA; 5th International Rice Congress held at Singapore; LSU AgCenter Rice Research Station field days. What do you plan to do during the next reporting period to accomplish the goals?Since the project has expired, we plan to prepare and publish few remaining manuscripts on whole genome and RNA-sequencing data analysis of donors, recurrent parents, and salt tolerant introgression lines for publication in peer-reviewed journals. In addition, we will make use of the genomic resources and advanced breeding lines with enhanced salt tolerance for breeding rice varieties with tolerance to multiple stresses and elucidation of salinity tolerance in future activities.

Impacts
What was accomplished under these goals? We have developed introgression line (IL) populations of salt tolerant donors (Pokkali, Nona Bokra, TCCP266) in the backgrounds of several high yielding US cultivars, Bengal, Jupiter, and Cheniere, respectively. These IL populations were genotyped either SNP or SSR markers and phenotyped for seedling stage salt tolerance leading to identification of salt tolerant ILs. Based on salt tolerance screening, the selected salt tolerant ILs of Pokkali and Nona Bokra were used in several combinations to develop F2 population for pyramiding the salt tolerant QTLs. for pyramiding the salt tolerant QTLs. Two different approaches were used to pyramid the desirable QTLs to improve salt tolerance. One approach involved using of salt tolerant ILs of Pokkali and Nona Bokra. The ILs were crossed and the F2 plants were genotyped with QTL linked markers and advanced to F3 generation for salt stress evaluation. In some salt tolerant ILs without the genotypic information, salt tolerant individuals were identified based on phenotyping and advanced to next generation. In another approach, a modified multi-parental advanced generation intercross (MAGIC) was used to exploit allelic diversity from multiple donors in combination with US adapted cultivars to develop salt tolerant lines. The procedure involved crossing of two F1s from the crosses to produce double cross hybrids, which were then backcrossed to recurrent parent 2-3 times. The resulting plants were selfed for two generations and selections were done for desirable agronomic traits. The multiparental populations were developed with following cross combinations: Mermentau/Hasawi//Mermentau/Pokkali and Hasawi/Cheniere//FL478/Dular. The seedling stage salt tolerance was evaluated in sand culture. Significant differences were observed among parents and breeding lines for all morphological traits such as root length, shoot length, dry weight, root-shoot ratio, and chlorophyll content. From the initial screening of 180 introgression lines (ILs) in sand culture, 36 tolerant and moderately tolerant ILs were selected and were again validated for salinity tolerance via hydroponics screening system. The selected tolerant lines showed no significant difference in terms of salt injury score with donors under hydroponics screening at 12 dS/m salt stress, indicating enhanced salt tolerance level in selected lines. Shoot and root Na/K ratio was lower in tolerant lines compared with recurrent parents suggesting homeostasis between Na and K or Na compartmentation as the mechanisms of tolerance. Response to salinity stress in selected lines at germination stage was evaluated using EC 12 salt solution. Salt stress reduced percent germination, speed of germination, coleoptile and shoot length, and increased mean germination time in selected ILs. Results of germination and seedling stage salinity tolerance evaluation of selected lines indicated involvement of different mechanism of salinity tolerance during germination and seedling stage. The combination of principal component analysis (PCA) and phenotypic clustering based on 12 morphophysiological traits revealed the grouping of positively correlated morphophysiological traits. The selected tolerant ILs were grouped with tolerant donors and showed lower root and shoot Na/K ratio compared with susceptible recurrent parents. Genotyping results with 100 polymorphic SSR markers indicated a low level of genetic diversity among genotypes and most salt tolerant donors clustered separately from the ILs in both populations and recurrent parents grouped with ILs suggesting the genetic background of the ILs with recurrent parents. The salt tolerant ILs identified in this study will be useful source for identification of superior salt tolerant alleles and development of climate resilient rice varieties. For pyramiding of salt tolerant QTLs, we used the salt tolerant ILs CLPK757 and CLPK683 (Pokkali in the background of CL151). JN100 and JN520 (Nona Bokra in the background of Jupiter). Two different approaches were used: phenotypic evaluation followed by genotyping and marker-assisted selection of QTLs followed by phenotypic evaluation. For phenotypic evaluation approaches, F3 progenies were screened for salinity stress in hydroponics. Two-week old seedlings were exposed to 12 EC salt stress for a week and tolerant lines were transferred to soil. F4 seeds were harvested from tolerant lines and continue to F5 generation. Again, F5 seeds were screened under salt stress to identify fixed tolerant lines. For genotypic selection, target QTLs were confirmed in F3 and F4 generation. F5 seeds were evaluated for salt stress at the seedling stage. The targeted QTLs (qNa2.2, qK2.1, and qK2.2, from JN100 and qK4.1 and qK8.1 from JN520) were detected in most salt tolerant lines. The visual salt injury score (SIS) and shoot Na and K concentration in selected lines indicated salt tolerance higher than the IL parents. Since few studies focused on the genome level variation in rice genotypes with contrasting response to salt stress, whole genome sequences of two salt tolerant (Pokkali and Nona Bokra) and three salt sensitive (Bengal, Cocodrie, and IR64) rice genotypes were analyzed to elucidate the molecular basis of salt tolerance mechanisms. A total of 413 million reads were generated with a mean genome coverage of 93% and mean sequencing depth of 18X. Analysis of the DNA polymorphisms revealed that 2347 nonsynonymous SNPs and 51 frameshift mutations could differentiate the salt tolerant from the salt sensitive genotypes. The genome-wide polymorphism information was integrated with the QTL mapping and expression profiling data and 396 differentially expressed genes with large effect variants in the coding regions were identified. These genes were involved in multiple salt tolerance mechanisms, such as ion transport, oxidative stress tolerance, signal transduction, and transcriptional regulation. The candidate genes identified in this study will be helpful for molecular breeding of salt tolerant rice varieties. Whole genome sequencing and RNA-sequencing were conducted to identify the genes responsible for salt tolerance. A salt tolerant introgression line (IL) was compared with its recurrent parent 'Jupiter' and salt tolerant donor 'Nona Bokra'. There were 23818 SNPs and InDels present in 487 genes differentiating the introgression line from the recurrent parent. These SNPs and InDels were present on three introgressed segments on chromosomes 1, 2, and 9. RNA-sequencing of salt stressed and controlled Leaf tissues in both parents and the selected IL revealed that fewer genes are differentially expressed under control condition than salt stress. Compared to Nona Bokra, fewer genes were expressed in the IL under salt stress. Although WGS indicated only 487 genes are introgressed from Nona Bokra, only 65 genes were differentially expressed and rest of the differentially expressed genes were from the recurrent parent. Some of these genes, which include some transcription factors ex. ZFP, BHLH, MYB and some ROS enzymes, membrane proteins, NADP reductase kinases, and some cytochrome, transport related proteins, had expression pattern same as the donor parent. Another group of genes had expression pattern different from Nona Bokra and these genes include basic helix-loop-helix , GDSL lipase, C4 dicrboxylate transporter, Hairpin-induced protein, peroxisomal membrane protein, CK1 Casein kinase, Isoflavone reductase, Pectinesterase, peptidyl-prolyl cis-trans isomerase, aminotransferase, ferredoxin--NADP reductase, IQ calmodulin-binding motif family protein, Catalase, terpene synthase, MYB family TF, and translation initiation factor IF-2. The above genes may be contributing toward enhanced salt tolerance in the selected ILs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Garg R, Subudhi PK, Varshney RK, Jain M (2023) Editorial: Abiotic stress: Molecular genetics and genomics, volume II. Front. Plant Sci. 13:1101139. doi: 10.3389/fpls.2022.1101139
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Singh LP, Coronejo S, Pruthi R, Chapagain S, Bhattarai U, Subudhi PK (2022) Genetic dissection of alkalinity tolerance at the seedling stage in rice using a high-resolution linkage map. Plants 11(23): 3347; https://doi.org/10.3390/plants11233347
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Pruthi R, Chapagain S, Coronejo S, Singh L, Subudhi P (2022) QTLs, candidate genes, and breeding lines to improve salt tolerance at the flowering and seedling stages in rice. Food and Energy Security DOI: 10.1002/fes3.433.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Singh LP, Coronejo S, Pruthi R, Chapagain S, Subudhi PK (2022) Integration of QTL Mapping and whole genome sequencing identified candidate genes for tolerance to alkaline stress at the seedling stage in rice (Oryza sativa L.). Intl J Mol Sci 23:11791. https://doi.org/10.3390/ijms231911791
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Pruthi R, Puram VRR, Ontoy J, Subudhi PK (2022) Genetics of yield component traits under salt stress at flowering stage and selection of salt tolerant pre-breeding lines for rice improvement Genetica 150:273-288. doi: 10.1007/s10709-022-00160-y
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Garcia R, Coronejo S, Concepcion J, Subudhi PK (2022) Whole genome sequencing and RNA-Seq reveal differences in genetic mechanism for flowering response between weedy rice and cultivated rice. Intl J Mol Sci 23(3):1608; https://doi.org/10.3390/ijms23031608
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Chapagain S, Concepcion J, Pruthi R, Singh LP, Famoso A, Subudhi PK (2022) Genetic variation among the salinity tolerant breeding lines identified from two multi-parent advanced generation introgression lines (MAGIL) populations in rice (Oryza sativa). J. Agron. Crop Sci. 208:295-313. https://doi.org/10.1111/jac.12581
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Chapagain S, Singh L, Garcia R, Pruthi R, Concepcion J, Coronejo S, Subudhi PK (2021) Molecular breeding for improving salinity tolerance in rice: Recent progress and future prospects. In: Molecular breeding for rice abiotic stress tolerance and nutritional quality, Hossain MA, Hassan L, Ifterkharuddaula KM, Kumar A, and Henry R (Eds). John Wiley & Sons, Inc. PP 26-52.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Subudhi PK, Concepcion J, Coronejo S, Garcia R (2021) QTL-Seq-based identification of genomic regions and candidate genes for nitrogen uptake in rice. ASA, CSSA, and SSSA International Annual Meeting, Salt Lake City, Utah, Nov 7-10, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Chapagain S, Pruthi R, Singh L, Subudhi PK (2021) Genetic dissection and QTL pyramiding using multiple donors to improve salt tolerance in rice. ASA, CSSA, and SSSA International Annual Meeting, Salt Lake City, Utah, Nov 7-10, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Pruthi R, Puram VRR, Ontoy J, Subudhi PK (2021) Genetic dissection of agronomic and yield related components under salinity stress in rice (Oryza sativa L.). ASA, CSSA, and SSSA International Annual Meeting, ASA, CSSA, and SSSA International Annual Meetings, Salt Lake City, Utah, Nov 7-10, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Subudhi PK (2021) Challenges and opportunities in designing salt tolerant rice. ISCA Webinar - International Symposium on Coastal Agriculture: Transforming Coastal Zone for Sustainable Food and Income Security. organized by Indian Society of Coastal Agricultural Research (ISCAR) in collaboration with ICAR-Central Soil Salinity Research Institute, Karnal, India, 16th March - 19th March 2021.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Subudhi PK, Shankar R, Jain M (2020) Whole genome sequence analysis of rice genotypes with contrasting response to salinity stress. Sci. Rep. 10:21259; doi: 10.1038/s41598-020-78256-8
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Chapagain S, Singh L, Subudhi PK (2020) Novel breeding approaches for developing climate-resilient rice. In: Quantitative Genetics, Genomics, and Plant Breeding (Second edition), M.S. Kang (Ed.) Publisher CAB International, Nosworthy Way, Wallingford OX10 8DE, United Kingdom. PP 259-275.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Chapagain S, Subudhi PK (2020) Screening and evaluation of multi-parental rice genotypes for seedling stage salinity tolerance. 2nd NAPA Biennial International Scientific Conference: Sept 25-28, 2020 (Virtual), Atlanta, GA 30344, USA (Oral).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: De Leon TB, Pruthi R, Jampala B, Subudhi PK (2020) Genetic determinants for agronomic traits localized on a GBS-SNP linkage map from a japonica x indica cross in rice (Oryza sativa). Plant Gene. https://doi.org/10.1016/j.plgene.2020.100249
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Subudhi PK, Garcia R, Coronejo S, De Leon T (2020) A novel mutation of the NARROW LEAF 1 gene adversely affects plant architecture in rice (Oryza sativa L.). Intl J Mol Sci 21(21):8106.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Islam S, Coronejo S, Subudhi PK (2020) Whole genome sequencing reveals uniqueness of black hulled and straw hulled weedy rice genomes. Theor Appl Genet https://doi.org/10.1007/s00122-020-03611-2
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Subudhi PK, Garcia RS, Coronejo S, Tapia R (2020) Comparative transcriptional profiling of root tissues in two rice genotypes reveals differential expressed genes associated with root architecture under nitrogen stress. Int J Mol Sci 21:5759; doi:10.3390/ijms21165759
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Subudhi PK, Famoso A, Chapagain S, Garcia R, Coronejo S, Concepcion J, Singh L, De Leon T, Puram VRR, Ontoy J (2020) Progress in development of salt tolerant rice varieties. The 38th Rice Technical Working Group Meeting, Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020 (Oral).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Garcia R, Coronejo S, Tapia R, Subudhi PK (2020) RNA-Seq reveals differential expressed genes in two rice genotypes with contrasting response to nitrogen stress. The 38th Rice Technical Working Group Meeting, Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020 (Poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Singh L, Bhattarai U, Chapagain, Subudhi PK (2020) Mapping quantitative trait loci for alkalinity stress tolerance in rice. The 38th Rice Technical Working Group Meeting, Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020 (Oral).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Chapagain S, Garcia R, Coronejo S, Concepcion J, Singh L, Pruthi R, Famoso A, Subudhi PK (2020) Characterization and identification of salt tolerant breeding lines from several mini MAGIC populations in rice. The 38th Rice Technical Working Group Meeting, Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020 (Poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Subudhi P, Garcia R, Chapagain S, Coronejo S, De Leon T, Ontoy J, Puram VR, Famoso A, Shankar R, Mukesh Jain M (2019) NIFA: Pyramiding QTLs/genes from multiple donors to enhance salt tolerance in rice. National Association of Plant Breeders 2019 Annual meeting and USDA-NIFA Project Directors meeting, Callaway Garden, Pine Mountain, Georgia, Aug 25-29, 2019
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Islam S, Ontoy J, Subudhi PK (2019) Meta-analysis of quantitative trait loci (QTLs) associated with seedling stage salt tolerance in rice (Oryza sativa L.). Plants 8(2):33. https://doi.org/10.3390/plants8020033
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Puram VRR, Ontoy J, Subudhi PK (2018) Identification of QTLs for salt tolerance traits and prebreeding lines with enhanced salt tolerance using a salt tolerant donor Nona Bokra. Plant Mol Biol Rep doi: 10.1007/s11105-018-1110-2.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Biradar H, Karan R, Subudhi PK (2018) Overexpression of a salt responsive protein3-1 as well as pyramiding with SaVHAc1 from Spartina alterniflora L. enhances salt tolerance in rice. Front Plant Sci 9:1304. doi: 10.3389/fpls.2018.01304


Progress 02/15/21 to 02/14/22

Outputs
Target Audience:Rice producers, plant breeders, geneticists, pathologists and other researchers working with rice and abiotic stresses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunities for training and professional development of twograduate students and one research associate. The students were involved in molecular analysis, whole genome sequencing, and evaluation of advanced introgression lines for salinity tolerance traits. How have the results been disseminated to communities of interest?Research findings from this project was presented at the ASA, CSSA, and SSSA International Annual Meeting held at Salt Lake City, Utah, November 7-10, 2021, and an International Symposium on Coastal Agriculture: Transforming Coastal Zone for Sustainable Food and Income Security, March 16-19, 2021. What do you plan to do during the next reporting period to accomplish the goals?We plan to identify salt tolerant lines using both phenotypic screening and marker-assisted pyramiding.We also plan to confirm the pyramided QTLs and conduct both whole genome sequencing and RNA-sequencing in recurrent parents, donors, and salt tolerant multiparental advanced introgression lines to identify superior salt tolerant alleles.

Impacts
What was accomplished under these goals? We developed two multiparental advanced generation introgression lines populations using salt tolerant donors Pokkali, Hasawi, and FL478 in US rice backgrounds Mermentau, Jupiter, and Cheniere and evaluated physiological and morphological responses of salinity stress in germination and seedling stages. From the initial screening of 180 introgression lines (ILs), 36 ILs were selected and were again validatedfor salinity tolerance via hydroponics screening system. These breedinglines were either tolerant andmoderately tolerant. The combination of principal component analysis (PCA) and phenotypic clustering based on 12 morphophysiological traits revealed the grouping of positively correlated morphophysiological traits. The selected tolerant ILs were grouped with tolerant donors and showed lower root and shoot Na/K ratio compared with susceptible recurrent parents. Genotyping results with 100 polymorphic SSR markers indicated a low level of genetic diversity among genotypes and most salt tolerant donors clustered separately from the ILs in both populations and recurrent parents grouped with ILs suggesting the genetic background of the ILs with recurrent parents. The salt tolerant ILs identified in this study will be useful source for investigating the molecular genetic basis of salt tolerance and physiological mechanism and agronomic evaluation for use as prebreeding lines or for future release as superior salt tolerant rice varieties. In our pyramiding project, we used the salt tolerant ILs identified from the IL populations which were developed using salt tolerant donors Pokkali and Nona Bokra and used for QTL mapping of salt tolerance. The ILs used for pyramiding were CLPK757 and CLPK683 (Pokkali in the background of CL151). JN100 and JN520 (Nona Bokra in the background of Jupiter). Two different approaches, phenotypic evaluation followed by genotyping and marker-assisted selection of QTLs followed by phenotypic evaluation, were implemented. First, we developed F1 and confirmed F1 using molecular markers. For phenotypic evaluation approaches, F3 progenies werescreenedfor salinity stress in hydroponics. Two-week oldseedlings were exposed to 12 EC salt stress for a week and tolerant lines were transferred to soil. F4 seeds were harvested from tolerant lines and continue toF5 generation. Again, F5 seeds were screened under salt stress to identify fixed tolerantlines. For genotypic selection, target QTLs were confirmed in F3 and F4 generation. F5 seeds were evaluated for salt stress at the seedling stage. The identified salt tolerant pyramided lines will be genotyped for target QTLs associated with salt tolerance. A biparental population developed from a cross between US cultivar Cheniere and a new salt tolerant donor 'TCCP' was evaluated for salt tolerance at germination, seedling, and reproductive stages to identify QTLs. The population has been genotyped with Genotyping by Sequencing. Phenotypic screening at all three stages has been completed and data is being analyzed. Our objective in this QTL mapping project is to identify advanced breeding lines with salt tolerance at all three stages.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Subudhi PK, Concepcion J, Coronejo S, Garcia R (2021) QTL-Seq-based identification of genomic regions and candidate genes for nitrogen uptake in rice. ASA, CSSA, and SSSA International Annual Meeting, Salt Lake City, Utah, Nov 7-10, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Chapagain S, Pruthi R, Singh L, Subudhi PK (2021) Genetic dissection and QTL pyramiding using multiple donors to improve salt tolerance in rice. ASA, CSSA, and SSSA International Annual Meeting, Salt Lake City, Utah, Nov 7-10, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Pruthi R, Puram VRR, Ontoy J, Subudhi PK (2021) Genetic dissection of agronomic and yield related components under salinity stress in rice (Oryza sativa L.)ASA, CSSA, and SSSA International Annual Meeting, ASA, CSSA, and SSSA International Annual Meetings, Salt Lake City, Utah, Nov 7-10, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Subudhi PK (2021) Challenges and opportunities in designing salt tolerant rice. ISCA Webinar - International Symposium on Coastal Agriculture: Transforming Coastal Zone for Sustainable Food and Income Security. organized by Indian Society of Coastal Agricultural Research (ISCAR) in collaboration with ICAR-Central Soil Salinity Research Institute, Karnal, India, 16th March  19th March 2021.
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Chapagain S, Singh L, Garcia R, Pruthi R, Concepcion J, Coronejo S, Subudhi PK (2021) Molecular breeding for improving salinity tolerance in rice: Recent progress and future prospects. In: Molecular breeding for Rice Abiotic Stress Tolerance and Nutritional Quality, Hossain MA, Hassan L, Ifterkharuddaula KM, Kumar A, and Henry R (Eds). John Wiley & Sons, Inc. PP 26-52.
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Chapagain S, Concepcion J, Pruthi R, Singh LP, Famoso A, Subudhi PK (2021) Genetic variation among the salinity tolerant breeding lines identified from two multi-parent advanced generation introgression lines (MAGIL) populations in rice (Oryza sativa). J. Agron. Crop Sci. (accepted)


Progress 02/15/20 to 02/14/21

Outputs
Target Audience:Rice producers, plant breeders, geneticists, pathologists and other researchers working with rice and abiotic stresses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunities for training and professional development of three graduate students and one research associate. The students were involved in molecular analysis, whole genome sequencing, and evaluation of advanced introgression lines for salinity tolerance traits. How have the results been disseminated to communities of interest?Research findings from this project was presented at the 38th Rice Technical Working Group Meeting held at Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020. What do you plan to do during the next reporting period to accomplish the goals?We plan to identify lines with multiple QTLs for salt tolerance and evaluate the salt tolerant multiparental lines, and analyze the whole genome sequencing data of donors and some selected lines.

Impacts
What was accomplished under these goals? Two different approaches were used to pyramid the desirable QTLs to improve salt tolerance. One approach involved a set of selected salt tolerant introgression lines (ILs) such as ClPK 683, ClPK 757, JN100, JN 520, ChN1446, ChN 1327, which were developed using known salt tolerant donors 'Pokkali' and 'Nona Bokra' in the backgrounds of three US varieties, Cl151, Cheniere, and Jupiter. The introgression lines were crossed and plants in the F2 generation were genotyped for QTL linked markers and advanced to F3 generation for salt stress evaluation. Since some salt tolerant ILs did not have any genotypic data, salt tolerant individuals were identified based on phenotyping in hydroponic system and advanced to next generation for selection of breeding lines with enhanced salt tolerance. In another approach, a modified multi-parental advanced generation intercross (MAGIC) was used to accumulate desirable alleles from multiple donors in combination with US adapted cultivars to develop salt tolerant lines. Two-week old seedlings were exposed to incremental salinity stress from electrical conductivity (EC) 6 dS/m up to EC 18 dS/m and salt injury was scored after 1 week. Two different multiparental populations (Mermentau/Hasawi//Mermentau/Pokkali and Hasawi/Cheniere//FL478/Dular///Jupiter) were evaluated for seedling stage salt tolerance in a sand culture experiment and 27% and 52% of breeding lines were highly tolerant, respectively. Significant differences were observed among parents and breeding lines for all morphological traits such as root length, shoot length, dry weight, root-shoot ratio, and chlorophyll content. The selected tolerant lines showed no significant difference in terms of salt injury score with donors under hydroponics screening at 12 dS/m salt stress, indicating enhanced salt tolerance level in selected lines. From these two populations, 36 tolerant to highly tolerant lines were selected for re-evaluation in hydroponics experiment. Our results suggested that different tolerance mechanisms inherited from different donors may be responsible for enhanced salt tolerance. These lines will be used for identification of superior salt tolerant alleles that can be used for rice improvement program to develop climate resilient rice varieties. Since few studies focused on the genome level variation in rice genotypes with contrasting response to salt stress, whole genome sequences of two salt tolerant (Pokkali and Nona Bokra) and three salt sensitive (Bengal, Cocodrie, and IR64) rice genotypes were analyzed to elucidate the molecular basis of salt tolerance mechanisms. A total of 413 million reads were generated with a mean genome coverage of 93% and mean sequencing depth of 18X. Analysis of the DNA polymorphisms revealed that 2347 nonsynonymous SNPs and 51 frameshift mutations could differentiate the salt tolerant from the salt sensitive genotypes. The genome-wide polymorphism information was integrated with the QTL mapping and expression profiling data and 396 differentially expressed genes with large effect variants in the coding regions were identified. These genes were involved in multiple salt tolerance mechanisms, such as ion transport, oxidative stress tolerance, signal transduction, and transcriptional regulation. The candidate genes identified in this study will be helpful for molecular breeding of salt tolerant rice varieties.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Subudhi PK, Shankar R, Jain M (2020) Whole genome sequence analysis of rice genotypes with contrasting response to salinity stress. Sci Reports; doi: 10.1038/s41598-020-78256-8
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Chapagain S, Singh L, Subudhi PK (2020) Novel breeding approaches for developing climate-resilient rice. In: Quantitative Genetics, Genomics and Plant Breeding (Second edition), M.S. Kang (Ed.) Publisher CAB International, Nosworthy Way, Wallingford OX10 8DE, United Kingdom. PP 259-275.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Chapagain S, Subudhi PK (2020) Screening and evaluation of multi-parental rice genotypes for seedling stage salinity tolerance. 2nd NAPA Biennial International Scientific Conference: Sept 25-28, 2020 (Virtual), Atlanta, GA 30344, USA (Oral).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: De Leon TB, Pruthi R, Jampala B, Subudhi PK (2020) Genetic determinants for agronomic traits localized on a GBS-SNP linkage map from a japonica x indica cross in rice (Oryza sativa). Plant Gene https://doi.org/10.1016/j.plgene.2020.100249
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Subudhi PK, Garcia R, Coronejo S, De Leon T (2020) A novel mutation of the NARROW LEAF 1 gene adversely affects plant architecture in rice (Oryza sativa L.). Intl J Mol Sci 21(21):8106.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Islam S, Coronejo S, Subudhi PK (2020) Whole genome sequencing reveals uniqueness of black hulled and straw hulled weedy rice genomes. Theor Appl Genet https://doi.org/10.1007/s00122-020-03611-2


Progress 02/15/19 to 02/14/20

Outputs
Target Audience:Rice producers, plant breeders, geneticists, pathologists and other researchers working with rice and abiotic stresses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunities for training and professional development of two graduate students and one research associate. The students were involved in development and molecular analysis, and evaluation of advanced introgression lines for salinity tolerance traits. How have the results been disseminated to communities of interest?Research findings from this project was presented at following national and international professional meetings and conferences: National Association of Plant Breeders 2019 Annual meeting held at Callaway Garden, Pine Mountain, Georgia, Aug 25-29, 2019 and the 38th Rice Technical Working Group Meeting held at Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020. What do you plan to do during the next reporting period to accomplish the goals?We plan to identify lines with multiple QTLs for salt tolerance and evaluate the introgression lines developed using multiple parents, and analyze the whole genome sequencing data.

Impacts
What was accomplished under these goals? The selected salt tolerant introgression lines (ILs) of salt tolerant donors Pokkali and Nona Bokra developed in the backgrounds of US cultivars, Jupiter, Cheniere and Bengal, were used in several combinations to develop F2 population for pyramiding the salt tolerant QTLs. To exploit allelic diversity present in exotic donors, we have developed breeding lines from different cross combinations involving multiple salt tolerant donors. During this reporting period, we evaluated seedling stage salt tolerance in populations developed from two different multiparental combinations using sand culture. These multiparental populations were at BC3F3 generations with following cross combinations: Mermentau/Hasawi//Mermentau/Pokkali (US cultivar Mermentau background) and Hasawi/Cheniere//FL478/Dular (Jupiter background). Two-week old seedlings were exposed to salt stress (6 EC) for 3 days followed by 12 EC for 7 days and then 18EC for 5 days before observations were taken. Salt injury score was scored in a scale of 1-9 and chlorophyll was measured 4 days after salinization using SPAD meter. Shoot length, root length, dry weight, and Na and K concentration in root and shoot were measured. Based on the sand culture screening, the seedling stage salt tolerant lines were again confirmed in hydroponic experiments. Shoot and root Na/K ratio was lower in tolerant lines compared with recurrent parents suggesting homeostasis between Na and K or Na compartmentation as the mechanisms of tolerance. Response to salinity stress in selected lines at germination stage was evaluated using EC 12 salt solution. Salt stress reduced percent germination, speed of germination, coleoptile and shoot length, and increased mean germination time in selected ILs. Results of germination and seedling stage salinity tolerance evaluation of selected lines indicated involvement of different mechanism of salinity tolerance during germination and seedling stage. Additional populations were also advanced and breeding lines with desirable agronomic attributes and improved seed fertility will be selected for salt tolerance screening in future. We are continuing the analysis of the whole genome resequencing data of salt tolerant donor lines (Pokkali, Nona Bokra) and salt susceptible US cultivars (Bengal, Cypress) to ascertain the differences among them with respect to genes contributing towards salt tolerance.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Subudhi P, Garcia R, Chapagain S, Coronejo S, De Leon T, Ontoy J, Puram VR, Famoso A, Shankar R, Mukesh Jain M (2019) NIFA: Pyramiding QTLs/genes from multiple donors to enhance salt tolerance in rice. National Association of Plant Breeders 2019 Annual meeting and USDA-NIFA Project Directors meeting, Callaway Garden, Pine Mountain, Georgia, Aug 25-29, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Subudhi PK, Famoso A, Chapagain S, Garcia R, Coronejo S, Concepcion J, Singh L, De Leon T, Puram VRR, Ontoy J (2020) Progress in development of salt tolerant rice varieties. The 38th Rice Technical Working Group Meeting, Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020 (Oral).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Chapagain S, Garcia R, Coronejo S, Concepcion J, Singh L, Pruthi R, Famoso A, Subudhi PK (2020) Characterization and identification of salt tolerant breeding lines from several mini MAGIC populations in rice. The 38th Rice Technical Working Group Meeting, Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020 (Poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Singh L, Bhattarai U, Chapagain, Subudhi PK (2020) Mapping quantitative trait loci for alkalinity stress tolerance in rice. The 38th Rice Technical Working Group Meeting, Perdido Beach Resort, Orange Beach, AL, Feb 24-27, 2020 (Oral).


Progress 02/15/18 to 02/14/19

Outputs
Target Audience:Rice producers, plant breeders, geneticists, pathologists and other researchers working with rice and abiotic stresses? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunities for training and professional development of two graduate students, one research associate, and one visiting research scholar. The students were involved in development and molecular analysis, and evaluation of advanced introgression lines for salinity tolerance traits. How have the results been disseminated to communities of interest?Research findings from this project was presented at following national and international professional meetings and conferences: 2018 Meeting of the Southern Section of the American Society of Plant Biologists held at New Orleans, LA, 37th Rice Technical Working Group Meeting held at Long Beach, CA, 5th International Rice Congress held at Singapore and LSU AgCenter Rice Research Station field days. What do you plan to do during the next reporting period to accomplish the goals?We plan to make crosses using introgression lines of different donors, advance the multiparental populations and publish few remaining manuscripts for publication in peer-reviewed journals.

Impacts
What was accomplished under these goals? We have developed introgression line (IL) populations of salt tolerant donors (Pokkali, Nona Bokra, TCCP266) in the backgrounds of several high yielding US cultivars, Bengal, Jupiter, and Cheniere, respectively. These IL populations were genotyped either SNP or SSR markers and phenotyped for seedling stage salt tolerance leading to identification of salt tolerant ILs with marker delimited introgressed donor segments. Salt tolerance screening was conducted in sand culture followed by evaluation in hydroponic experiments. The selected salt tolerant ILs of Pokkali and Nona Bokra are planted in greenhouse for pyramiding the salt tolerant QTLs. To exploit allelic diversity present in exotic donors, we used multiple donors in different cross combinations. The procedure involved crossing of two F1s from the crosses, recurrent parent x Donor 1 and same RP x Donor 2, followed by selfing to produce double cross hybrids, which were then backcrossed to recurrent parent 2-3 times. The resulting backcross plants were selfed for two generations and selections were done for desirable agronomic traits. Populations were developed from several cross combinations involving multiple donors and multiple US cultivars. These populations will be advanced and breeding lines with desirable agronomic attributes and improved seed fertility will be selected for salt tolerance screening. We have resequenced some salt tolerant donor lines (Pokkali, Nona Bokra) and salt susceptible US cultivars (Bengal, Cypress). We are currently analyzing this data to compare the similarities and differences among the donors as well as the US adapted susceptible cultivars.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Islam S, Ontoy J, Subudhi PK (2019) Meta-analysis of quantitative trait loci (QTLs) associated with seedling stage salt tolerance in rice (Oryza sativa L.). Plants 8(2):33. https://doi.org/10.3390/plants8020033
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Puram VRR, Ontoy J, Subudhi PK (2018) Identification of QTLs for salt tolerance traits and prebreeding lines with enhanced salt tolerance using a salt tolerant donor Nona Bokra. Plant Mol Biol Rep doi: 10.1007/s11105-018-1110-2.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Biradar H, Karan R, Subudhi PK (2018) Overexpression of a salt responsive protein3-1 as well as pyramiding with SaVHAc1 from Spartina alterniflora L. enhances salt tolerance in rice. Front Plant Sci 9:1304. doi: 10.3389/fpls.2018.01304