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Introgression of Sub1 Qtl Into Br22 Using Marker Assisted Backcrossing

Al Amin
Plant Breeding Division, Bangladesh Rice Research Institute, Bangladesh

Khandakar Md. Iftekharuddaula
Plant Breeding Division, Bangladesh Rice Research Institute, Bangladesh

Animesh Sarker
Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Scienceand Technology University, Bangladesh

Sharmistha Ghoshal
Plant Breeding Division, Bangladesh Rice Research Institute, Bangladesh

Tamal L. Aditya
Plant Breeding Division, Bangladesh Rice Research Institute, Bangladesh

Ashraf H. Talukder
Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Scienceand Technology University, Bangladesh

Farah Sabrin
Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Scienceand Technology University, Bangladesh

Morsaline Md. Billah
Department of Biotechnology and Genetic Engineering, Khulna University, Bangladesh

Bertrand Collard
Sugar Research Australia, Australia

Abstract/ Executive Summary:

Submergence is one of the important abiotic stresses incurring substantial loss to rice production in Bangladesh. With a view to improving submergence tolerance in BR22 (popular name Kiron) is a promising antioxidant enriched, high yielding and strongly photosensitive transplanted Aman rice variety, BRRI dhan 51 was used as a donor parent to introgress the SUB1 QTL conferring submergence tolerance. Molecular marker within submergence tolerant QTL (SUB1), flanking markers and evenly distributed background markers were utilized in a marker aided selection program to develop advanced breeding lines with broad-spectrum tolerance to flash flooding submergence. Indel and simple sequence repeats (SSRs) markers were used in the marker assisted backcrossing scheme in BC4F1 and BC5F1 generation. Sub1C173 a gene specific indel marker SUB1 QTL was used to select plants possessing the tolerant genes (foreground selection). The result of recombinant selection also revealed that the size of the SUB1 QTL introgression from the donor parent was estimated as 2.5 Mb. The percentage of recipient genome recovery in the best plant viz. BR10190-3-1-20-3 was 95%. This research work illustrates the successful application of marker- assisted breeding to introgress the submergence tolerant QTLs into the genetic background of BR22.

Keywords: Submergence; Abiotic stress; SUB1 QTL; Molecular marker; Marker assisted selection; Indel, Microsatellite markers

Location: Plant breeding division, Bangladesh Rice Research Institute, Gazipur-1701

Start Date: 00-00-2014

End Date: 00-00-2015

Program Area: Variety and Species

Commodity: Rice

Objectives:

To introgress the submergence tolerant QTLs into the genetic background of BR22.

Methodology:

The experiment was designed and carried out in order to introgress SUB1 QTL into the genetic background of BR22 (recurrent parent). BR22 is a T. aman variety having 150 days growth duration, high yielding capability, strong photosensitivity, short and bold grain and does not possess SUB1 QTL. Need to refer to this; cite appropriate reference, carrying the submergence tolerant QTL served as the donor parent during the cross. The parental crosses were initiated in T. Aman, 2011 (Kharif, 2011) season and crossing as well as marker assisted selection was attempted in every T. Aman season. This research work was started from BC4F₁ to BC₅F₁ generation as a part of development of breeding pipelines of plant breeding division, Bangladesh Rice Research Institute, Gazipur-1701, using marker assisted selection techniques. Leaf samples were collected following ten stick method (leaf picking-up from every ten plants tagged by sticks). DNA was extracted following modified Mini scale method [13] as well as CTAB method. The DNA concentration of samples was adjusted to 25 ng/μl. PCR for Simple Sequence Repeats (SSRs) was performed in 10 μl reactions containing around 25 ng/μl of DNA template (3 μl DNA), 1 μl 10X TB buffer (containing 200 mM Tris–HCl pH 8.3, 500 mM KCl), 0.2 μl of 10 mM dNTP, 1.35 μl 25 mM MgCl₂, 0.5 μl each of 10 μM forward and reverse primers and 0.2 μl of Taq DNA polymerase (5 U/μl) using G-STORM thermal cycler [15,16] and Gene Atlas thermal cycler. CR for STS (Sequence Tagged Sites) Primer Sub1C173 was performed in 20 μl reactions containing around 25 ng/μl of DNA template (4 μl DNA with 10-20X dilution factor), 2 μl 10X TB, 2.7 μl 25mM MgCl₂, 0.4 μl of 10 mM dNTP, 1 μl each of 10 μM forward and reverse primers, 1 μl DMSO (Dimethyl Sulphoxide) and 0.4 μl of Taq DNA polymerase (5 U/μl) using G-STORM and Gene Atlas thermal cycler. The PCR products of SSR and STS markers were resoluted using Polyacrylamide gel electrophoresis. The size (in nucleotide base pairs) of the most intensely amplified band for each microsatellite marker was measured based on its migration in relation to a molecular-weight size marker (1 Kb⁺ DNA Ladder) with Alpha Ease 4.0 software. The homozygous allele of the recipient parent for the particular SSR marker was scored as ‘A’. Again, the homozygous allele of the donor parent for the particular SSR marker was scored as ‘B’. However, heterozygous alleles were scored as ‘H’. The molecular data was analyzed with Graphical Genotyper (GGT 2.0) software. The software produced graphical sketch of the percentage of recurrent parent chromosomal segments in the selected backcross population. Selection approaches of backcross population- Simple sequence repeats (SSRs) and indel markers were used for selection. The different selection levels followed in this backcross breeding is discussed as follows: Foreground selection- In BC₃F₁ & BC₄F₁ generations of T. Aman 2014 & 2015 season, the foreground selection was done over 77 and 175 plants respectively using Sub1C173, an indel marker specific to SUB1 QTL. After foreground selection, the heterozygous plants were marked by sticks in the field. Phenotypic selection was applied during active tillering stage based on resemblance to recipient parent BR22. Different characters considered during phenotypic selection were tiller numbers, tillering pattern, flag leaf angle, leaf shape, leaf length, leaf breadth, leaf color etc. In this study, gene-based indel marker Sub1C173 specific to the putative gene of Sub1C of SUB1 QTL was used in the foreground selection. The individuals that were heterozygous for the foreground marker were selected in this selection step. Recombinant selection-In this selection level, individual plants that were homozygous for the recipient allele at the marker loci distally or proximally flanking the Sub1 locus (i.e. recombinants) were identified. This was titled as “recombinant selection” . The objective of this selection was to minimize the linkage drag. The flanking markers selected were as close as possible. Population sizes depend on distance of flanking markers from target locus. Flanking markers used for recombinant selection were selected from the tip of chromosome 9 around the Sub1 region was targeted. In this study, four markers were selected as flanking markers like RM5799, RM23843, RM8300 and RM23915. Background selection- Microsatellite or Simple Sequence Repeat (SSR) markers covering all the chromosomes that were polymorphic between the donor and recurrent parent were used for background selection to recover the recipient genome. Out of 403 SSR primers surveyed, a total of 97 microsatellite markers were used for background selection initially. Maximum number of background markers used was 10 for chromosome 1 and chromosome 9. The minimum number of background markers used was 5 for chromosome 3. The average number of background markers over all the 12 chromosomes was 8.50. The microsatellite markers that revealed fixed (homozygous) alleles at non-target loci at one generation are not screened at the next BC generation. Only those markers that were not fixed for the recurrent parent allele are analyzed in the following generations. The segregates with fixed donor alleles were discarded from the selection in backcross-F₁ generation.

Source of Information: Int J Plant Biol Res 6(5): 1103 (2018)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The newly developed BR22-Sub1 lines could be useful as a strongly photosensitive submergence tolerant rice line for the multiple flash flood affected greater Northern region of Bangladesh. However, the submergence tolerance, yield and other attributes of the newly developed BR22 line will be evaluated later.

Knowledge Management: Journal