Mapping of Qtl for Anaerobic Germination Using the Donor Ac39416a in the Genetic Background of Swarna Sub-1 (Oryza Sativa L.)

Background: Anaerobic germination is an important trait in particular for cultivation under direct seeding method in kharif season, as well as during nursery rising for transplant rice, as sometimes unexpected rains immediately after sowing will drastically reduce the plant population. Methods and Results: In the present investigation phenotypic screening for Anaerobic germination (AG) was carried out using 188 F 2:3 population of Swarna Sub1/AC39416A at RARS (APRRI), Maruteru. The mean anaerobic germination per cent recorded after the two weeks of submergence ranged from 0% to 95% with overall mean of 47.51% whereas, for three weeks of submergence, the mean anaerobic germination per cent recorded between 0 and 95%, with overall mean of 37.66%. 134 (19.42%) out of 687 Simple Sequence Repeats (SSR) markers surveyed were polymorphic between the parents. Linkage analysis was done with 83 SSR markers showing polymorphism clearly using the integrated software called QTL IciMapping software version 4.1.0. The length of linkage map constructed across whole genome was 3600.8 cM and identied seven QTLs viz., qAG2, qAG3, qAG7-1, qAG7-2, qAG9, qAG10 and qAG12. All these seven QTLs explained phenotypic variance of about 37.47% collectively for AG trait, with their individual contributions ranging from 3.5% to 8.67% of phenotypic variation and LOD scores of 2.6 to 5.86. The LOD score and phenotypic variance is 5.86 and 8.67% respectively for qAG10 a novel QTL identied in the present study using ICIM method. Conclusion: QTL “qAG12-1” identied in this study may be considered for introgression into popular elite rice varieties otherwise susceptible for anaerobic germination after ne mapping studies.


Introduction
Rice production is in uenced by many of the biotic and abiotic stresses throughout the world, where abiotic stresses alone contribute to nearly 50% of the total losses in the yield. Under coastal irrigated ecosystem major abiotic stresses viz., oods, cyclones (causes lodging of the crop) and salinity resulting in decline in the productivity of rice. Severe and unexpected heavy rains leaves no time to leach excess water in to ground which leads to ooding, a major climate change challenge severely affecting productivity and often place a major limitation on the cultivation. Three types of oods viz., submergence during germination (germination under anaerobic conditions), ash oods (complete submergence up to 2 weeks) and stagnant ooding (30-50cm water depth) are the most prevailing types of oods in coastal Andhra Pradesh (Reddy et al., 2015).
Flooding during seed germination might be a consequence of unevenly levelled elds or early and unforeseen rains, or even, when rice elds are purposely ooded after sowing to combat weeds. Among all abiotic stress, tolerance to ooding during the process of seed germination i.e anaerobic germination (AG) is the rarest phenomenon (Zhang et al., 2018). Rice is the only chief cereal that exhibits a degree of tolerance to anaerobic conditions during germination, which is limited to coleoptiles emergence and partial growth, but not adequate to triumph over the stress (Miro et al., 2017). Semi-aquatic nature of rice made it to survive few days of submergence and broad genetic diversity among the rice landraces and traditional varieties has enabled its cultivation in different agro ecological zones and water regimes.
Although rice could tolerate ooding, its germination is limited to coleoptiles elongation as in susceptible genotypes; however root and primary leaf fall short to develop normally (Kumar et al., 2018). Tolerance of rice crop to ooding stress through enhanced germination and early growth of the seedlings is a prerequisite for successful cultivation in regions where oods is a recurrent problem.
Anaerobic germination is characterized by rapid elongation of coleoptile under submergence, with concomitant delay in development of radical (Kretzschmar et al., 2015). A series of biochemical properties such as, changes in the enzymatic activities of α-amylase, peroxidase and alcohol dehydrogenase in uences anaerobic stress tolerance in rice. Positive in uence of α-amylase in improving the germination of the seed is by converting starch into sugars (Perata et al., 1993). The tolerance mechanism that enables rice to germinate in the absence of O 2 is based mainly on the fact that rice seeds are able to degrade their starchy reserves under anoxia also (Magneschi and Perata. (2009) and Nghi et al., 2019). The ability of the rice coleoptile to elongate under anoxia represents an unveiled enigma, whereas the mechanisms and genes involved in adaptation of rice ora to submergence have newly been discovered.
Identi cation of the molecular markers linked to QTLs or genes controlling tolerance to submergence during germination would assist selection for this character, which have low heritability (Angaji et al., 2010). Screening of markers for polymorphism between the parents forms the basis for tagging of the desired gene, ne mapping of the gene in the rice chromosome and in the subsequent Marker Assisted Selection (MAS) programmes (Reddy et al., 2018). Of the various types of DNA markers, PCR-based markers called simple sequence repeats (SSRs) or microsatellites are used widely due to their high degree of polymorphism, technically simple method of nding and are cost e cient (Gonzaga et al., 2015). The quantitative trait locus (QTL) analysis and other molecular methods are employed in order to nd the genetic locus that underlies the trait of interest. If a genetic locus has been discovered and characterized has major effect on the trait, it can be transferred subsequently into modern high-yielding cultivars, but are stress-sensitive using marker-assisted breeding technology to achieve stress-tolerant cultivars e ciently (Mustroph, 2018). QTL mapping for AG in rice has begun to identify the promising loci that  (Table. 4).
Breeding for increased anaerobic germination or ooding tolerance during germination has been attempted previously by many workers, but the progress is little due to the limitation of donors with AG i.e genetic diversity, limited knowledge on the genetics and complex mechanisms of tolerance and methods used for screening or measurement of tolerance (Jiang et al., 2004). Keeping in vision the importance of anaerobic germination the present investigation was planned and executed using the parents Swarna Sub1 and AC39416A for generating F 2:3 mapping population in lieu of identi cation of QTLs responsible for AG.

Plant material and Mapping population:
The experimental plant material consisting of 188 F 2:3 mapping population was developed by crossing Swarna Sub1 and AC39416A at RARS (formerly APRRI), Maruteru, West Godavari district of Andhra Pradesh. Swarna sub1 is a variety developed by IRRI, Philippines by introgression of sub1gene into a mega rice variety Swarna. It has submergence tolerance during vegetative stage for 7-10 days, but lacks tolerance to submergence during germination. Several donors were identi ed for anaerobic germination tolerance across the world. In RARS, Maruteru the cultures, AC39416A and AC39397 were identi ed as good donors for the anaerobic germination trait. Hence, in the present study the parents Swarna sub1 and AC39416A were used for generating mapping population. The 188 F 2:3 lines along with their corresponding contrast parents screened phenotypically and genotypically to develop reliable data in an attempt to unravel the tolerance of submergence during germination.
Screening for tolerance to anaerobic conditions during germination: Phenotypic Screening of 188 F 2:3 population of Swarna Sub1 / AC39416A along with parents was conducted at RARS, Maruteru, West Godavari district of Andhra Pradesh, located at an altitude of 5m above MSL, 81.44 0 E longitude and 26.38 0 N latitude, using complete randomized design with two replications in concrete tank as per Septiningsih et al. (2013b). Initially anaerobic stress is created and then level of their tolerance was recorded. From each line about 25 healthy seeds were soaked for a period of about 24 hours and incubated in dark or closed chamber yet again for 24 hours so that seeds will start germinating. Then pre-germinated seeds were sown in pro-trays which are lled with well puddled soil in such a way that the sprouted portion facing upwards or top end. And nally the pro-trays were arranged randomly inside the concrete tank and submergence is imposed by lling water up to 15 cm above the trays. Constant depth of water is maintained throughout the submergence treatment i.e two weeks and three weeks separate experiments. After submergence treatment for two weeks and three weeks pro-trays were kept outside of the concrete tank for about one week of de-submergence treatment during which watered daily, nally record the data from survived lines.

Genotyping of mapping population:
Fresh, young leaf samples were collected from all 188 F 2:3 lines and parents Swarna Sub1 and AC39416A at tillering stage (45 DAS) during early hours of a day and stored at -20ºC. Genomic DNA isolation was done using the modi ed Cetyl Tri Methyl Ammonium Bromide (CTAB) method Zheng et al. (1995). Parental polymorphism survey was conducted using a total of 687 SSR (Microsatellite) markers. The SSR primer sequences and other information like physical position, annealing temperature, expected PCR product size and sequences were obtained from Gramene markers database (http://www.gramene.org.in). Genotyping of entire population was done using the polymorphic SSR markers. 7.5 µl of master mix was added to each well of PCR plate having 2.5 µl of template DNA to make the nal volume to 10 µl per cell. Then PCR plate was kept in a thermal cycler for the reaction to take place.PCR ampli ed products i.e DNA samples (10 µl) were loaded into wells of 3 %agarose gels and run for 2 hours at constant mode with 110 volts where, DNA molecules with in an agarose gel matrix were subjected to steady electric eld; it will migrate through the gel towards the positive electrode, anode since DNA has a strong negative charge at neutral P H . The pores in the gel separate the linear fragments of DNA according to their size. The DNA fragments were then visualized under UV-trans-illuminator as bands and documented using gel documentation system (SYNGENE Gene ash U.K.).

Linkage map construction:
Linkage between the markers and QTL was detected by a statistical test called the Logarithm of Odds

Results
Phenotypic screening of mapping population: Early generation biparental mapping population consisting of 188 F 2:3 lines developed with contrasting parents Swarna Sub1 / AC39416A was screened for AG. The number of seedlings survived after submergence treatment was counted and expressed as anaerobic germination percent. The mean anaerobic germination per cent recorded after the two weeks of submergence among the population of 188 lines ranged from 0-95% with overall mean of 47.51% whereas mean AG per cent of the contrasting parents SwarnaSub1 and AC39416A was 40% and 85% respectively indicating signi cant differences for the trait. For three weeks of submergence treatment, the mean anaerobic germination per cent recorded between 0 and 95%, with overall mean of 37.66% and the mean AG per cent of the two contrasting parents was 27% and 75.6%, respectively, indicating that there was wider variation in the mapping population for anaerobic germination. (Fig. 1) Plant survival rate was calculated by counting the seedlings survived after one week of de-submergence.
The average survival rate of Swarna Sub1 and AC39416A was 35% and 80% respectively after two weeks of submergence, whereas for F 2:3 mapping population it ranged from 0 to 95% with overall mean of 36.74%. The plant survival rate after three weeks of submergence for Swarna Sub1 was 17.6% whereas 72% for the donor AC39416A. The average survival rate of population was 15.5% which indicated clear cut variation and following normal distribution. (Fig. 2) SSR marker based linkage map construction:   A total of seven putative QTLs viz., qAG2, qAG3, qAG7-1, qAG7-2, qAG9, qAG10 and qAG12 were identi ed and mapped using Icimapping (ICIM) method with manual input threshold LOD score of 2.5. (Table 3) Out of 7 QTLs found, 2 QTLs were located on chromosome 7, 1 QTL on each of the chromosomes 2, 3, 9, 10 and 12. All these 7 QTLs explained phenotypic variance of about 37.47% collectively for AG trait, with their individual contributions ranging from 2.99-8.67% of phenotypic variation and LOD scores of 2.65 to 5.86. The phenotypic variance explained by qAG2, was highest (8.66%) followed by qAG10 (8.60%) which were mapped on chromosome 2 and 10 respectively. Whereas, the highest LOD score (5.86) was shown by qAG10and qAG-7-2.   In general, rice seeds contain the complete set of enzymes needed for the degradation and use of starch for the growth and maintenance of the growing embryo; however, the activities of these enzymes are affected by anaerobic conditions due to the low availability of oxygen (Ismail et al., 2012). Some of these enzymes, especially alcohol dehydrogenase 1 (ADH1), rice alpha amylase (RAmy3D), and sucrose synthase, are more active in anoxia-tolerant rice genotypes under low-oxygen conditions during germination but are inhibited in sensitive genotypes, RAmy3D encoding starch-degrading enzymes, upregulated during germination under anaerobic conditions. This increased gene expression under anaerobic conditions leads to higher amylase activity for starch hydrolysis, which in turn enhances the activity of ADH1, a key enzyme involved in alcohol fermentation that is crucial for rice seed germination under anaerobic conditions. Upon germination, ethylene produced by the growing embryo may further promote cell expansion and starch hydrolysis, along with reduced abscisic acid (ABA) biosynthesis and increased gibberellic acid (GA) biosynthesis (Rauf et al., 2019). Hence, tolerance of anaerobic conditions during germination is an essential trait for direct-seeded rice cultivation in both rainfed and irrigated ecosystems (Septiningsih et al., 2013b).
Polymorphism is a measure of genetic diversity and varies with the parental combinations. The contrasting parents Swarna Sub1 and AC39416A selected for development of mapping population were initially surveyed for polymorphism using SSR markers to identify polymorphic markers between them.
In conclusion, the QTLs identi ed in the study majorly qAG12-1 may be considered for introgression into popular elite rice varieties otherwise susceptible for anaerobic germination after characterization of the mechanism underlying anaerobic germination and ne mapping.        Depiction of LOD score for AG on Chromosome 7 (SMA).