during anaerobic germination
OE Manangkil, AB Rafael, WV Barroga, PNM Marcelo
In the Philippines, rice varieties are mainly bred for transplanting method due to its various advantages such as good seedling establishment for weed competition, low seed requirement, and controlled plant density.
However, the system requires a lot of labor inputs which includes seedling pulling, distribution and transplanting. In the present time, such tedious endeavor is getting inadequate owing to decreasing manpower. As a result, direct seeding as an alternative method of establishing rice is gaining more acceptances to the farmers due to reduced labor, hence, reduced cost of farm inputs, labor and capital compared to transplanted rice. Farmers have been practicing direct seeding system using rice varieties for transplanted system. Owing to this, high mortality rate of germinating seeds during monsoon season affected the takings of rice farmers. Recent results in low seed requirement using mechanical seeder further added to the acceptance of the method by farmers. Yield is almost similar to the transplanted rice,
however there are limited varieties suited for direct seeding. Knowing the intolerance of our modern cultivars to anaerobic germination making it not suited for direct-seeded cultivation and knowing the harsh environmental conditions of direct seeded rice, high mortality of seedlings or germinating seeds are observed in direct seeding rice varieties bred for transplanted system. However, introgression of genes that control anaerobic germination tolerance in rice, anaerobic germination 1 gene (AG1) is a way to alter our modern varieties in their reaction to impeded gas exchange in submerged or anaerobic condition. In this way, varieties bred for transplanted system could perform better to direct seeded cultivation.
The ability of rice seeds to germinate even in the presence of water is important for the plants’ establishment and its eventual success. Aside from avoiding high mortality of the seeds, anaerobic germination is also seen as way to control the growth of weeds and pest infestation like birds and rodents.
Tolerance under anoxic condition of seeds can be considered as one component trait of seedling vigor in rice. It was proven that during anoxia or absence of oxygen, intolerance of certain genotypes exhibited low sugar mobilization than malfunctioning of metabolic machinery for glycolysis and ethanol production (Huang et al. 2003). Mustroph et al.
(2006) demonstrated that rice shoots prevented excessive production of acetaldehyde and subsequent post-anoxic cell injuries and leaf damage. The observation can be due to the induction of aldehyde dehydrogenase (ALDH) during anaerobiosis (Tsuji et al. 2003; Nakazono et al. 2000). Consequently, the lower acetaldehyde formation is thus proposed to be another reason for the high tolerance of rice plants to anaerobic and post-anaerobic conditions (Mustroph et al. 2006).
This study attempts to introgress anaerobic germination tolerance with submergence tolerance genes, Sub1 + AG1 (Parent: Ciherang Sub1+AG1) to NSIC Rc222. It will determine the genetic and molecular mechanism of anaerobic germination tolerance to rice transplanted varieties and its relationship with agronomic performance in wet-direct seeded rice genotypes
Highlights:
• F1 seeds of Ciherang (Sub1+ Ag1) X NSIC Rc222 introgressed with anaerobic germination tolerance were planted in the glasshouse in 2014DS. Thirty-seven individual F1 plants were backcrossed to NSIC Rc222 and 1,363 BC1F1 seeds were produced (Table 15 and Figure 25).
• Leaf samples of Ciherang (Sub1+Ag1) and NSIC Rc222 were
collected for DNA extraction. DNA polymorphism survey of the parents (Ciherang (Sub1+Ag1) and NSIC Rc222) was conducted using 459 simple sequence repeats (SSR) markers covering the 12 chromosomes of rice (Figure 26). Of these, 123 SSRs showed polymorphism between the parents.
• Optimization of primers using ART5, RM8300, RM105 and RM24161 were done. Both ART5 and RM8300 were found polymorphic to Ciherang (Sub1+Ag1) and NSIC Rc222 but RM105 and RM24161 were found to be monomorphic (Figure 27).
• In 2014 WS, 1,025 BC1F1 seeds were established in the glasshouse and only 422 BC1F1 plants survived. The surviving BC1F1 plants were individually collected of leaf samples for DNA extraction and were subjected to target gene assay.
Published markers for submergence tolerance genes (ART5 and RM8300) and SSR markers for anaerobic germination (RM3769 and RM24141) were used for heterozygosity test (Figure 28).
• In Table 16, out of 1,025 BC1F1 plants, 46 BC1F1 plants with the Sub1 and Ag1 genes were identified. Total of 575 BC2F1 seeds were generated from 12 BC1F1 plants (no. 20-5, 20-21, 32-14, 32-16, 33-20, 41-2, 45A-14, 53a-3, 53B-1, 62-16, 62-20, 62-22). Background selection using polymorphic SSR markers were ongoing to select the best 3 BC1F1 plants to be backcrossed to NSIC Rc222.
Table 15. Total number of generated BC1F1 during 2014 DS.
Cross Combination Generated BC1F1
F1-14/NSIC Rc222-65 115
F1-17/NSIC Rc222-91 31
F1-18/NSIC Rc222-66 51
F1-18/NSIC Rc222-80 46
F1-20/NSIC Rc222-52 60
F1-21/NSIC Rc222-58 33
F1-24/NSIC Rc222-38 24
F1-27/NSIC Rc222-52 13
F1-29/NSIC Rc222-70 59
F1-30/NSIC Rc222-82 67
F1-32/NSIC Rc222-79 49
F1-33/NSIC Rc222-77 79
F1-34/NSIC Rc222-81 79
F1-37/NSIC Rc222-65 34
F1-39/NSIC Rc222-65 78
F1-40/NSIC Rc222-81 30
F1-41/NSIC Rc222-81 20
F1-42/NSIC Rc222-99 9
F1-43/NSIC Rc222-75 18
F1-44/NSIC Rc222-162 3
F1-45/NSIC Rc222-85 81
F1-45/NSIC Rc222-6 5
F1-46/NSIC Rc222-6 37
F1-47/NSIC Rc222-92 16
F1-47/NSIC Rc222-84 9
F1-48/NSIC Rc222-90 42
F1-48/NSIC Rc222-110 24
F1-49/NSIC Rc222-82 22
F1-51/NSIC Rc222-145 36
F1-53/NSIC Rc222-74 24
F1-53/NSIC Rc222-66 8
F1-53/NSIC Rc222-80 4
F1-55/NSIC Rc222-179 30
F1-56/NSIC Rc222-153 30
F1-61/NSIC Rc222-110 71
F1-62/NSIC Rc222-99 26
Figure 25. Establishment of anaerobic germination and crossing activities in the glasshouse during 2014 DS.
LADDER CIHERANG sub 1 NSIC Rc222 NSIC Rc160 ITALICA LIVORNO IR64 N22 PSB Rc82 IR52 CIHERANG sub 1 NSIC Rc222 NSIC Rc160 ITALICA LIVORNO IR64 N22 PSB Rc82 IR52 CIHERANG sub 1 NSIC Rc222 NSIC Rc160 ITALICA LIVORNO IR64 N22 PSB Rc82 IR52 CIHERANG sub 1 NSIC Rc222 NSIC Rc160 ITALICA LIVORNO IR64 N22 PSB Rc82 IR52 CIHERANG sub 1 NSIC Rc222 NSIC Rc160 ITALICA LIVORNO IR64 N22 PSB Rc82 IR52 LADDER A B A A B A B A A A A A A B A B A B B A B A B A A A B B A B A B A B A A A B A B
RM584 RM176 RM7193 RM549 RM197
Figure 26. Polymorphism survey between Ciherang (Sub1+Ag1) and NSIC Rc222 using SSR markers.
20bp ladder ciherang sub1 NSIC Rc222 NSIC Rc160 Italica livorno IR64 N22 PSB RC82 IR52 ciherang sub1 NSIC Rc222 NSIC Rc160 Italica livorno IR64 N22 PSB RC82 IR52 ciherang sub1 NSIC Rc222 NSIC Rc160 Italica livorno IR64 N22 PSB RC82 IR52 20bp ladder A A A B A B A A A B A B A B A B A B B A B B B A
RM105 ART5 RM8300
Figure 27. Optimization of primers using RM105, ART5 and RM8300.
1Kb LADDER CIHERANG CIHERANG NSIC Rc222 NSIC Rc222 BC1F1 62-10 BC1F1 62-11 BC1F1 62-12 BC1F1 62-13 BC1F1 62-14 BC1F1 62-15 BC1F1 62-16 BC1F1 62-17 BC1F1 62-18 BC1F1 62-19 BC1F1 62-20 BC1F1 62-21 BC1F1 62-22 1Kb LADDER
ART5 A A NA B B H B H H B H H A B H H H
1Kb LADDER CIHERANG CIHERANG NSIC Rc222 NSIC Rc222 BC1F1 62-10 BC1F1 62-11 BC1F1 62-12 BC1F1 62-13 BC1F1 62-14 BC1F1 62-15 BC1F1 62-16 BC1F1 62-17 BC1F1 62-18 BC1F1 62-19 BC1F1 62-20 BC1F1 62-21 BC1F1 62-22 1Kb LADDER
RM3769 A A B B B B H B H B H B H B H U H
1Kb LADDER CIHERANG CIHERANG NSIC Rc222 NSIC Rc222 BC1F1 62-10 BC1F1 62-11 BC1F1 62-12 BC1F1 62-13 BC1F1 62-14 BC1F1 62-15 BC1F1 62-16 BC1F1 62-17 BC1F1 62-18 BC1F1 62-19 BC1F1 62-20 BC1F1 62-21 BC1F1 62-22 1Kb LADDER
RM24141 A A B B B B U U H B H B H B H H H
1Kb LADDER CIHERANG CIHERANG NSIC Rc222 NSIC Rc222 BC1F1 62-10 BC1F1 62-11 BC1F1 62-12 BC1F1 62-13 BC1F1 62-14 BC1F1 62-15 BC1F1 62-16 BC1F1 62-17 BC1F1 62-18 BC1F1 62-19 BC1F1 62-20 BC1F1 62-21 BC1F1 62-22 1Kb LADDER
RM8300 A A B B B H B H H B U H A B H H H
Figure 28. Heterozygosity test in F1 plants using markers for Sub1 and Ag1 genes (A=Ciherang (Sub1+Ag1) and B= NSIC Rc222) showing the introgression of both genes. Note: “A” denotes allele pattern similar to donor parent, “H” denotes heterozygous allele, “B“ denotes allele pattern similar to
recurrent parent, “U” denotes outlier and “NA” denotes no amplicon.
Table 16. Total number of generated BC2F1 during 2014WS.
Cross Combination Generated BC2F1
BC1F1-20-5/NSIC Rc222-65 16
BC1F1-20-21/NSIC Rc222-65 33
BC1F1-32-14/NSIC Rc222-79 72
BC1F1-32-14/NSIC Rc222-79 22
BC1F1-33-20/NSIC Rc222-77 28
BC1F1-41-2/NSIC Rc222-81 67
BC1F1-45A-14/NSIC Rc222-85 89
BC1F1-53A-3/NSIC Rc222-74 58
BC1F1-53B-1/NSIC Rc222-66 39
BC1F1-62-16/NSIC Rc222-99 68
BC1F1-62-20/NSIC Rc222-99 14
BC1F1-62-22/NSIC Rc222-99 69