Pyramiding of three bacterial blight resistance genes for broad-spectrum resistance in deepwater rice variety, Jalmagna

Pyramiding of bacterial blight resistance genes

The parent polymorphism was detected for the donor (CRMAS 2232–85) and recurrent parent
(Jalmagna) with the markers pTA 248, RG 136 and xa5S, R (multiplex) for the genes
Xa 21, xa13 and xa 5 respectively (Table 1). The parents were polymorphic with respect to these genes. In addition, the parents
were screened with 236 rice microsatellite markers (Table 2) of which 120 were polymorphic and 60 were used for background selection.

Table 1. Markers used for foreground selection of three bacterial blight resistance genes in
marker-assisted backcross breeding

Table 2. Microsatellite markers those are polymorphic between Jalmagna and CRMAS 2232-85

Molecular markers were integrated in the backcross breeding programme upto BC₃F₂ generation. During the breeding procedure, foreground selection was practiced from
F₁ generation till BC₃F₃ generation at each stage to select the plants having resistance alleles of the three
target genes and only progenies having the resistance alleles were advanced for the
next generation (Figures 1, 2 and 3). Background selection was started from BC₁F₁ to BC₃F₁ generation and in each step genotype possessing highest genome content of the recipient
parent was selected to hybridize for next backcross. A total of 650 F₁ plants were produced and 150 F₁ plants were tested for the hybridity and confirmed by their heterozygosity for the
resistance gene linked markers of which 143 plants were observed to be true F₁s. The true F₁s were backcrossed using Jalmagna as a recurrent parent. These crossed seeds were
raised (360 BC₁F₁ seeds) for further backcrossing with Jalmagna. Ninety three BC₁F₁ plants showed the presence of Xa21 resistance gene specific bands (1000 bp) while 91 plants showed the presence of xa13 resistance gene specific bands (490 bp and 530 bp). One hundred sixteen BC₁F₁ plants showed the presence of xa5 resistance gene specific bands (160 bp). Based on the amplification of resistance
specific bands, 31 BC₁F₁ plants showed the presence of Xa21 and xa13 resistance genes while 42 BC₁F₁ plants showed the presence of Xa21 and xa5 resistance genes. Forty six BC₁F₁ plants showed the presence of xa13 and xa5 resistance genes. Only fourteen plants showed the presence of three BB resistance
genes Xa21, xa13 and xa5. Out of these 14 BC₁F₁ progenies, plant showing 77.5% of recurrent genome (Plant No.53) was backcrossed
with recurrent parent Jalmagna (Table 3).

Figure 1. PCR amplification of markers linked to resistance genes Xa21, xa13 and xa5 using primers A) pAT248, B) RG136 and C) xa5S and xa5R of BC₁F₁. Lanes on the top of the gel shows the BC₁F₁ plant no., CM- CRMAS 2232-85, J-Jalmagna, M1-Molecular weight marker (100bp plus
ladder), M2-Molecular weight marker (50bp ladder), Arrows indicate the resistance
specific markers.

Figure 2. PCR amplification of markers linked to resistance genes Xa21, xa13 and xa5 using primers A) pAT248, B) RG136 and C) xa5S and xa5R of BC₂F₁. Lanes on the top of the gel shows the BC₂F₁ plant no., CM- CRMAS 2232-85, J-Jalmagna, M1-Molecular weight marker (100bp plus
ladder), M2-Molecular weight marker (50bp ladder).

Figure 3. PCR amplification of markers linked to resistance genes, Xa21, xa13 and xa5 using primers A) pAT248, B) RG136 and C) xa5S and xa5R of BC₃F₁ plants. Lanes on the top of the gel shows the BC₃F₁ plant no. CM- CRMAS 2232-85, J-Jalmagna, M1-Molecular weight marker (100bp plus ladder),
M2-Molecular weight marker (50bp ladder).

Table 3. Number of triple resistant gene heterozygotes identified and estimation of recurrent
parent genome contribution

Total of 122 BC₂F₁ progenies were produced of which, twenty one, thirty three and thirty six BC₂F₁ plants showed presence of resistance genes, Xa21, xa13 and xa5, respectively. Based on the amplification pattern, 11 BC₂F₁ plants showed the presence of Xa21 and xa13 resistance genes while 13 BC₂F₁ plants showed the presence of Xa21 and xa5 resistance genes. Twenty three BC₂F₁ plants showed the presence of xa13 and xa5 resistance genes. Only nine plants exhibited the amplification of three resistance
genes Xa21, xa13 and xa5. The background selection of these nine BC₂F₁ plants with sixty polymorphic SSR markers exhibited the presence of 88.13 % to 91.82
% with an average of 90.95% of recurrent genome content. The plant containing 91.82%
genome content of Jalmagna (Plant No.53-21) was used for backcrossing (Table 3).

A total of 285 BC₃F₁ backcross derivative progenies were produced by backcrossing the plant showing 91.82%
recurrent genome with the recipient parent, Jalmagna. Twenty eight BC₃F₁ plants were positive for Xa 21, 35 for xa 5 and 14 for xa 13. Eighteen BC₃F₁ plants showed the presence of Xa21 and xa13 resistance genes while 14 plants showed the presence of Xa21 and xa5 resistance genes and 14 plants showed the presence of xa13 and xa5 resistance genes. Only fourteen plants showed the presence of three resistance genes
Xa21, xa13 and xa5. These BC₃F₁ plants showed recurrent genome content of Jalmagna ranging from 91 to 97% with an
average of 92.38% (Table 3). BC₃F₁ derivative SPJ53-21-77 and and SPJ53-21- 25 showed more than 95% genome content of
recipient parent were self pollinated to obtain the derivatives of BC₃F₂ generation. In BC₃F₂ generation, plants homozygous for three and two bacterial blight resistance gene
combinations were identified. It is observed that 26 plants containing Xa21, xa13 and xa5 genes; 31 plants with Xa21 and xa5; 31 plants with Xa21, xa13 and 30 with xa13 and xa5 amongst the BC₃F₂ derivatives. The plants with three and two genes were grown as BC₃F₃ lines.

Bioassays

Bioassays conducted against eight isolates of Xoo confirmed the resistance and susceptible reaction of the donor (CRMAS 2232–85) and
the recurrent (Jalmagna) respectively with the donor showing smaller range of average
lesion lengths (2.1-2.8 cm) while on Jalmagna, the lesion lengths were longer (9.4-12.8 cm)
(Table 4). The results indicated that the pyramided lines were better as compared to recurrent
parent, Jalmagna with regard to bacterial leaf blight tolerance. Screening of the
BC₃F₃ pyramided lines against Xoo isolates exhibited that all the pyramid lines were more effective in comparison to
the recipient parent. The lesion lengths observed on the lines containing Xa21?+?xa13 gene combination varied from 3.1 to 3.9 cm ; for Xa21?+?xa5 combination, 3.5-4.8 cm
; for xa5?+?xa13 combination 4.9 to 5.7 while 1.4 to 2.9 cm lesion length present in pyramided line
containing xa5?+?xa13?+?Xa21 combination. The individual values for the donor parent and recurrent parent are
in the range of 1.7–3.3 and 9.0–13.3 cm respectively. Though all the gene combinations
tested did not show any susceptible reaction to any of the eight isolates employed,
the gene pyramids with three genes displayed higher levels of disease resistance with
shorter lesion lengths against all BB isolates. Results indicated that the degree
of severity of the disease from the data, the order of gene combinations in conferring
resistance was: xa5?+?xa13??xa5?+?Xa21??xa13?+?Xa21??xa5?+?xa13?+?Xa21.

Table 4. Bacterial blight reaction of parental and BC₃F₃pyramided lines against differentXoostrains

Yield and agro-morphological traits of the pyramided lines

Fourteen three-gene pyramid and six two genes pyramid lines at BC₃F₃ generation along with the donor and recipient parents were evaluated during wet season,
2013 at CRRI, Cuttack. The recipient parent, Jalmagna recorded mean grain yield of
17.35 g/plant, while the donor parent (Swarna BB pyramided line) recorded 20.5 g/plant.
The test entries viz., SPJ23, SPJ25, SPJ50, SPJ51, SPJ52 and SPJ77 showed grain yields higher than recurrent
parent, Jalmagna (Table 5). Many test entries did not show any significant variation as compared to Jalmagna
in terms of flowering duration, panicles/m², plant stature as well as other characters that are considered under distinctness,
uniformity and stability (DUS) tests. The genetic distance coefficient on 14 agro-morphologic
traits of 20 pyramids and two parental lines revealed that two clusters were observed
and it is interesting to note that all the pyramided lines are similar to the recipient
parent, Jalmagna and are clubbed in cluster1 while in cluster 2, only solitary line
the donor parent is accommodated. (Table 5; Figure 4A).

Table 5. Agro-morphologic traits of pyramided and parental lines in BC₃F₃generation

Figure 4. Dendrograms illustrating the genetic relationship between parents and pyramided lines
(A) based on 14 agro-morphological traits; (B) based on microsatellite markers.

Background selection

The background selection was carried out for estimating the recurrent parent’s genome
content in the pyramided lines. Background selection was performed by using 60 SSR
markers among the lines possessing three resistance gene combinations in BC₁F₁, BC₂F₁ and BC₃F₁ generations. At BC₃F₁ generation, a total of 120 alleles from 60 markers were observed. The similarity
co-efficiency among all lines ranged from 0.791 to 0.952 suggesting a high level of
genetic similarity between the pyramids and Jalmagna. The dendrogram generated using
the SSR data grouped the 14 three-gene pyramid lines into two major clusters (Figure 4B) with cluster I having CRMAS 2232–85 and rest 13 pyramided lines were clubbed in
cluster II along with Jalmagna. Few members of cluster II viz., SPJ53-21-77, SPJ53-21-25, SPJ53-21-52 and SPJ53-21-18 were very close to Jalmagna
with 97, 95.33, 92.48 and 92.48% respectively.

Analysis of genome introgression on the carrier and non-carrier chromosomes

In CRMAS2232-85/Jalmagna combination, 5–6 microsatellite markers on each of three
carrier chromosomes in the genomic region flanking to xa5, xa13 and Xa21 were polymorphic. Based on six markers analysis, all the 14 lines showed heterozygosity
for donor segment introgression of xa5 between marker HYV59 and HYV5-37 in BC₃F₁ generation while exhibited homozygocity for Jalmagna genome and no drag to xa5 gene was observed. In the flanking region of xa13, for five polymorphic markers, nine lines showed introgression of the donor segment
of marker HYV14. In case of Xa21, six pyramid lines showed genetic drag of donor segment with the marker segment RM144
(Figure 5).

Figure 5. Analysis of genome introgression of 14 pyramided lines associated with resistance
genes (A) xa5 on chromosome 5; (B) xa13 on chromosome 8 and (C) Xa21 on chromosome 11 in Jalmagna and CR MAS 2232-85 BC₃F₃ derivatives.