Phenotyping for powdery mildew disease
Two trial sites were ideal experimental locations for testing PM disease resistant trait because of their higher disease pressure due to consecutively growth bean for more than five years resulting in the accumulation of E. polygoni inoculum season after season. The higher rainfall (>1000 mm) and higher relative humidity (>80%) also created conducive environment for disease development during these two bean growing seasons [27]. The environment was used to fully assess the PM resistance potentials of common bean genotypes in nature. As a result, highly significant (p<0.001) differences of resistant trait were observed among the 211 genotypes for powdery mildew in both 2017 and 2018 growing season [27]. Also, high significant interactions of genotype x environment (location and year) were observed, indicating the resistance to PM disease is affected by environments (Table 1). Disease severity was higher in 2018 with maximum disease severity score of 9 compared to 2017 with a maximum disease severity score of 6 among 206 genotypes (5 genotypes were lost during experiments) (Table 2). However, based on analysis of variance (ANOVA), the heritability of PM resistant trait was 0.69 in this collection, which is consistent with the result of 0.66 and 0.71 in two F2 populations reported by [28]. Disease severity of PM under natural infection was negatively correlated with yield at r=-0.24 (data not shown), indicating that PM had a significant effect on yield of common bean.
Table 1: Analysis of variance in the collection of common bean genotypes for PM disease.
Source of variation df SS MS p-value
Replication 1 29.4603 29.4603
Genotype (G) 210 668.3436 3.1826 <.001***
Environment (E) 1 624.8395 624.8395 <.001***
Season (S) 1 779.3892 779.3892 <.001***
GxE 210 337.5355 1.6073 <.001***
GxS 210 292.9858 1.3952 <.001***
ExS 1 105.0006 105.0006 <.001***
GxExS 210 224.8744 1.0708 0.093 ns
Residual 843 785.0397 0.9312
Note: df = degree of freedom; SS=Sum of Square; MS=Mean Square; ***=highly significant; ns=not significant. Significant level (p=0.05).
Table 2 Means and ranges of scores for disease severity of powdery mildew infection among 206 bean genotypes in two environments of northern Tanzania during 2017 and 2018 years.
Trait
|
Season
|
Min.
|
Max.
|
Mean of SE
|
P-value
|
Resistance to Powdery mildew (PM)
|
2017
|
1.00
|
6.00
|
1.51±0.02
|
<0.001
|
2018
|
1.00
|
9.00
|
2.87±0.05
|
<0.001
|
Population structure
Genetic relationship was revealed in the collection of 206 common bean lines using STRUCTURE software. Based on Bayesian model, the genetic population structure was captured by describing the molecular variation in each subpopulation using a separate joint probability distribution over the observed sequence sites or loci. The model was used for association analysis to reduce false association due to unequal distribution of alleles among subpopulation. The model grouped 206 genotypes into three clusters (K=3). The first cluster (K=1) consisted of 113 genotypes belonging to the Mesoamerican gene pool; the second cluster (K=2) consisted 72 genotypes belonging to the Andean gene pool and third cluster (K=3) consisted of 21 genotypes belonging to the admixture gene pool (Figure 1). Since this collection included some breeding lines derived from different sources of parents, there was a possibility that offspring combined genetic background from both Mesoamerican and Andean gene pools leading to this heterozygosity in the third cluster. The result suggested these subpopulations were associated with their genetic background. Interestingly, statistical analysis of disease severity scores based on genotypes in three structure groups showed the PM resistance was significantly different (p<0.01) between two gene pools, i.e. genotypes of Mesoamerican gene pool have significant higher resistances than those from Andean gene pool. However, genotypes in the admixture group have similar resistance with either gene pools depending on location and year, suggesting the expression of resistant trait in admixture genotypes was easier influenced by environment.
Marker trait associations for powdery mildew disease
Genome-wide association analysis of the resistance to PM disease showed comparable results of significant SNPs between two the year’s data. The most significant markers linked to PM resistance were located on chromosome 4 and 10 for both years, indicating the reliability of data set. A total of nine SNPs was detected as association with common bean powdery mildew resistance on the Pv04, Pv10, and Pv11 (Figure 2; Table 3). The SNP (ss715647913) at Pv10 was most significant (p = 7.22 x 10-07) contributing the highest phenotypic variation of 12.28%. While the SNPs ss715650009 and ss715639212 had the highest significance levels representing 10.69% and 10.42% of phenotypic variation, respectively, on Pv04. There were two peaks of significant SNPs located in the range of 2.01-4.30 Mb and 45.01-45.02 Mb on Pv04, while only one significant SNP peak was detected from 40.57-41.13 Mb on Pv10. Significant SNPs were observed between 43.67-43.79 Mb on Pv11 only in 2018 (Table 3, Figure 2).
Table 3 Marker-trait association showing significant SNPs, chromosome position, and contribution to phenotypic variation for powdery mildew disease.
SNP*
|
Chr.
|
Position (bp)
|
P-value
|
R2 (%)
|
ss715647913
|
10
|
40,571,646
|
7.22 x 10-07
|
12.28
|
ss715647918
|
10
|
40,624,302
|
9.36 x 10-07
|
12.07
|
ss715645507
|
10
|
40,937,977
|
3.73 x 10-06
|
10.93
|
ss715650009
|
4
|
2,013,385
|
5.22 x 10-06
|
10.69
|
ss715648114
|
4
|
4,303,144
|
9.54 x 10-05
|
8.29
|
ss715639212
|
4
|
45,014,435
|
1.11 x 10-6
|
10.42
|
ss715648250
|
11
|
43,773,598
|
7.23 x 10-06
|
11.01
|
ss715648263
|
11
|
43,673,759
|
2.29 x 10-05
|
9.87
|
ss715648249
|
11
|
43,792,293
|
5.35 x 10-05
|
10.81
|
SNP*=Single Nucleotide Polymorphism code; Chr.=Chromosome; R2 =phenotypic variation.
Identification of candidate genes associated with powdery mildew resistance
There were 181 coding genes observed within the interval of significant SNPs on Pv04, 46 on Pv10, and 24 on Pv11. Using the BLAST analysis of these coding genes nearby the significant SNPs against protein database in GenBank, nine coding genes were considered as candidate resistance genes for the PM disease (Table 4). On the Pv04, three coding genes located at the telomeres were homologous to resistance genes RPP13, TMV-N, and LRR receptor-like serine/threonine protein kinase (LRR-RLK) RPK2, while two closing to the opposite telomere were homologous to one LRR-RLK and the transcription factor (TF) MYB 87. Two coding genes on Pv10 hit RLM1A-like and At4g11170 putative resistance genes. Additional two coding genes on Pv11 were similar with LRR-RLK. The sequences of these candidate resistance genes were listed in Additional file 1.
Table 4 Identified positional candidate genes linked to powdery mildew diseases resistance in dry bean.
Candidate gene
|
Position in Pv chromosome (bp)
|
Similarity
|
Gene accession
|
Gene Annotation
|
Phavu_004G036200g
|
Pv04:4014080
-4016937
|
95%
|
XM_017584784.1
|
RPP13-like
|
Phavu_004G028900g
|
Pv04:3126300
-3130090
|
99%
|
XM_017584233.1
|
TMV N-like
|
Phavu_004G037500g
|
Pv04:4141435
-4145345
|
99%
|
XM_014658246.2
|
LRR-RLK (RPK2)
|
Phavu_004G173300g
|
Pv04:45377489-45382880
|
94%
|
XM_028069263.1
|
LRR-RLK
|
Phavu_004G173500g
|
Pv04:45394112-45395393
|
100%
|
XM_014638647.2
|
TF-MYB87
|
Phavu_010G1320001g
|
Pv10:40237562-40238278
|
95%
|
XM_022786275.1
|
RLM1A-like
|
Phavu_010G136800g
|
Pv10:40920056-40929831
|
95%
|
XM_014660831.2
|
At4g11170.1
|
Phavu_011G167800g
|
Pv11:4379752
-43809211
|
98%
|
XM_028053325.1
|
LRR-RLK
|
Phavu_011G169300g
|
Pv11:4398238
-43999989
|
97%
|
XM_028054653.1
|
LRR-RLK
|