# Presentation: 412936

## Linkage Among Stem and Crown Rust Resistance Genes

- Diego Vilaró, Angela Sebelius, and Michael McMullen
- NDSU, Plant Sciences

## Basis for the Study

- Lines with *pg-a *and B in homozygous condition have not been identified
- Linkage relationship of  N and *pg-a*

**Notes:**
The basis for the study was that in previous evaluations for different populations involving breeding for both pg-a stem rust resistance and IAB crown rust resistance together, there have been no lines identified for homozygous resistant condition.
The identification of such a line would prove to be useful for future breeding efforts for the development of a CRR-SSR line.
However previous evaluations have determined that IAB crown rust resistance is associated with one of the components of the pg-a complex.
And there is evidence of a linkage relationship for naked character and pg-a SRR

## Objective

- Determine *pg-a* stem rust resistance and B crown rust resistance relationship:
- ND873126//ND873126/Morton
- Paul//Morton/Paul
- Identify a homozygous line for *pg-a* and B

**Notes:**
The objectives of the study were to determine the linkage relationship between pg-a stem rust resistance and IAB crown rust resistance in BC1 population ND873126//ND873126/Morton and the relationship of a possible linkage for Naked character, CRR, and SRR in the BC1 population Paul//Morton/Paul
The second objective is to possibly identify a homozygous line for pg-a and IAB resistance

## Genetic Material

**ND873126 (*****pg-a*****)**
- Resistance conferred by at least two complementary recessive genes
**‘****Morton’ (B)**
- CRR derived from IAB605X
**‘****Paul’ (*****pg-a*****, NN)**
- A component of the *pg-a *complex appears associated with a gene conferring the naked character

**Notes:**
The three lines selected for the study were ND873126, Morton, and Paul.
ND873126 is a NDSU breeding line that possesses pg-a resistance. Previous studies have determined that pg-a resistance is conferred by at least two complementary recessive genes
Morton possesses crown rust resistance derived from the Iowa source IAB605X in which we defined as B in the study. 
The third line used was Paul a naked line that possesses pg-a stem rust resistance. Prior evaluations have found that one component of the pg-a complex is associated with the gene conferring the naked character. 

## SR and CR Isolates

- Stem rust race NA67
- (*16,a/1,2,3,4,8,9,13*)
- Composite of crown rust races
- Avirulent on Morton (B)
- Virulent on most CR resistance genes

**Notes:**
The two sources of inoculum used in the study were stem rust race NA67 which is avirulent on pg-a, and a composite of crown rust races that are virulent on most resistant gene yet avirulent on B

## Relationship of pg-a and B

- Materials and Methods
- ND873126/Morton
- F2, BC1F1 and BC1F2 generations
- Two sets from different F1 plants
- Composite CR and NA67
- Seedling evaluations
- Two classes of readings

**Notes:**
The relationship we first looked at was between pg-a and B. The evaluation involved the two sets derived from different F1 plants at the F2 generation for the cross ND873126/Morton and BC1 ND873126//ND873126/Morton at the F1 and F2 generations.
CR and SR evaluations were done at the seedling stage in the greenhouse and inoculated with Comp and NA67 isolates.
The IT exhibited were classified into two classes Resistant and Susceptible

## ND873126/Morton F2 Seedling CR IT

- Set #1
- 85R:29S
- Data fit the ratio 3R:1S
- Set #2
- 195R:60S
- Data fit the ratio 3R:1S

**Notes:**
The CR IT from the two sets of F2’s for the cross ND873126/Morton fit the ratio 3R:1S. Crown rust resistance was conferred by a single dominant gene

## ND873126/Morton F2 Seedling SR IT

- Set #1
- 29R:85S
- Data fit the ratio 1R:3S
- One recombinant (CRR-SRR) plant
- Set #2
- 69R:186S
- Data fit the ratio 1R:3S

**Notes:**
For SR IT both sets fit the ratio 1R:3S.  SRR was coferred by a single recessive gene. ND873126 and Morton differ by one factor for pg-a. In Set #1, one recombinant CRR-SRR line was recovered indicating that B and A1 are tightly linked.

## ND873126//ND873126/Morton BC1F1 CR and SR IT

| Classes | No. of BC1F1 seedlings |  |
| --- | --- | --- |
|  | Set # 1 | Set # 2 |
| CRR-SRS | 53 | 33 |
| CRS-SRR | 72 | 32 |
| CRS-SRS | 19 | 0 |
| CRR-SRR | 0 | 0 |
| Total | 144 | 65 |

**Notes:**
The two sets of BC1F1 seedlings from different F1 plants were evaluated for CR and ST IT’s. As expected there was no recombinant for Set #2 B_A1. 
However, in set 1 19 CRS:SRS lines were recovered. These lines may have been recombinants for B and A1, but no CRR:SRR lines were recovered. After many discussions with Howard Rines, it was proposed the possible presence of an inhibtor gene acting on B.

## ND873126//ND873126/Morton BC1F1 CR and SR IT

| Classes | No. of BC1F1 seedlings |  |
| --- | --- | --- |
|  | Set # 1 | Set # 2 |
| CRR-SRS | 53 | 33 |
| CRS-SRR | 72 | 32 |
| CRS-SRS | 19 | 0 |
| CRR-SRR | 0 | 0 |
| Total | 144 | 65 |

**Notes:**
The two sets of BC1F1 seedlings from different F1 plants were evaluated for CR and ST IT’s. As expected there was no recombinant for Set #2 B_A1. 
However, in set 1 19 CRS:SRS lines were recovered. These lines may have been recombinants for B and A1, but no CRR:SRR lines were recovered. After many discussions with Howard Rines, it was proposed the possible presence of an inhibtor gene acting on B.

## Proposed Inhibitor Gene Linked to a1

| Classes | No.  BC1F1 plants | F1 gamete |
| --- | --- | --- |
| CRR-SRS | 53 | B-A-I |
| CRS-SRR | 72 | b-a-I |
| CRS-SRS | 19 | B-A-i |
| CRR-SRR | 0 | B-a-i |
| Total | 144 |  |

- 19/144 + 19/144 = 0.26

**Notes:**
From the 19 CRS-SRS, it was postulated that there were 19 CRR-SRR within the 72 class. Total of 38 recombinant lines were proposed, the inhibitor was proposed to be located 0.26  or 26 centi morgans from A1.

Recessive inhibitor linked to a

## ND873126//ND873126/Morton BC1F1 CR Seedling IT

| Set | Total | R | S | Ho.† |
| --- | --- | --- | --- | --- |
| 1 | 144 | 53 | 91 |  |
| Test 1:1 |  |  |  | Reject |
| Test 1:3 |  |  |  | Reject |
| 2 | 65 | 33 | 32 |  |
| Test 1:1 |  |  |  | Accept |
|  |  |  |  |  |

- † χ2 to fit various ratios (*P*=0.05, 1 df).
-    Rejected if χ2 value ≥ 3.84

**Notes:**
When we looked at the results for BC1F1 CR IT, Set 1 did not fit the ratios 1:1 for a single gene or 1:3 for presence of an inhibitor. If the inhibitor is linked to pg-a than this can explain the deviation from the expected.
Set 2 fit the ratio 1:1 for a single dominant gene for conferring CR resistance.

## ND873126//ND873126/Morton BC1F1 SR Seedling IT

| Set | Total | R | S | Ho.† |
| --- | --- | --- | --- | --- |
| 1 | 144 | 72 | 72 |  |
| 2 | 65 | 32 | 33 |  |
| Total | 209 | 104 | 105 |  |
| Test 1:1 |  |  |  | Accept |

**†** χ2 to fit a 1:1 ratio (*P*=0.05, 1 df). Rejected if χ2 value ≥ 3.84

**Notes:**
BC1F1 SR IT, both sets fit the ratio 1:1 indicating single recessive gene conferring SR resistance.

## ND873126//ND873126/Morton Set #1 BC1F2 CR Seedling IT

|  | No. of plants |  |  |  |
| --- | --- | --- | --- | --- |
|  | Total | R | S | Ho.† |
| Test | 1248 | 960 | 288 |  |
|  9 : 7 |  |  |  | Reject |
|  3 : 1 |  |  |  | Accept |
| 2.55 : 1.45 ¶ |  |  |  | Reject |

**† **χ2 for various ratios (*P*=0.05, 1df). Rejected if χ2 value ≥ 3.84
- ¶ Model is B_A1 – I;  I  26 cM  from A1
- Note: No CRS-SRS lines observed

**Notes:**
BC1F1 CRR lines were further evaluated at the BC1F2 generation. We expect them to be heterozygous for both B and A.The data set fits the ratio 3R:1S for a single dominant gene conferring Crown Rust Resistance. There were no CRS:SRS lines recovered for the BC1F2 generation. 

## ND873126//ND873126/Morton Set #1 BC1F2 SR Seedling IT

|  | No. of plants |  |  |  |
| --- | --- | --- | --- | --- |
|  | Total | R | S | Ho.† |
| Test | 1314 | 356 | 958 |  |
| 1 : 3 |  |  |  | Accept |

- † χ2 (*P*=0.05, 1 df). Rejected if χ2 value ≥ 3.84

**Notes:**
Results from BC1F2 SR Seedling IT fit the ratio 1R:3S for a single gene model. 

## ND873126//ND873126/Morton Set #2 BC1F2 CR Seedling IT

|  | No. of plants |  |  |  |
| --- | --- | --- | --- | --- |
|  | Total | R | S | Ho.† |
| Tests | 252 | 172 | 80 |  |
|  9 : 7 |  |  |  | Reject |
|  3 : 1 |  |  |  | Reject |
| 2.55 : 1.45¶ |  |  |  | Accept |

- † χ2 for various ratios (*P*=0.05, 1df). Rejected if χ2 value ≥ 3.84
- ¶ Model is B_A1 - I ;  I 26 cM  from A1
- Note: 23 CRS-SRS lines observed

**Notes:**
BC1F2  from BC1F1 CRR lines were evaluated for CR and SRR. Remember that the BC1F1 data set best fitted a single gene model for CR.
However the data fits the ratio 2.55R:1.45S. The results indicated that an inhibitor gene 0.26 from A1. The set was inoculated also with NA67. From the set, 23 CRS-SRS lines were observed indicating the possible presence of an inhibitor gene. 

## ND873126//ND873126/Morton Set #2 BC1F2 SR Seedling IT

|  | No. of plants |  |  |  |
| --- | --- | --- | --- | --- |
|  | Total | R | S | Ho.† |
| Test | 249 | 77 | 172 |  |
| 1 : 3 |  |  |  | Reject
0.031 |

- † χ2 (*P*=0.05, 1 df). Rejected if χ2 value ≥ 3. 84

**Notes:**
The data for SR IT BC1F2 seedlings did not fit the ratio 1:3 for a single gene. Excess resistant lines were observed supporting that the data is closer to fitting a single gene than a two gene model suggested for pg-a resistance. 

## Relationship of pg-a and B Conclusions

- SRR single recessive gene.
- CRR single dominant gene
- B and A1 are tightly linked.
- The model B_A1 – I  26 cM between A1 and I  and the simple B_A1 model are proposed from BC1F1 and BC1F2 data.

**Notes:**
The overall conclusions for the population were that CRR and SRR both are conferred by single genes. The genes B and A1 are completely linked because only one recombinant CRR-SRR line was identified. BC1F1 and BC1F2 data were in-conclusive in the possible presence of an inhibitor gene 0.26 from A1. 

## Relationship of N, B, and a 

- Genetic Material
- Paul – NNbbaa
- Morton- nnBBAA
- Materials and Methods
- BC1 Paul//Morton/Paul
- Two sets BC1F1 and one BC1F2
- Composite and NA67
- Two classes
- Naked phenotype class

**Notes:**
To determine the relationship of Naked character, IAB CRR and pg-a SRR created the Morton/Paul F1 and using Paul as the recurrent parent we created BC1. Two sets of BC1F1 seedlings were inoculated with Comp and NA67.Only one set was further evaluated at the BC1F2 generation. Like the first population the IT’s were classified as either Resistant or Susceptible.

## Paul//Morton/Paul BC1F1 SR and CR Seedling IT

- Data fit the expected 1R:1S ratio for both CRR and SRR

**Notes:**
BC1F1 SR seedling evaluation for both data sets fit the ratio 1R:1S for a single recessive gene conferring stem rust resistance.

## Paul//Morton/Paul  BC1F1 Gametic Array and Genotypic Frequencies

| Spikelet –B – pg-a |  | BC1F1 genotypes |
| --- | --- | --- |
| N-B-A | XO | 6 |
| N-B-a | XO | 0 |
| n-B-A | PT | 12 |
| n-B-a | XO | 0 |
| N-b-A | XO | 0 |
| N-b-a | PT | 17 |
| n-b-A | XO | 0 |
| n-b-a | XO | 11 |

**Notes:**
Here we are looking at the gametic array for three traits spikelet type, IAB resistance, and pg-a resistance . 17 showed the Paul PT N-b-a , 12 fell into the Morton PT class n-B-A , 6 fell into the recombinant class N-B-A, while the other 11 lines fell into the recombinant class n-b-a.

## Linkage	

- Recombinant classes N-B-A and n-b-a
- 6/46 + 11/46 = 0.37
- b_a

**Notes:**
From here we calculated linkage involving the two recombinant classes N-B_A and n-b_a.  From the data it appeared that a distance 37cM between N and a1 indicating the genes are loosely linked. No recombinants were recovered for b and a1 indicating the genes are completely linked.

## Paul//Morton/Paul BC1F1 Naked or Intermediate Spikelet Classes

| Set | Total  | NN | Nn | Ho.† |
| --- | --- | --- | --- | --- |
| 1 | 46 | 27 | 19 |  |
| 2 | 44 | 17 | 27 |  |
| Total | 90 | 44 | 46 |  |
| Test 1:1 |  |  |  | Accept |

- † χ2 to fit the ratio (*P*=0.05, 1 df). Rejected if χ2 value ≥ 3.84

**Notes:**
We looked at both sets of  BC1F1’s for type of spikelets produced. Both sets of data fit the ratio 1NN:1Nn indicating a single partial dominant gene controlling the naked character in this material. 

## Paul//Morton/Paul BC1F2 N, B, a

|   BC1F1 phenotype | BC1F1 genotype | No. 
families |  |
| --- | --- | --- | --- |
| CRS-SRR |  |  |  |
|  | NN bb aa | 17 | PT |
|  | Nn bb aa  | 11 | XO |
| CRR-SRS |  |  |  |
|  | Nn Bb Aa | 12 | PT |
|  | NN Bb Aa | 06 | XO |

**Notes:**
The first set were evaluated at the BC1F2 generation for spikelet, IAB resistance, and pg-a resistance. The results from the evaluation verified the BC1F1 genotypes. Recombinants between N and B were observed

## Relationship of N, B, a  Conclusions

- Morton and Paul differ by one factor for *pg*-*a*
- b is completely linked to a
- N approx. 37 cM from B

**Notes:**
The data from this population suggested that Morton and Paul differ by one factor for pg-a.
The recessive form of b is completely linked to the recessive form a1.
The BC1F1 data suggested the gene for multifloret is loosely linked to a1.

## Overall Conclusions

- SRR conferred by a single gene
- CRR conferred by a single gene
- B and A and b and a are completely linked
- Possible presence of an inhibitor gene 26 cM from A
- One CRR-SRR plant was recovered

**Notes:**
The overall conclusions from the study were that:
In the population ND873126/Morton there was indication the possible presence of an inhibitor gene 0.26 from A1 in two of four BC1 analysis however the results were not conclusive even though the simple model B_A1 is favored.
The dominant forms of B and A1 and the recessive forms of b and a1 are completely linked.
In both populations it was determined that pg-a resistance was conferred by a single gene

## Acknowledgments

- UDSA-ARS National Oat Germplasm Enhancement Project
- PepsiCo/Quaker Oats
- Pioneer Hi-Bred International, Inc.