# Presentation: 017590

## Interspecific Testing of Gender-associated SCAR Markers in Salix

- Oak Ridge National Laboratory
- State University of New York-Syracuse
- BFDP Subcontractor’s Workshop
- November 8, 2001

## Background

**Discovery of RAPD markers associated with femaleness in certain families of *****S. viminalis***
**Modification of quantitative genetics model of sex determination in *****S. viminalis***
**Two loci, each with 4 alleles**
**Epistatic interaction**
**RAPD markers associated with one allele of hypostatic locus **

**Notes:**
Cilla proposed a working model of sex determination, suggesting a 2 locus, 4 allele epistatic interaction. Predicted 3/13 families should segregate at a single locus for an allele determining gender.
Identification of a  RAPD marker linked to femaleness in certain genetic backgrounds. Most known markers linked to males (except kiwi). Marker is biparentally inherited and linked to a nuclear gene, but subject to expression of at least one alternative, unknown locus.

## Putative sex-determination locus in S. viminalis

**Putative sex-determination locus in *****S. viminalis***

**Notes:**
RAPD marker sequenced and converted to SCAR; additional, marker developed.
Estimates of crossing over used to calculate mapping distance from putative sex determination locus.

## Objectives

**Task 1: Test small segregating F1 populations of *****S. eriocephala***** progeny for the presence of the SCAR markers developed for *****S. viminalis***** **
**Task 2: Determine significance of association of markers to gender in *****S. eriocephala***

## Methods

**Survey parental genotypes of *****S. eriocephala***** F1 families**
**Select families for marker analysis**
**Perform segregation analysis of markers within and among families**

## Survey of S. eriocephala P1

**Identify alternate allelic combination of both markers**

**Notes:**
Gender phenotype vs. marker phenotype
Because SCAR markers dominant, no a priori knowledge of marker presence signifying homozygous or heterozygous allelic state.
Look at specific parents to identify 1) families representing alternate allelic combination for both markers; that is one parent has both markers and the other parent lacks both.

## S. eriocephala Family Selection

| P1 | ♂ |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| ♀ | 646 | 95061 | 95316 | 95019 | 95024 | 95022 | 95054 | 287 |
| 96305 | 98-1 |  | 98-3 | 98-4 | 98-5 |  |  |  |
| S652 | 98-106 | 98-6 | 98-7 | 98-8 | 98-9 | 98-10 |  |  |
| S25 |  |  | 98-11 | 98-12 | 98-13 | 98-14 | 98-15 | 98-42 |
| 95331 |   |  |  | 98-16 | 98-17 | 98-18 | 98-19 | 98-43 |
| 95064 | 98-26 | 98-27 |  |  |  | 98-28 | 98-29 | 98-44 |
| 95306 | 98-31 | 98-32 |  |  |  |  | 98-34 | 98-45 |
| 95311 |  | 98-37 | 98-38 | 98-39 |  |  |  | 98-46 |

## Segregation Analysis:SCAR 354520

**41 F1 genotypes in 5 *****S. eriocephala***** families**
**19 females, 20 males, 2 unknown gender**
**No association with gender**

**Notes:**
Family ID above and below section of gel associated with family.
Female parent green, male parent
For 354-520, none of the families satisfy both criteria of alternate homozygous allelic form and expression of hypostatic locus

## Segregation Analysis:SCAR AE08780

**In Family 984, marker associated 100% with female progeny**
**Test other families sharing mother 96305:**
**981**
**983**
**985**

**Notes:**
Other families represent an additional 26 genotypes.

## Segregation Analysis:SCAR AE08780

**Goodness-of-fit: progeny of 96305 families 98-1, 98-3,98-4**
**15/15 females**
**Χ****2****=15.0, p<0.005, df=1**
**1/9 males**
**χ****2**** =5.4, p<0.025, df=1 **

**Notes:**
Know mother is het for marker based on 98-4 segregation of marker with gender
Father in 98-1 is also het; marker not associated with gender
Father in 98-3 doesn’t have marker
Father in 98-5 hom +
Due to recombination, not unexpected that some male progeny exhibit marker

## Results

**Collected gender and marker data on additional 43 (31 female and 12 male) progeny in *****S. eriocephala***** family 99-4**
**No association of marker with gender for 354****520**
**Goodness-of-fit for AEO8****780**** ****showed a significant association of marker with gender for female progeny of 994 and 984 families combined (n = 36 X****2**** = 13.4 p < 0.005 d.f. =1) as well as for males of these families (n = 15 X****2**** = 15 p < 0.005 d.f. =1)**

## Results

**Recombination based on single crossover estimates SCAR ****AEO8****780**** marker at 13.7 **_**+**_** 3.4 cM from locus involved in gender determination**
**If mapping distance in *****Salix *****is***** *****similar to *****Populus***** (~220 kb/cM), then distance from marker to locus is ~3.0 Mb **_**+**_** 750 kb.**

## Conclusions

**Marker developed for *****S. viminalis***** is associated with femaleness in *****S. eriocephala, *****suggests****:**
**Genetic basis for gender**
**Multilocus system common in family**
**Putative epistatic interaction allows us to observe association of marker with femaleness in other families**
**Additional work needed to enable use of markers to predict gender in broader genetic backgrounds**

**Notes:**
Suggests multilocus sex determination system common in family
No relation not unexpected given recombination

## A RAPD Marker Associated With Twisted Stem Form in Corkscrew Willow F1 Hybrids

- Oak Ridge National Laboratory
- State University of New York-Syracuse
- BFDP Subcontractor’s Workshop
- November 8, 2001

## Background

**Corkscrew willow (*****S. matsudana***** ‘Tortuosa’) exhibits an unusual phenomenon–tortuosity, or spiral growth along the branches and petioles—that is common to only a few species of woody plants. **
**Is the phenotype exhibited is due to a single gene trait?**
**Could finding a gene controlling this trait help us understand one of the mechanisms involved in secondary growth? **

## Objectives

**Task 1: Obtain interspecific F1 hybrid progeny segregating for tortuosity and collect phenotypic data associated with growth and development. **
**Task 2: Develop and analyze molecular marker associations between the phenotype of the progeny and genetic loci associated with tortuosity.**

## Methods

***S. matsudana***** ‘Tortuosa’ female crossed to male *****S. alba***** clone 99010 at SUNY**
**85 family 99270 F1 hybrids assessed for degree of stem twisting in field**
**Bulked segregant analysis of straight-stemmed vs. corkscrew-stemmed full sibs used to look for RAPD makers**

## Results

**85 F1 hybrids**
**41 straight-stemmed**
**40 twisted-stemmed**
**  ****4 uncertain**

## Results

**DNA extracted and bulked for 10 corkscrew and 10 straight-stemmed progeny of family 99270**
**640 RAPD primers screened**
**530 RAPD primers amplified**
**32 primers polymorphic between bulks**

## Primer OPQ-07 amplified 1.2 kb band with 100% fidelity in corkscrew F1 phenotypes

Marker absent from straight-stemmed F1 phenotypes  

**Marker absent from straight-stemmed F1 phenotypes  **

**Results**

## Conclusions

**Stem phenotype and marker exhibit 1:1 segregation in F1 hybrids**
**Need to test additional F1 to determine strength of relationship**
**Sequence marker to seek possible functional role of DNA**
**Need to develop additional markers**

## Acknowledgments

**Office of Transportation Technology BioPower Program, DOE**
**Bioenergy Feedstock Development Program, ORNL**
***Salix***** Consortium, SUNY**
**Rachel McCord, Davidson College**