1. Nilesh Dighe and David M. Stelly
Recombination of DNA Markers for Reniform and Root-knot Nematode Resistance in LONREN x Acala NemX Hybrids
Alois A. Bell
USDA-ARS, College Station, TX
Nilesh Dighe and David M. Stelly
Dept. Soil and Crop Sciences, Texas A&M University, College Station, TX

2. from G. longicalyx (F) into G. hirsutum (AD)
Creation of Triple-Species Tetraploid Hybrids Used to Introgress Resistance
from G. longicalyx (F) into G. hirsutum (AD)
1. HLA hybrid (strategy: substitute F for A)
AADD x FF (FAD)2 x DD (FADD)
2. HHL hybrid (strategy: substitute F for D)
AADD x AA (AAD)2 x FF (AADF)
DD = G. armourianum
AA = G. herbaceum

3. LONREN Backcross-1 Families
1. HLA-A Group (4 random BC1 male parents)
8 families derived from backcross-1 plants:
2, 77A, 83, 84*, 85, 103A, 122, and 132
2. HLA-B Group (Acala NemX BC1 male parent)
14 families derived from backcross-1 plants:
4, 8, 21, 26, 34, 35, 45, 61, 75, 77B, 81, 91, 99, 103B
3. HHL Group (Acala NemX BC1 male parent)
6 families derived from backcross plants:
3, 5, 7, 11, 14, and 17
* LONREN-1 and -2 germplasm lines released from this family in April 2007.

4. Effects of Longicalyx Resistance in Deltapine 458 B/R Isolines
Reniform Nematode Populations in BC6S1 Progeny
Seven Weeks After Inoculation
Resistance
Status* n
Nematode Numbers (% DP 16)
Mean
Range RR 12
0.8
0 – 2 Rr 14
3.7
0 – 16 rr 8
106.1
5 – 201
* Determined from test crosses and six SSR markers.

5. Seed Cotton Yields of LONREN Progeny Rows Texas A&M Brazos River Plantation (2007)
Line / Cultivar
No. of Rows*
Yield
Mean
Range
(Kg/Row)
LONREN-1 (Composite of BC7S2 seed from 17 Sibs):
Susceptible Sibs 3
3.34
Resistant Sibs 16
3.22
LONREN-2 (Composite of BC7S2 seed from 20 Sibs):
3.42 20
3.75
Fibermax 958 6
3.19
PSC 355 1
3.65
* 17 Sib lines exceeded the mean of Fibermax 958, the last backcross parent;
10 Sibs exceeded the mean for PSC 355, the performance standard.

6. Objectives 1. Combine resistance to reniform nematodes
from LONREN lines with resistance to root
knot nematodes from Acala NemX in
agronomically desirable germplasm lines.
2. Develop additional LONREN germplasm
lines from other families in the HLA-A group.
3. Develop new sources of root knot resistance
with improved yield and fiber quality.

7. Parents Used in Crosses
LONREN Parents:
BC6 plant from MB Line (77A family)
BC6 plant from MD Line (83 family)
BC7 plant from ME Line (84 family)
BC6 plant from ML Line (103A family)
BC6 plant from MM Line (122 family)
BC6 plant from MO Line (132 family)
Acala NemX Parents:
Type 1 Atypical CIR 316 and BNL 1231
and typical BNL 1066 markers
Type 2 Atypical CIR 316 and BNL 1066
and typical BNL 1231 markers

8. Fiber Quality of CIR 316_202 Sources
Cultivar/Line
MIC
UHM
STR UI
Acala NemX-1
4.7
1.17
33.9
87.5
Acala Nemx-2
4.9
1.22
33.2
87.2
Auburn 623
5.3
1.11
29.5
85.3
M-315
4.5
1.13
29.3
86.0
STV 5599
5.7
1.16
29.6
Clevewilt 6
3.9
1.24
28.6

9. Markers on Long Arm of Chromosome 11 Used to Characterize Parents and Follow Crosses
Acala NemX
LONREN Lines
MD, ME, MM, MO
Type 1
Type 2
fzg (white)
fzg (white)
Fzglon (green)
Mi2
CIR 316_202
CIR 316_202
CIR 316_191
rkn1 ML
cut
Area of
Possible
Recombination
Rrlon
BNL 1231_197
BNL 1231_192
BNL 1231_null
BNL 3279_112
BNL 3279_112
BNL 3279_114 MB
cut
BNL 1066_130
BNL 1066_139
BNL 1066_156
BNL 836_207
BNL 836_207
BNL 836_215

10. Markers on Long Arm of Chromosome 11 Used to Characterize Parents and Follow Crosses
Acala NemX
LONREN Lines
MD, ME, MM, MO
Type 1
Type 2
fzg (white)
fzg (white)
Fzglon (green)
Mi2
CIR 316_202
CIR 316_202
CIR 316_191
rkn1 ML
cut
Area of
Possible
Recombination
Rrlon
BNL 1231_197
BNL 1231_192
BNL 1231_null
BNL 3279_112
BNL 3279_112
BNL 3279_114 MB
cut
BNL 1066_130
BNL 1066_139
BNL 1066_156
BNL 836_207
BNL 836_207
BNL 836_215

11. Marker BNL 3279 Scan on 34 BC6S1 Plants (Coupling phase linkage)
bp 114
bp 112 D
HLA F1
AD1 A
Homozygous Resistant
Heterozygous Resistant
Susceptible
Marker BNL 1231 Scan on 34 BC6S1 Plants (Repulsion phase linkage)
AD1 D M F1
HLA
Homozygous Resistant
Heterozygous Resistant
Susceptible
bp 197
bp 192 B

12. BNL1231 Profile of 20 Random Acala NemX Plants from USDA Cotton Germplasm Collection
Type 1
bp 197
bp 192
Type 2

13. Seed Showing Effect of Fzglon Gene on Linter Color
G. longicalyx
fzglon fzglon
Fzglon fzglon
Fzglon Fzglon

14. Protocols 1. F1 hybrids heterozygous for BNL 3279_114
and CIR 316_202 were selected for selfing.
Then F2 progeny were screened for
BNL 3279, CIR 316, BNL 1066, and BNL 1231
markers to identify recombinants.
2. F2 recombinants were selfed to produce F3
generations and were backcrossed to
produce BC1 and BC2 generations.
3. Plants homozygous for BNL3279_114 only,
CIR 316_202 only, or both are being
evaluated for agronomic performance and
resistance to nematodes.

15. Segregation of F2 Progeny LONREN x Acala NemX F1
Genotype
LONREN Parent Line
Observed
Total
Theoretical
Unlinked
3279*
316* MB MD ME ML MM MO AA BB 18 Bb 1 2 4 36 bb 8 9 13 7 15 65 Aa 3 5
Bb† 25 26 20 23
144
172 aa 11 12 64
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202 from Acala NemX
compared to b = CIR 316_191 in LONREN lines.
† Parental genotype

16. Segregation of F2 Progeny LONREN x Acala NemX F1
Genotype
LONREN Parent Line
Observed
Total
Theoretical
Unlinked
3279*
316* MB MD ME ML MM MO AA BB 18 Bb 1 2 4 36 bb 8 9 13 7 15 65 Aa 3 5
Bb† 25 26 20 23
144
172 aa 11 12 64
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202 from Acala NemX
compared to b = CIR 316_191 in LONREN lines.
† Parental genotype

17. (No. F3 Progeny with Genotype)
Segregation of F3 Progeny from LONREN x Acala NemX F2 Recombinants (AABb)
Genotype*
F2 Recombinant Parent
3279
316
ME-25
ML-20
ML-37
MO-33
(No. F3 Progeny with Genotype) AA BB 5 4 8 Bb 28 10 20 bb 7 6 12
Chi Square (1:2:1)
6.6
2.4
0.4
0.8
P Value
0.04
0.30
0.82
0.46
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202;
and b = CIR 316_191 markers.

18. (No. F3 Progeny with Genotype)
Segregation of F3 Progeny from LONREN x Acala NemX F2 Recombinants (AaBB)
Genotype*
F2 Recombinant Parent
3279
316
ML-24
ML-42
ML-43
MM-9
(No. F3 Progeny with Genotype) AA BB 6 3 4 Aa 8 7 27 aa 9
Chi Square (1:2:1)
0.8
3.7
0.7
7.6
P Value
0.67
0.16
0.72
0.02
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202;
and b = CIR 316_191 markers.

19. Charactersistics of Homozygous Stacked F3 Progeny (AABB) Compared to Parents
Acala NemX
Recombinant
F3 Progenies
LONREN
Lines
Fuzz Color
White (fzg)
Green (Fzglon)
except ML (fzg)
CIR 316
_202
_191
BNL 1231
_197 Type 1
_null
_192 Type 2
BNL 3279
_112
_114
BNL 1066
_130 Type 1
_156
_139 Type 2
BNL 836
_207
_215
CONCLUSION: Crossovers in all 8 recombinants occurred
between CIR 316 and BNL 1231.

20. Segregation of Backcross-1 Progeny from F2 Recombinant Plants
Genotype*
F2 Parent Plant†
3279
316
ME-25
ME-20
ML-24
ML-37
MO-33
(No. BC1 Plants in Genotype) Aa Bb 4 5 2 bb 6 - 8 aa 3
Chi Square (1:1)
0.4
0.5
3.6
P Value
0.53
0.48
0.06
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202; and b = CIR 316_191.
† ME-25, ME-20, and ML-24 backcrossed to Deltapine 493;
ML-37 and MO-30 backcrossed to Tamcot CAMD-E.

21. Segregation of BC2 Progeny from Doubly Heterozygous BC1 Progeny of Recombinant ME-25
Genotype*
aa bb
Aa bb
aa Bb
Aa Bb
Observed 18 7 9 16
Expected
12.5
Chi Square (1:1:1:1) = 6.8
Recombinant % = 32
P Value = 0.08
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202;
and b = CIR 316_191.

22. Fiber Quality of Selfed Progeny and Homozygous Sibs of BC7S1 Recombinant ME-25
Genotype*
MIC
UHM
STR UI
AABb (ME-25)
4.60
1.22
36.1
88.9
aaBB CIR 316_202 Sib
4.18
1.26
28.5
88.1
AAbb BNL 3279_114 Sib
3.84
1.25
31.6
87.8
AAbb BNL 3279_114 Progeny
4.16
33.4
88.3
AABB Stacked Progeny
4.44
1.27
32.3
88.5
aa BB Acala NemX (Type 2)
4.9
33.2
87.2
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202; and b = CIR 316_191.
CONCLUSION: ME-25 Stacked has excellent fiber quality.

23. Fiber Quality of Selfed Progeny and Homozygous Sibs of BC7S1 Recombinant MO-30
Genotype*
MIC
UHM
STR UI
AABb (MO-30)
5.10
1.17
34.4
87.3
aaBB CIR 316_202 Sib
5.03
1.18
30.5
87.0
AAbb BNL 3279_114 Sib
3.86
1.21
29.6
AAbb BNL 3279_114 Progeny
4.06
1.23
29.2
86.6
AABB Stacked Progeny
4.75
1.25
30.1
86.7
aa BB Acala NemX (Type 1)
4.65
33.9
87.5
* A = BNL 3279_114; a = BNL 3279_112; B = CIR 316_202; and b = CIR 316_191.
CONCLUSION: MO-30 Stacked has very good fiber quality.

24. Comparison of Fiber Quality of Stacked Lines with the White Fuzzed ML Line
MIC
UHM
STR UI
LONREN ML
4.31
1.06
30.5
86.1
ME-25 Stack
4.44
1.27
32.3
88.5
MO-30 Stack
4.75
1.25
30.1
86.7
CONCLUSION: Stacked white fuzz lines have fiber quality superior to that
of LONREN ML Line.

25. Conclusions 1. Eight plants with a recombination between
CIR 316_202 from Acala NemX and BNL 1232_null
from four LONREN lines have been obtained along
with more that 60 sib plants homozygous for either
marker.
2. Homozygous recombinants from ME-25 (84 family)
and MO-30 (122 family) have excellent fiber quality
and will be further evaluated and probably released
as new germplasm as early as the spring of 2008.
3. These recombinants and their sibs allow a critical
evaluation of the proposed map location of the
root-knot resistance genes rkn-1 and Mi2, and the
reniform resistance gene Rrlon.

26. Acknowledgements Jose Quintana - Performing DNA marker analyses
and preparing illustrations.
Brandy Velten and Andrew Long - Performing
DNA preparation and laboratory work.
Dr. Forest A. Robinson - Determining the
resistance of the LONREN parents used in
these studies.
Dr. Robert Nichols and Cotton Inc. - Providing
financial assistance and fiber analyses.