Is simultaneous improvement of fall dormancy (FD) and winter hardiness (WH)

possible in alfalfa? QTL mapping and inheritance of FD & WHLaxman Adhikari and Ali M. Missaoui

Institute of Plant Breeding, Genetics & Genomics, University of Georgia, Athens, GA 30602

Background & objective

[1] E.C. Brummer, M.M. Shah, D. Luth, Reexamining the relationship

between fall dormancy and winter hardiness in alfalfa, Crop Science

40(4) (2000) 971-977.

[2] Teuber L, Taggard K, Gibbs L, McCaslin M, Peterson M, Barnes D.

Fall dormancy. Standard tests to characterize alfalfa cultivars North

American Alfalfa Improve Conf, 36th, Bozeman, MT1998. p. 2-6.

[3] Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler

ES, et al. A robust, simple genotyping-by-sequencing (GBS) approach

for high diversity species. PLoS ONE. 2011;6(5):e19379.

FD was assessed using regression equations derived from check plants

canopy height measured at 4 weeks after clipping on 21 September and

their standard FDR. Dormancy was assessed after winter clipping. WH was

visually scored (1-5), 1 being winter-hardy and 5 being very winter

susceptible.

References

Dormant (left) and non-dormant (right) F1 progeny rows of the

F1 population (3010 × CW1010) after frost occurrence in early

March, 2017 at the JPC environment.

mapping population development &

experimental design

Fig 3. Mapping population (JPC farm) planted in August,

2014. F1, parents and checks for FD and WH using were

planted in a RCBD design in 3 reps at two locations:

Watkinsville and Blairsville, GA. Four clones were planted

per row plot.

Dormant and

winter-hardyNon-dormant and

winter-sensitive

CW1010, ♂3010, ♀

184 F1 confirmed using SSRs

Made clones by stem cuttings

x

Marker discovery using genotyping by sequencing (GBS)

ApeKI enzyme

Barcode adapter Common

adapterDNA

DNA extraction & quantification

GBS SNPs

Fig 4. Workflow displaying GBS marker discovery. We followed GBS protocol by [3].

X

1/21/2

Segregation of a SDM

Constructing genetic map using single dose marker (SDM)

Phenotyping fall dormancy & winter-hardiness

TP

57355

0.0

TP

20165

11.0

TP

58948

17.5

TP

38700

20.9

TP

61179

25.1

TP

70863

32.9

TP

70879

38.2

TP

67965

43.3

TP

43172

47.3

TP

37893

53.9

TP

62715

55.1

TP

30295

55.9

TP

55470

57.1

TP

47965

61.6

TP

12175

63.9

TP

86018

66.6

TP

59131

67.4

TP

15098

68.6

TP

73186

70.3

TP

70400

71.3

TP

72089

72.0

TP

46942

72.6

TP

73780

73.2

TP

89716

73.9

TP

7175

76.9

MR

G_294866

79.2

TP

32958

81.6

TP

22446

83.6

TP

58124

84.5

TP

75440

85.1

TP

78320

85.3

TP

5775

85.7

TP

67270

86.1

TP

67699

86.3

MR

G_27654922

86.6

MR

G_27654958

86.7

MR

G_39914804

86.9

MR

G_37602036

TP

37501

TP

49846

87.0

TP

17276

87.1

TP

65414

87.7

TP

62622

87.9

MR

G_11082360

88.3

TP

52576

88.9

TP

35274

89.8

TP

995

92.2

TP

78651

92.8

TP

6492

93.2

TP

53543

93.4

TP

15998

94.5

TP

5699

95.3

TP

85729

96.1

TP

36877

96.7

TP

49657

97.6

TP

65855

98.5

TP

86274

99.5

TP

34618

104.9

TP

79985

108.5

1A

Saturated genetic maps for both 3010 & CW1010

Table 1 Phenotypic correlations (r) among traits based on JPC environment data collected on

segregating F1. Dormancy was assessed in the fall 2015 & 2016 and in the winter 2016 and

2017. WH data was collected in 3 consecutive winters (WH2015, WH2016 and WH2017).

FD & WH phenotypic correlation

FD2015 FD2016 WD2016 WD2017 WH2015 WH2016 WH2017

FD2015 0.50 ** 0.62 ** 0.60 ** 0.39 ** 0.52 ** 0.57 **

FD2016 0.39 ** 0.43 ** 0.12NS 0.31 ** 0.50 **

WD2016 0.92 ** 0.22** 0.65 ** 0.80 **

WD2017 0.23** 0.71 ** 0.85 **

WH2015 0.16* 0.10NS

WH2016 0.68 **

WH2017

TP64234 0.0TP28256 4.1TP80202 6.3TP39176 7.2TP33825 7.8TP77211 8.4

TP1366 8.8TP31419 9.4TP48606 9.6TP38182 9.9TP48110 10.3TP71409 10.7TP89030 10.9TP84072 11.2

MRG_37413589 11.6TP80271 12.1TP58773 12.4TP88794 12.5TP41376 12.8

MRG_27247717 13.1MRG_27247742 13.4MRG_35867245 13.7

TP1340 14.1TP21757 15.1TP87956 16.5TP82621 18.0TP25395 20.7TP81771 23.7TP69248 25.0TP58371 28.9TP34795 29.4

TP2134 31.2TP51882 31.6TP46097 32.0TP13829 32.5TP35133 32.9TP72218 34.0TP58925 34.2TP51881 34.8TP87228 36.0TP22536 36.8TP55743 37.5TP24733 38.3TP34483 39.0TP30610 41.5

MRG_10667023 42.4MRG_10666983 42.5MRG_10666968 42.8

TP52373 43.8TP50807 46.1TP59349 47.3TP69889 47.7TP41963 48.2TP43142 48.7

TP6189 49.2TP46619 49.9TP15177 50.5TP71458 51.0

dorm3

dorm6

wh2

wh7

3010(7A) [1]

TP67945 52.5

TP34796 55.4

TP16932 56.8

TP63331 58.3

TP16757 59.8

TP10992 61.6

TP67963 62.7

TP87504 64.3

TP63918 65.9

MRG_27142324 69.2

MRG_27142336 70.6

MRG_24987546 74.4

TP51377 77.8

TP47813 82.0

TP68486 85.0

TP18137 92.3

3010(7A) [2]

Acknowledgement

Common & separate QTLs for FD & WH

Conclusions

Linkage groups & synteny with M. truncatula

Fig 5. (Left) displaying segregation pattern of single dose markers (SDM); (right) markers data

for CW1010 parent on JoinMap 5.0 to determine linkage group (LG) and genetic maps.

Fig 7. Genetic linkage map of homolog 1A of 3010 parent. The paternal map retained 1377

SNPs and maternal maps retained 1837 SDA SNPs for 32 LGs with an average map density of

1.5 cM/SNP for both parental maps. Positions of SNP are shown in Kosambi centiMorgan(cM).

Fig 2. Seasonal forage gaps & forage production patterns

of cool season and warm season forage species. The gap in

autumn-winter can be overcome by developing non-

dormant & winter hardy alfalfa.

Fig 9. (Left) alfalfa FD (black bar) and WH (red bar) stable QTLs mapped

on homolog 7A for 3010 parent. The QTL bars have two intervals, an

inner (1-LOD support) interval and an outer (2-LOD support) interval,

where the rectangle represents inner interval and the line represents

the outer. Some stable QTLs for dormancy were co-localized with winter

hardiness in the same genomic regions.(Right) a dormancy QTL for 3010

parent on 7B. The QTL mapping was done using QTL cartographer

(version 2.5).

Fig 8. Dot plot displaying the chromosome grouping pattern and

positions of SNPs on 32 LG of 3010 linkage map. Of the 32 groups, each

4 homology groups were assigned to a chromosome based on synteny

with M. truncatula genome. Based on BLAST, we unanimously assigned 2

homologs to 8 chromosomes.

The authors acknowledge the research technician Joseph

Young, Franco V. Chirinos and Shiva Makaju for their

assistance in the field work and data collection. Our special

thanks goes to Mr. Dev Paudel for his assistance with data

analysis in bioinformatics. This research was supported by

UGA CRDP.

45 significant (P≤ 0.05) QTLs for FD and 35 QTLs for WH

were detected on both 3010 and CW1010 linkage maps.

Of which, 18 dormancy QTLs and 17 WH QTLs were

stable QTLs and remaining QTLs were potential QTLs.

> 75 % (22/28) of the dormancy QTL detected for 3010

parent did not share genomic regions with WH QTLs and

> 70% (12/17) dormancy QTLs detected from CW1010

parent were also localized in different genomic regions

from WH QTLs.

Results of this study suggest that FD and WH in alfalfa

have independent inheritance and therefore can be

improved separately in breeding programs.

The QTLs detected in this study will be valuable addition

to the genomic resources for alfalfa breeding programs

and to the understanding of the genetic basis of seasonal

dormancy and winter-hardiness.

Two 96-plexed libraries were submitted to Georgia Genomics and

Bioinformatics Core (GGBC), UGA, for SPRI cleanup and sequencing. Raw

data were processed using Tassel 3.0 UNEAK (bytebucket.org) and GBS-

SNP-CROP (github.com) pipelines. Raw SNPs were filtered in MS Excel.

Single dose allele (SDA) marker were confirmed using Chi square test, and

genetic maps were constructed in JoinMap 5.0 (www.kyazma.nl). The SDA

SNPs were grouped using minimum independence LOD of 10 and mapped

using regression mapping.

Fig 6. (Left) measuring alfalfa height after autumn clipping; (right) winter impacted alfalfa

at Watkinsville farm were visually rated.The objective of this study was to understand

the genetic basis of FD and WH in an alfalfa

F1 pseudotestcross population through

quantitative trait loci (QTL) mapping.

Fall dormancy (FD) reduces growth and yield of

certain alfalfa (Medicago sativa L.) genotypes in

response to decreased temperature and day

length [1]. There are 11 different classes of FD in

alfalfa ranging from very dormant to non-

dormant [2]. Winter-hardiness (WH) is another

trait associated with alfalfa yield, survival, and

ecological distribution [1].

FD and WH create a seasonal forage gap

(www.nrcs.usda.gov) from mid-autumn to the

end of winter, which can be overcome by

developing non-dormant winter-hardy alfalfa

cultivars. Therefore, understating the genetic

basis of the FD and WH is essential.

Contact @ - cssamm@uga.edu or laxman.adhikari25@uga.edu

Detail: https://www.frontiersin.org/articles/10.3389/fpls.2018.00934/full

Pro

ducti

on

Cool

season

forages

Warm

season

grasses

Forage gaps

J F M A M J J A S O N D