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1

Shan Wu

Fei Lab Boyce Thompson Institute

PAG 2018

Cucurbita Genome

Sequences Provide

Insights into Polyploid

Genome Evolution

and Heterosis in

Interspecific Hybrid

Outline

Introduction

- The origins and domestication of C. maxima and C. moschata

- The uses of C. maxima and C. moschata

The genome sequences of C. maxima and C. moschata

- De novo genome assembly, anchoring and quality evaluation

- The two distinguishable paleo-subgenomes in Cucurbita

- Genome and gene evolution after allotetraploidization

Gene expression alteration in the interspecific F1 hybrid

- Higher total carotenoid content in the F1 fruit

Origins and domestication of Cucurbita species

C. moschata

Lowlands of Mexico and

Northern South America China and Japan

https://www.marthastewart.com

C

c

c C. pepo

C. moschata

C. argyrosperma

C. maxima

Esquinas-Alcazar and Gulick, 1983

Genetic resources of Cucurbitaceae: a global report

C. maximaSouthern South America India and Myanmar

Harry Potter and the Prisoner of Azkaban

- The Cucurbita crops are consumed all over the world and are a staple food in

many developing countries.

- The fruits are used as ornaments and carved into decorative lanterns around

Halloween.

- C. maxima and C. moschata are also used as rootstocks for other cucurbit crops,

including watermelon, cucumber and melon, to enhance tolerance to soilborne

diseases and abiotic stresses.

The uses of Cucurbita crops

C. maxima, Rimu C. moschata, Rifu

- The interspecific hybrid developed from a cross between C. maxima cv. Rimu

and C. moschata cv. Rifu, ‘Shintosa’, is a popular rootstock for different

cucurbits, and especially preferred in watermelon grafting for its Fusarium wilt

resistance, cold-tolerance, and the ability to increase fruit weight, fruit quality and

plant vigor.

C. maxima and C. moschata genome assemblies

High quality cleaned Illumina readsC. maxima: 109.3 Gb, 283× coverage

C. moschata: 80.2 Gb, 215× coverage

93%

2%

5%

Complete BUSCOs

Fragmented BUSCOs

Missing BUSCOs

93%

1%

6%

Complete BUSCOs

Fragmented BUSCOs

Missing BUSCOs

Assessment of completeness using BUSCO

C. maxima C. moschata

Summary statistics of the assemblies Scaffold Contig

Size (bp) Number Size (bp) Number

C. maxima

Longest 11,871,986 1 400,020 1

N50 3,717,157 24 40,681 1,813

N90 52,757 295 6,851 7,555

Total 271,413,401 8,299 265,448,559 25,524

C. moschata

Longest 11,258,782 1 292,205 1

N50 3,995,720 24 40,480 1,897

N90 593,097 93 10,017 6,788

Total 269,943,085 3,500 262,991,909 17,340

Estimated genome size: 386.8 Mb

Estimated genome size: 372.0 Mb

Repeat sequences accounts for 40.3% and 40.6% of C. maxima and C. moschata genomes, respectively.

C. maxima, RimuC. maxima, SQ026 C. moschata, Rifu C. moschata,

Honey jujube

Anchoring the scaffolds into pseudomolecules

× × ×

Intraspecific F2Intraspecific F2

Interspecific F1BC1

C. maxima map2,030 SNPs

C. moschata map3,487 SNPsInterspecific map

13,783 SNPs

C.maxima C.moschataNum.ofanchoredscaffolds 92 98

Num.oforientedscaffolds 64 71Totalsize(Mb) 211.4 235

%ofassembledgenomeanchored 77.9 88.4Num.oflinkagegroups 20 20

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Extensive

synteny between

the two Cucurbita

species

01 02 06 07 08 11 12 18 04 03 05 09 10 13 14 15 16 17 19 20

Subgenome A Subgenome BC. maxima≤0.35 ≥0.40

KsChr

01

02

03

04

05

06

07

08

09

10

11

Melo

n

12

MelonChr02

Chr11 Chr10C. maxima

Two distinguishable paleo-subgenomes of Cucurbita

0.029 Mya

Cucumber

Melon

Watermelon

C. maxima B

C. moschata B

C. maxima A

C. moschata A

Bitter gourd

Walnut

6.51

19.06

3.34

3.37

26.28

30.75

36.13

82.04

[6.06, 6.94]

[3.04, 3.82]

[3.09, 3.84]

[18.34,19.75]

[25.54,27.00]

[29.86,31.60]

[34.94,37.24]

[79.37,84.59]

Divergence of progenitor A from the common ancestor of

progenitor B and Benincaseae

Divergence of progenitor B from

Benincaseae

Divergence between C. maxima and

C. moschata

Allotetraploidization 31 26 3 Mya

Paleo-allotetraploidization in Cucurbita Fractionation bias and genome dominance

WGD

×× ××

××

Radom

fractionation

××× × ××

Biased

fractionation

The dominant

subgenome

Schnable et al., 2011 PNAS

Maize1 dominates expression

Maize2 dominates expression

Radom fractionation and lack of

genome-wide expression bias

0

5

10

15

20

25

30

Fruit Leaf Stem Root

Pe

rce

nt o

f ge

ne

pa

irs (

N=

5,5

81

)

Subgenome A dominance Subgenome B dominance

C. maxima

TE insertions in the 5’ upstream regions of genes

Hypothesis:

"The diploid parent of a tetraploid with the lowest transposon load was

to become the dominant subgenome." -Woodhouse et al., 2014 PNAS

The diploid parental genomes of Cucurbita could have a similar total load of TEs near genes,

leading to the lack of genome dominance and subsequent random fractionation in the polyploid.

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Gene expression alteration in the interspecific F1 hybrid

C. maxima ♀ C. moschata ♂

The interspecific F1 ‘Shintosa’

×

P

1

F1 P

2

P

1

F1 P

2

Dominant (equal to one of the parents) and Transgressive (higher than the high parent or lower than

the low parent) patterns might underlie heterotic phenotypes and combined favorable characters from parents.

Exp

ressio

n l

eve

l

In the Shintosa, 4,002 (fruit), 6,718 (leaf), 6,732 (stem) and 7,067 (root) genes were dominantly or

transgressively expressed, representing 12.5-22.0% of the total genes in the genome. The expression patterns

of some of these genes are correlated with disease resistance, higher growth vigor and increased

carotenoid biosynthesis in Shintosa.

Expression patterns of carotenoid biosynthetic genes

C. maxima Shintosa C. moschata

0

200

400

600

800

Cma F1 Cmo

Lutein

0

100

200

300

400

500

600

700

Cma F1 Cmo

α-carotene

0

0.5

1

1.5

2

Cma F1 Cmo

β-carotene

0

10

20

30

40

50

60

Cma F1 Cmo

Lycopene

μg/1

00gF

W

mg/1

00gF

W

μg/1

00gF

W

μg/1

00gF

W

FPKM

<1

1-10

11-20

21-30

31-40

41-50

>50

A B

Cma F1 Cmo

GGPP

Phytoene

ζ-carotene

Lycopene

α-carotene β-carotene

Lutein Zeaxanthin

Carlactone

PSY

PDS

Z-ISO

ZDS

CRTISO

LYCE

LYCB LYCB

CHYB

CYP97A3

CYP97C1

CHYB B-ISO

CCD7

CCD8

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Subgenome

Summary

We assembled high-quality genome sequences of C. maxima and C. moschata,

which are a valuable resource for genetic improvement of the crop.

We provide evidence supporting an lineage-specific ancient allotetraploidization

event in Cucurbita. The two diploid progenitors of Cucurbita successively diverged

from Benincaseae around 31 and 26 million years ago (Mya), and the

allotetraploidization happened earlier than 3 Mya, when C. maxima and C. moschata

diverged.

The subgenomes have largely maintained the chromosome structures of their diploid

progenitors. Such long-term karyotype stability after polyploidization has not been

commonly observed in plant polyploids. The two subgenomes have retained similar

numbers of genes, and neither subgenome is globally dominant in gene expression.

We detected transgressive gene expression changes in the F1 hybrid of C. maxima

and C. moschata correlated with heterosis in important agronomic traits.

Acknowledgement

Beijing Academy of

Agriculture and

Forestry Sciences

Zhangjun Fei

Honghe Sun

Chen Jiao

Yong Xu

Haizhen Li

Guoyu Zhang

Shaogui Guo

Yi Ren

Jeff Doyle

William Lucas

Supported by grants from the Beijing Scholar Program, the Beijing

Excellent Talents Program, the Ministry of Agriculture of China, the

Beijing Natural Science Foundation, US NSF and USDA NIFA SCRI.