Literature DB >> 35551526

Genetic diversity analysis and marker-trait associations in Amaranthus species.

Norain Jamalluddin1, Festo J Massawe1, Sean Mayes1,2,3, Wai Kuan Ho1,3, Rachael C Symonds4.   

Abstract

Amaranth (Amaranthus spp.) is a highly nutritious, underutilized vegetable and pseudo-cereal crop. It possesses diverse abiotic stress tolerance traits, is genetically diverse and highly phenotypically plastic, making it an ideal crop to thrive in a rapidly changing climate. Despite considerable genetic diversity there is a lack of detailed characterization of germplasm or population structures. The present study utilized the DArTSeq platform to determine the genetic relationships and population structure between 188 amaranth accessions from 18 agronomically important vegetable, grain, and weedy species. A total of 74, 303 SNP alleles were generated of which 63, 821 were physically mapped to the genome of the grain species A. hypochondriacus. Population structure was inferred in two steps. First, all 188 amaranth accessions comprised of 18 species and second, only 120 A. tricolor accessions. After SNP filtering, a total of 8,688 SNPs were generated on 181 amaranth accessions of 16 species and 9,789 SNPs generated on 118 A. tricolor accessions. Both SNP datasets produced three major sub-populations (K = 3) and generate consistent taxonomic classification of the amaranth sub-genera (Amaranthus Amaranthus, Amaranthus Acnida and Amaranthus albersia), although the accessions were poorly demarcated by geographical origin and morphological traits. A. tricolor accessions were well discriminated from other amaranth species. A genome-wide association study (GWAS) of 10 qualitative traits revealed an association between specific phenotypes and genetic variants within the genome and identified 22 marker trait associations (MTAs) and 100 MTAs (P≤0.01, P≤0.001) on 16 amaranth species and 118 A.tricolor datasets, respectively. The release of SNP markers from this panel has produced invaluable preliminary genetic information for phenotyping and cultivar improvement in amaranth species.

Entities:  

Mesh：

Year:  2022        PMID： 35551526      PMCID： PMC9098028          DOI： 10.1371/journal.pone.0267752

Source DB:  PubMed          Journal:  PLoS One        ISSN： 1932-6203            Impact factor:   3.240


Introduction

Climate predictions indicate that the agriculture sector in many parts of the world will be subjected to increasingly detrimental weather conditions such as droughts and elevated temperatures, directly impacting global food supply chains. A strategy to mitigate climate related agricultural losses is to diversify the food basket with a wide range of underutilized crop species with increased abiotic stress tolerance traits [1]. Amaranth (Amaranthus spp.), an ancient, nutrient-dense and climate-smart crop has high degree of genetic variation, environmental adaptability and phenotypic plasticity [2, 3]. Amaranth belongs to the Amaranthaceae family and is a C4 dicotyledonous plant [4]. It consists of approximately 60–70 species grouped into three sub-genera; Amaranthus Albersia (vegetable amaranth), Amaranthus Amaranthus (cultivated grain amaranth) and Amaranthus Acnida (weedy amaranth) [5]. Amaranthus tricolor is a leafy vegetable amaranth species, widely cultivated in South Asia and Africa [6, 7], and is an excellent source of vitamins, protein, carotenoid, minerals and antioxidants, greater than other leafy vegetables such as lettuce and spinach [2, 8]. A. tricolor has the capacity to alter its physiological characteristics in response to environmental changes, for instance, increasing transpiration efficiency [9] and accumulation of compatible solutes such as proline, in response to drought stress [10]. It also had high genetic and phenotypic diversity which may provide an excellent opportunity for varietal development with increased drought tolerance characteristics [11-13]. Correct genotypic identification and preservation of genetic variation in amaranth is important to maintain ecotypes with desired traits useful for breeding programmes. The assembly of very high-quality grain amaranth, Amaranthus hypochondriacus (“Plainsman” cultivar) sequence genome by [14] allows anchoring of genotyping-by-sequencing (GBS) markers for all the SNP loci and allele sequences discovered, and GBS has proven to be the most efficient method to evaluate genetic diversity of grain amaranth as well as to validate the phylogeny of the genus [15-17]. This genome assembly was used as a reference genome for an annotation framework and gene discovery of MYB-like transcription factor genes that regulate the betalain red pigment pathway, which gives rise to stem and seed colour variations [18] through traditional bi-parental mapping [14] and now through genome-wide association studies (GWAS) [19]. More recently, this plainsman reference genome together with low-coverage PacBio reads and the contigs of amaranth draft genome [20] were used to assemble A. hypochondriacus (A.hyp_K_white), a landrace cultivated in India [21]. This assembly offers a better reference genome for the improvement of grain and vegetable amaranth crops in South Asia as it is genetically closer to most landraces and accessions originated from India and South Asia. Nevertheless, vegetable species of amaranth have been less studied by molecular means than pseudo-cereal grain amaranths and weed species, especially when both are phylogenically related and the domestication events separating them have been revealed [15, 19, 22, 23]. Limited knowledge of the genetic diversity in these leafy vegetable amaranth species and the lack of availability of suitable molecular markers hamper breeding efforts. Cultivar development and improvement relies on access to a well characterised, genetically diverse pool of material and so a comprehensive knowledge of these genetic relationships is essential. To date, there is only one molecular study that exploits a large number of A. tricolor accessions using simple sequence repeat (SSR) and matK protein-coding chloroplast gene, which concluded that the genetic diversity in Vietnamese amaranths was established by dispersal events mainly from East Asia and adaptation to local environments [24]. While the amaranth marker studies have been useful for evolutionary and phylogenetic studies, further germplasm characterization and marker validation is needed. GBS offers a number of potential advantages to SSR markers; it is more practical, inexpensive and has driven genotyping to be applied for non-model organisms [25, 26]. DArTSeqTM technology based on GBS methods is a platform developed by Diversity Arrays Technology Pty Ltd. (Canberra, Australia) for high-throughput genotyping via an intelligent selection of genome fraction by targeting active genes and low copy DNA areas [27, 28]. This present study is the first to utilize the DArTSeq platforms in amaranth to determine the genetic relationships and population structure between 188 amaranth accessions from 18 agronomically important vegetable, grain, and weedy species. This study also aimed to investigate the genetic relationship among a numerically larger group of A. tricolor accessions. The development of SNP markers from this panel has allowed a GWAS analysis on morphological traits such as shape, size, and colour of the leaf, stem, and inflorescence. These traits are fast and easy to assess for direct use by farmers and are of great help to plant breeders when selecting potential parental lines [12, 29]. This will facilitate understanding of the genetic bases and dissection of complex genes controlling economic traits such as drought tolerance and provides useful information on the degree of genetic variation and its correlations with agronomic traits.

Materials and methods

Plant materials, growing conditions and morphological assessment

A total of 188 amaranth accessions, comprising 18 species originating worldwide were used for genetic diversity analysis. Out of 188 accessions, 131 accessions were obtained from the World Vegetable Center Genebank, Taiwan (AVRDC), 52 accessions from the United State Department of Agriculture Genebank (USDA) and five commercial varieties were included as checks, of which three African varieties from East-West Seed, Thailand, were included and two local varieties from Serbajadi Gardening, Malaysia (Table 1).
Table 1

List of 188 amaranth accessions and their morphological traits observed under shade-house conditions.

EntryAccessionsIDSpeciesOrigin countryGermplasmGrowth habitBranching indexStem colourLeaf colourPetiole colourInflorescence colourLeaf shapeLeaf marginTerminal inflorescence shapeTerminal inflorescence attitude
1 AV ATRVI044435AtropurperusIndonesiaAVRDCErectAlong the stemPinkNormal greenGreenGreenLanceolateEntireShort branchesErect
2 AV GRAVI036225GraecizansHungaryAVRDCErectAlong the stemPurple/PinkNormal greenWhitePinkLanceolateEntireOtherErect
3 AV GRA SILVI044403Graecizans sspIndiaAVRDCErectMany branchesPurple/PinkNormal greenPink/GreenPinkCuneateEntireOtherErect
4 AV GRA ASCVI044388Graecizans sspIndiaAVRDCErectMany branchesPurple/PinkNormal greenPink/GreenGreenObovateUndulateOtherErect
5 AV MANVI044427MantegazzianusUSAAVRDCErectFew branchesGreenNormal greenGreenYellowLanceolateEntireClub-shapedErect
6 AV BLITOVI036227BlitoidesHungaryAVRDCProstateMany branchesPurple/PinkNormal greenGreenGreenCuneateEntireOtherErect
7 AV LEUVI044445LeucocarpusIndiaAVRDCErectAlong the stemGreenNormal greenGreenGreenEllipticalEntireSpike (dense)Erect
8 AV PALVI044473PalmeriSenegalAVRDCErectFew branchesPurple/PinkNormal greenGreenGreenOvatainateEntireOtherErect
9 AV RET 1VI048310RetroflexusViet NamAVRDCErectFew branchesPurple/PinkMargin/VeinPurpleRedOvatainateEntireSpike (dense)Erect
10 AV RET 2VI048311RetroflexusViet NamAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireSpike (dense)Erect
11 AV RET 3VI033461RetroflexusVenezuelaAVRDCErectMany branchesPurple/Pinkchlorotic stripeGreenGreenRhombicUndulateSpike (dense)Erect
12 AV RET 4VI048391RetroflexusViet NamAVRDCErectAlong the stemPurple/PinkCentral spotPurpleRedRhombicEntireSpike (dense)Erect
13 US RET 1Ames 26236RetroflexusChinaUSDAErectAlong the stemPinkNormal greenGreenGreenEllipticalEntireLong branchesErect
14 AV SPI 1VI044410SpinosusPuerto RicoAVRDCErectAlong the stemPurple/PinkNormal greenGreenGreenLanceolateEntireShort branchesDrooping
15 AV SPI 4VI040944SpinosusThailandAVRDCErectMany branchesGreenNormal greenGreenGreenRhombicEntireShort branchesErect
16 AV SPI 5VI048723SpinosusThailandAVRDCErectAlong the stemGreenOne stripeGreenGreenRhombicUndulateShort branchesErect
17 AV SPI 6VI046123SpinosusLaosAVRDCErectMany branchesPurple/PinkNormal greenGreenGreenLanceolateUndulateSpike (dense)Erect
18 AV SP 1VI050253SpTaiwanAVRDCErectMany branchesGreenOthersGreenGreenRhombicUndulateOtherErect
19 AV SP 2VI049530SpThailandAVRDCErectAlong the stemPurple/PinkNormal greenGreenOtherEllipticalEntireShort branchesErect
20 AV SP 3VI054799SpLaosAVRDCErectAlong the stemGreenNormal greenGreenGreenCuneateUndulateShort branchesErect
21 AV SP 4VI033471SpMalaysiaAVRDCErectMany branchesGreenNormal greenGreenGreenLanceolateEntireOtherErect
22 AV SP 5VI044448SpIndiaAVRDCErectMany branchesPurpleMargin/VeinPurpleGreen/RedRhombicUndulateOtherErect
23 AV SP 6VI056563SpBangladeshAVRDCErectMany branchesPurple/PinkEntire laminaDark purpleRedRhombicEntireSpike (dense)Drooping
24 AV SP 7VI056560SpBangladeshAVRDCErectMany branchesGreenDark greenGreenGreenRhombicEntireSpike (Dense)Drooping
25 AV VIR 1VI049893ViridisThailandAVRDCProstateMany branchesPurple/PinkNormal greenGreenPinkRhombicEntireShort branchesErect
26 AV VIR 4VI049001ViridisThailandAVRDCErectAlong the stemPurple/PinkNormal greenPurpleGreenCuneateEntireShort branchesErect
27 AV VIR 6VI048697ViridisThailandAVRDCErectMany branchesPurple/PinkNormal greenGreenPinkOvatainateEntireShort branchesErect
28 AV VIR 9VI055027ViridisMalaysiaAVRDCErectFew branchesGreenNormal greenGreenGreenOvatainateUndulateShort branchesErect
29 AV VIR 12VI046127ViridisLaosAVRDCErectAlong the stemPurple/PinkNormal greenPurplePinkOvatainateEntireShort branchesDrooping
30 AV VIR 14VI044432ViridisIndonesiaAVRDCErectAlong the stemPurple/PinkNormal greenGreenOtherRhombicEntireShort branchesDrooping
31 AV CRU 1VI036230CruentusAustriaAVRDCErectAlong the stemPurple/PinkNormal greenPinkGreenEllipticalEntireLong branchesErect
32 AV CRU 2VI044366CruentusEthiopiaAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateUndulateShort branchesErect
33 AV CRU 3VI036231CruentusAustriaAVRDCErectAlong the stemPurple/PinkMargin/VeinPinkGreen/RedOvatainateEntireShort branchesErect
34 AV CRU 5VI044453CruentusMexicoAVRDCErectFew branchesPurple/Pinkchlorotic stripeGreenYellowOvatainateUndulateClub-shapedErect
35 AV CRU 6VI050473CruentusSudanAVRDCErectMany branchesPurple/PinkMargin/VeinPurplePinkOvatainateEntireShort branchesErect
36 AV CRU 12VI044457CruentusZimbabweAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireShort branchesErect
37 AV CRU 14VI033487CruentusMalaysiaAVRDCErectAlong the stemGreenCentral spotPurplePinkOtherUndulateShort branchesErect
38 AV CRU 15VI044449CruentusGuatemalaAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateUndulateClub-shapedErect
39 AV HYB 1VI044419HybridusUSAAVRDCErectFew branchesPurple/PinkNormal greenGreenGreenRhombicUndulateSpike (dense)Erect
40 AV HYB 2VI044421HybridusUSAAVRDCProstateFew branchesPurple/PinkNormal greenGreenGreenCuneateUndulateSpike (dense)Erect
41 AV HYB 3VI051004HybridusKenyaAVRDCErectAlong the stemPurple/PinkNormal greenPurpleGreenLanceolateEntireSpike (dense)Drooping
42 US HYB 2PI 641052HybridusNigeriaUSDAErectAlong the stemPurpleEntire laminaPurpleRedEllipticalEntireSpike (dense)Erect
43 AV GRA 1VI056002GracilisCambodiaAVRDCErectAlong the stemPurpleCentral spotGreenGreenOvatainateUndulateSpike (dense)Drooping
44 EW CRU#20866CruentusTanzaniaE-WESTErectAlong the stemGreenNormal greenGreenGreenOvatainateUndulateClub-shapedErect
45 AV HYP 2VI044454HypochondriacusMexicoAVRDCErectFew branchesPurple/PinkNormal greenGreenGreenOvateEntireLong branchesErect
46 AV HYP 3VI044414HypochondriacusIndiaAVRDCErectFew branchesGreenNormal greenGreenPinkEllipticalUndulateLong branchesErect
47 AV HYP 5VI036229HypochondriacusHungaryAVRDCErectAlong the stemPurple/PinkMargin/VeinPinkPinkLanceolateEntireLong branchesDrooping
48 AV HYP 6VI044479HypochondriacusNepalAVRDCErectFew branchesPurple/PinkCentral spotGreenRedLanceolateUndulateLong branchesErect
49 AV HYP 10VI044365-AHypochondriacusGhanaAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireSpike (dense)Erect
50 AV HYP 13VI047551HypochondriacusViet NamAVRDCErectAlong the stemPurple/PinkEntire laminaDark purpleRedLanceolateEntireSpike (Dense)Erect
51 AV HYP 14VI033462-AHypochondriacusEcuadorAVRDCErectFew branchesPurple/PinkMargin/VeinPurple/PinkRedEllipticalUndulateLong branchesDrooping
52 AV HYP 16VI044395HypochondriacusAfghanistanAVRDCErectMany branchesPurple/PinkMargin/VeinPurple/PinkPinkOvatainateEntireSpike (Dense)Drooping
53 AV BLI 1VI044404Blitum cvg alecereusIndiaAVRDCErectFew branchesPurple/PinkNormal greenGreenGreenObovateUndulateSpike (dense)Erect
54 AV BLI 3VI055755Blitum cvg alecereusLaosAVRDCErectAlong the stemGreenNormal greenWhiteGreenEllipticalUndulateSpike (dense)Erect
55 AV BLI 4VI055121Blitum cvg alecereusMalaysiaAVRDCErectFew branchesPurple/PinkNormal greenDark purpleRedEllipticalEntireSpike (dense)Erect
56 AV BLI 7VI049036BlitumThailandAVRDCErectMany branchesWhiteNormal greenPink/GreenGreenOvatainateUndulateLong branchesErect
57 AV BLI 10VI044447BlitumKoreaAVRDCProstateAlong the stemPurple/PinkCentral spotGreenGreenOvatainateEntireShort branchesErect
58 AV BLI 12VI044423BlitumIndiaAVRDCErectMany branchesPurple/PinkEntire laminaPurpleRedCuneateEntireOtherErect
59 AV BLI 13VI056127BlitumCambodiaAVRDCErectAlong the stemWhitechlorotic stripeWhiteGreenOvatainateUndulateShort branchesDrooping
60 AV THU 1VI050456ThunbergiiUnknownAVRDCErectFew branchesPurple/PinkEntire laminaPurpleRedRhombicUndulateShort branchesErect
61 AV THU 2VI050467ThunbergiiUnknownAVRDCErectAlong the stemPurple/PinkEntire laminaPurplePinkOvatainateUndulateSpike (dense)Erect
62 AV THU 3VI050468ThunbergiiUnknownAVRDCErectAlong the stemPurple/PinkMargin/VeinPurpleGreenLanceolateUndulateSpike (dense)Erect
63 AV DUB 1VI047576DubiusViet NamAVRDCErectAlong the stemGreenCentral spotGreenGreenOvatainateEntireSpike (Dense)Erect
64 AV DUB 2VI047537DubiusViet NamAVRDCErectMany branchesPink/GreenCentral spotPink/GreenGreenOvatainateEntireSpike (Dense)Erect
65 AV DUB 6VI048985DubiusThailandAVRDCErectAlong the stemPinkNormal greenPinkPinkCuneateUndulateShort branchesErect
66 AV DUB 7VI050464DubiusTanzaniaAVRDCErectAlong the stemPurple/PinkNormal greenPurpleGreenEllipticalUndulateSpike (Dense)Erect
67 AV DUB 13VI044377DubiusSurinamAVRDCErectAlong the stemPinkNormal greenPinkGreenOvatainateUndulateSpike (Dense)Erect
68 AV DUB 15VI057160DubiusCambodiaAVRDCErectAlong the stemPurple/PinkCentral spotPurpleGreenLanceolateUndulateSpike (Dense)Drooping
69 AV TRI 1VI038237TricolorBangladeshAVRDCErectMany branchesPurple/PinkEntire laminaDark purpleRedRhombicUndulateShort branchesErect
70 AV TRI 2VI055356TricolorBangladeshAVRDCErectNo branchesPurple/Pinkchlorotic stripePurpleGreenRhombicUndulateShort branchesDrooping
71 AV TRI 3VI055353TricolorBangladeshAVRDCErectAlong the stemPurple/Pinkchlorotic stripePurpleGreen/RedEllipticalUndulateShort branchesDrooping
72 AV TRI 4VI055350TricolorBangladeshAVRDCErectMany branchesPurple/PinkEntire laminaPurpleRedOvatainateUndulateLong branchesErect
73 AV TRI 5VI048269TricolorBangladeshAVRDCErectAlong the stemPurple/PinkCentral spotPurpleRedOvatainateUndulateSpike (dense)Erect
74 AV TRI 6VI048233-ATricolorBangladeshAVRDCErectAlong the stemGreenNormal greenGreenGreenOvatainateEntireSpike (dense)Erect
75 AV TRI 7VI048200TricolorBangladeshAVRDCErectAlong the stemPurple/PinkEntire laminaPurpleGreen/RedOvatainateEntireShort branchesErect
76 AV TRI 8VI048146TricolorBangladeshAVRDCErectAlong the stemPurple/PinkEntire laminaPurpleGreen/RedOvatainateEntireShort branchesErect
77 AV TRI 9VI048089TricolorBangladeshAVRDCErectAlong the stemPurple/PinkEntire laminaPurpleGreen/RedOvatainateEntireSpike (dense)Erect
78 AV TRI 10VI047848TricolorBangladeshAVRDCErectAlong the stemPurple/PinkEntire laminaPurpleGreen/RedOvatainateEntireShort branchesErect
79 AV TRI 11VI047795TricolorBangladeshAVRDCErectFew branchesPurple/PinkEntire laminaPurpleGreen/RedRhombicEntireShort branchesErect
80 AV TRI 12VI044420TricolorChinaAVRDCErectFew branchesPurple/PinkCentral spotGreenGreenCuneateEntireSpike (dense)Erect
81 EW TRI ThidaThidaTricolorMalaysiaE-WESTErectAlong the stemGreenNormal greenGreenGreenOvatainateEntireSpike (dense)Erect
82 EW TRI ZeyaZeyaTricolorMalaysiaE-WESTErectAlong the stemGreenCentral spotGreenGreenOvatainateEntireSpike (dense)Erect
83 AV TRI 15VI042983TricolorIndonesiaAVRDCErectAlong the stemGreenNormal greenGreenGreenRhombicEntireSpike (dense)Erect
84 AV TRI 16VI047439TricolorIndiaAVRDCErectAlong the stemPurple/PinkCentral spotPurpleGreenOvatainateUndulateOtherErect
85 AV TRI 17VI048528TricolorJapanAVRDCErectMany branchesGreenOthersGreenGreenRhombicUndulateShort branchesErect
86 AV TRI 18VI044446TricolorIndiaAVRDCErectAlong the stemGreenTwo stripesGreenGreenLanceolateUndulateShort branchesErect
87 AV TRI 19VI044443TricolorIndiaAVRDCErectFew branchesGreenNormal greenWhiteYellowRhombicEntireClub-shapedErect
88 AV TRI 20VI043725TricolorMalaysiaAVRDCErectAlong the stemPurple/PinkCentral spotPurpleRedOtherUndulateShort branchesErect
89 AV TRI 21VI043724TricolorMalaysiaAVRDCErectAlong the stemPurple/PinkCentral spotPurpleGreenOvatainateEntireLong branchesDrooping
90 AV TRI 22VI044438-ATricolorNigeriaAVRDCErectFew branchesGreenNormal greenGreenGreenRhombicUndulateLong branchesErect
91 AV TRI 23VI055809TricolorLaosAVRDCErectAlong the stemGreenCentral spotGreenGreenRhombicUndulateLong branchesErect
92 AV TRI 24VI044396-ATricolorPakistanAVRDCErectAlong the stemGreenNormal greenGreenGreenMixtureEntire/UndulateLong branchesErect
93 AV TRI 25VI049129TricolorThailandAVRDCErectAlong the stemPurple/PinkCentral spotGreenGreenEllipticalUndulateLong branchesDrooping
94 AV TRI 26VI049006TricolorThailandAVRDCErectFew branchesPurple/PinkCentral spotGreenGreenOvatainateEntireLong branchesErect
95 AV TRI 27VI049004TricolorThailandAVRDCErectMany branchesGreenNormal greenGreenGreenLanceolateUndulateShort branchesErect
96 AV TRI 28VI044389TricolorTurkeyAVRDCErectNo branchesPurple/PinkNormal greenGreenGreenOvatainateEntireSpike (dense)Erect
97 AV TRI 29VI044470TricolorUSAAVRDCErectFew branchesGreenNormal greenGreenGreenLanceolateUndulateSpike (dense)Erect
98 AV TRI 30VI047747TricolorViet NamAVRDCErectAlong the stemGreenchlorotic stripeGreenGreenRhombicUndulateSpike (dense)Drooping
99 AV TRI 31VI050615-ATricolorViet NamAVRDCErectAlong the stemPurple/PinkEntire laminaDark purpleRedOvatainateUndulateSpike (dense)Erect
100 AV TRI 32VI050613TricolorViet NamAVRDCErectMany branchesGreenNormal greenGreenGreenOvatainateEntireShort branchesErect
101 AV TRI 33VI050610-ATricolorViet NamAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateUndulateShort branchesErect
102 AV TRI 34VI050609-ATricolorViet NamAVRDCErectMany branchesPurple/PinkCentral spotPurpleGreenRhombicEntireShort branchesErect
103 AV TRI 35VI047603TricolorViet NamAVRDCErectAlong the stemPurple/PinkEntire laminaPurplePinkOvatainateEntireSpike (dense)Erect
104 Local PRvar. BBS014TricolorUnknownLOCALErectAlong the stemPurple/PinkEntire laminaPurpleRedOvatainateEntireShort branchesErect
105 AV TRI 37VI054536TricolorTaiwanAVRDCErectFew branchesPinkCentral spotGreenGreenOvatainateUndulateLong branchesDrooping
106 AV TRI 38VI050214TricolorTaiwanAVRDCErectAlong the stemPinkCentral spotGreenGreenEllipticalEntireLong branchesErect
107 AV TRI 39VI054572TricolorPhilippinesAVRDCErectMany branchesPurple/PinkEntire laminaDark purpleRedRhombicUndulateLong branchesDrooping
108 AV TRI 40VI054571TricolorPhilippinesAVRDCErectAlong the stemPurple/PinkEntire laminaDark purpleRedOvatainateUndulateLong branchesErect
109 AV TRI 41VI044450TricolorPapua New GuineaAVRDCErectAlong the stemGreenSpotted purplePurpleGreenLanceolateUndulateOtherErect
110 AV TRI 42VI044407TricolorPapua New GuineaAVRDCErectAlong the stemGreenSpotted purplePurpleGreenEllipticalEntireOtherOther
111 AV TRI 43VI048301TricolorBangladeshAVRDCErectAlong the stemPurpleEntire laminaDark purpleRedOvatainateUndulateShort branchesDrooping
112 AV TRI 44VI048286TricolorBangladeshAVRDCErectAlong the stemPurple/PinkMargin/VeinPink/GreenOtherMixtureEntireShort branchesErect
113 AV TRI 45VI048021TricolorBangladeshAVRDCErectFew branchesPurpleEntire laminaPurpleRedRhombicEntireShort branchesDrooping
114 AV TRI 46VI047929TricolorBangladeshAVRDCErectAlong the stemPurple/PinkEntire laminaPurpleGreen/RedOvatainateUndulateShort branchesErect
115 AV TRI 47VI047682TricolorBangladeshAVRDCErectAlong the stemPink/GreenMargin/VeinGreenPinkRhombicUndulateSpike (dense)Drooping
116 AV TRI 48VI047681TricolorBangladeshAVRDCErectFew branchesGreenNormal greenGreenGreenOvatainateUndulateSpike (dense)Erect
117 AV TRI 49VI047504TricolorBangladeshAVRDCErectFew branchesPurple/PinkEntire laminaDark purpleRedRhombicEntireSpike (dense)Erect
118 AV TRI 50VI047501TricolorBangladeshAVRDCErectFew branchesPurpleEntire laminaPurpleRedMixtureEntireErect
119 AV TRI 51VI057270TricolorCambodiaAVRDCErectFew branchesGreenNormal greenGreenGreenRhombicEntireShort branchesDrooping
120 AV TRI 52VI056168TricolorCambodiaAVRDCErectFew branchesPurple/PinkNormal greenGreenGreenOtherUndulateShort branchesErect
121 AV TRI 53VI042979TricolorIndonesiaAVRDCErectFew branchesGreenNormal greenGreenGreenOvateUndulateSpike (dense)Drooping
122 AV TRI 54VI042978TricolorIndonesiaAVRDCErectFew branchesGreenNormal greenGreenGreenOvateUndulateSpike (dense)Erect
123 AV TRI 55VI059413TricolorIndiaAVRDCErectAlong the stemGreenNormal greenGreenGreenRhombicUndulateOtherErect
124 AV TRI 56VI058498TricolorIndiaAVRDCErectAlong the stemPurple/PinkMargin/VeinPurplePinkLanceolateUndulateOtherErect
125 AV TRI 57VI044426TricolorMalaysiaAVRDCErectFew branchesGreenNormal greenGreenGreenRhombicUndulateOtherDrooping
126 AV TRI 58VI055139TricolorMalaysiaAVRDCErectFew branchesPurple/PinkCentral spotPurpleGreenRhombicEntireOtherDrooping
127 AV TRI 59VI055062TricolorMalaysiaAVRDCErectAlong the stemGreenchlorotic stripeGreenGreenLanceolateEntireOtherErect
128 AV TRI 60VI033490TricolorMalaysiaAVRDCErectAlong the stemPurple/PinkCentral spotPurpleGreenRhombicUndulateOtherErect
129 AV TRI 61VI033480TricolorMalaysiaAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateUndulateOtherErect
130 AV TRI 62VI033474TricolorMalaysiaAVRDCErectAlong the stemGreenOthersGreenGreenOtherUndulateOtherErect
131 AV TRI 63VI033473TricolorMalaysiaAVRDCErectAlong the stemPink/GreenCentral spotPink/GreenGreenRhombicUndulateShort branchesDrooping
132 AV TRI 64VI049005TricolorThailandAVRDCErectAlong the stemGreenchlorotic stripeGreenGreenOtherUndulateOtherDrooping
133 AV TRI 65VI044379-ATricolorUSAAVRDCErectFew branchesGreenNormal greenGreenGreenRhombicUndulateOtherErect
134 AV TRI 66VI047526-ATricolorViet NamAVRDCErectAlong the stemPurple/PinkMargin/VeinPurplePinkRhombicEntireOtherErect
135 AV TRI 67VI047387TricolorViet NamAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireSpike (dense)Erect
136 AV TRI 68VI050111TricolorTaiwanAVRDCErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireShort branchesErect
137 AV TRI 69VI049431TricolorTaiwanAVRDCErectAlong the stemGreenNormal greenGreenGreenObovateUndulateShort branchesErect
138 US TRI 1Ames 5368TricolorBangladeshUSDAErectAlong the stemPinkEntire laminaPurpleRedEllipticalEntireLong branchesErect
139 US TRI 2Ames 29504TricolorBrazilUSDAErectAlong the stemPinkEntire laminaPurpleRedRhombicEntireSpike (dense)Erect
140 US TRI 3Ames 29505TricolorBrazilUSDAErectAlong the stemPinkEntire laminaPurpleRedRhombicEntireSpike (dense)Drooping
141 US TRI 4Ames 2017TricolorChinaUSDAErectAlong the stemGreenNormal greenGreenGreenOvatainateEntireSpike (dense)Erect
142 US TRI 5PI 419121TricolorChinaUSDAErectAlong the stemPurpleEntire laminaPurpleRedOvatainateEntireShort branchesErect
143 US TRI 6PI 478310TricolorChinaUSDAErectAlong the stemPurple/PinkMixturePink/GreenGreen/RedOvatainateEntireSpike (dense)Drooping
144 US TRI 7Ames 2204TricolorHong KongUSDAErectAlong the stemPinkCentral spotGreenGreenOvatainateUndulateShort branchesErect
145 US TRI 8Ames 2205TricolorHong KongUSDAErectAlong the stemGreenNormal greenGreenGreenOvatainateEntireShort branchesErect
146 US TRI 9Ames 2040TricolorIndiaUSDAErectAlong the stemGreenNormal greenGreenGreenOvatainateUndulateShort branchesErect
147 US TRI 10Ames 2145TricolorIndiaUSDAErectAlong the stemPinkNormal greenGreenGreenOvateUndulateShort branchesErect
148 US TRI 11PI 669847TricolorIndiaUSDAErectAlong the stemPurpleEntire laminaPurpleRedOvatainateUndulateShort branchesErect
149 US TRI 12PI 674261TricolorIndiaUSDAErectAlong the stemPurpleEntire laminaPurpleRedOvatainateUndulateShort branchesErect
150 US TRI 13Ames 2039TricolorIndonesiaUSDAErectAlong the stemGreenNormal greenGreenGreenRhombicCrenateShort branchesErect
151 US TRI 14Ames 5354TricolorMadagascarUSDAErectAlong the stemGreenBasal areaGreenGreenOvateEntireLong branchesErect
152 US TRI 15Ames 2029TricolorMalaysiaUSDAErectAlong the stemPinkCentral spotPurpleGreen/RedEllipticalEntireSpike (dense)Erect
153 US TRI 16Ames 29034TricolorMalaysiaUSDAErectAlong the stemOthersOthersOthersGreenRhombicUndulateShort branchesErect
154 US TRI 17Ames 5111TricolorPapua New GuineaUSDAErectAlong the stemGreenNormal greenGreenGreenOvateEntireSpike (dense)Erect
155 US TRI 18PI 349553TricolorPapua New GuineaUSDAErectAlong the stemGreenSpotted purplePurpleGreenOvateCrenateOtherOther
156 US TRI 19Ames 2199TricolorTaiwanUSDAErectAlong the stemGreenCentral spotPurple/PinkGreenOvatainateEntireLong branchesErect
157 US TRI 20Ames 2024TricolorThailandUSDAErectAlong the stemGreenNormal greenGreenGreenRhombicUndulateLong branchesErect
158 US TRI 21PI 607446TricolorThailandUSDAErectAlong the stemGreenNormal greenWhiteGreenOvatainateEntireShort branchesErect
159 US TRI 22PI 603897TricolorUSAUSDAErectAlong the stemPurpleEntire laminaPurpleRedEllipticalEntireOtherOther
160 US TRI 23PI 603898TricolorUSAUSDAErectAlong the stemPurpleMargin/VeinPurpleRedEllipticalCrenateOtherOther
161 US TRI 24PI 632237TricolorUSAUSDAErectAlong the stemPurpleCentral spotGreenGreenRhombicEntireLong branchesErect
162 US TRI 25Ames 5110TricolorWest AfricaUSDAErectAlong the stemGreenNormal greenGreenGreenEllipticalEntireLong branchesErect
163 US TRI 26Ames 1980TricolorZaireUSDAErectAlong the stemGreenNormal greenGreenGreenOvatainateEntireLong branchesErect
164 US TRI 27Ames 26209TricolorChinaUSDAErectAlong the stemPinkBasal areaGreenGreenRhombicEntireSpike (dense)Erect
165 Local Redvar. BBS027TricolorMalaysiaLOCALErectAlong the stemGreenCentral spotGreenGreenOvatainateEntireShort branchesErect
166 US TRI 29Ames 26216TricolorChinaUSDAErectAlong the stemPurpleEntire laminaPurpleRedLanceolateEntireSpike (dense)Erect
167 US TRI 30Ames 5102TricolorHong KongUSDAErectAlong the stemPurple/PinkCentral spotPink/GreenGreenLanceolateEntireOtherDrooping
168 US TRI 31Ames 5317TricolorHong KongUSDAErectAlong the stemGreenNormal greenGreenGreenOtherEntireOtherErect
169 US TRI 32PI 674260TricolorHong KongUSDAErectAlong the stemPink/GreenCentral spotGreenGreenOtherEntireSpike (dense)Erect
170 US TRI 33Ames 2100TricolorIndiaUSDAErectAlong the stemPurpleEntire laminaPurple/PinkRedLanceolateUndulateSpike (dense)Erect
171 US TRI 34Ames 2101TricolorIndiaUSDAErectAlong the stemPurple/PinkMargin/VeinPinkRedLanceolateEntireShort branchesErect
172 US TRI 35Ames 2102TricolorIndiaUSDAErectAlong the stemPurple/PinkMargin/VeinPurple/PinkRedRhombicEntireShort branchesErect
173 US TRI 36Ames 2119TricolorIndiaUSDAErectAlong the stemGreenNormal greenGreenGreenOvatainateEntireOtherErect
174 US TRI 37Ames 2120TricolorIndiaUSDAErectAlong the stemPink/GreenMargin/VeinPinkGreenOtherUndulateOtherErect
175 US TRI 38Ames 2121TricolorIndiaUSDAErectAlong the stemPinkNormal greenGreenGreenEllipticalEntireShort branchesErect
176 US TRI 39Ames 2132TricolorIndiaUSDAErectAlong the stemPurpleMargin/VeinPurpleRedLanceolateEntireShort branchesErect
177 US TRI 40Ames 2134TricolorIndiaUSDAErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireShort branchesErect
178 US TRI 41Ames 2135TricolorIndiaUSDAErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireShort branchesErect
179 US TRI 42Ames 2138TricolorIndiaUSDAErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireShort branchesErect
180 US TRI 43Ames 2223TricolorIndiaUSDAErectAlong the stemPurple/PinkMargin/VeinPinkGreenLanceolateEntireShort branchesErect
181 US TRI 44Ames 2224TricolorIndiaUSDAErectAlong the stemPink/GreenCentral spotPink/GreenLanceolateEntireShort branchesErect
182 US TRI 45Ames 5117TricolorPuerto RicoUSDAErectAlong the stemPinkNormal greenPinkGreenLanceolateEntireOtherErect
183 US TRI 46Ames 5118TricolorPuerto RicoUSDAErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireShort branchesErect
184 US TRI 47Ames 1993TricolorTaiwanUSDAErectAlong the stemPink/GreenCentral spotGreenGreen/RedOvatainateEntireShort branchesErect
185 US TRI 48Ames 1998TricolorTaiwanUSDAErectAlong the stemGreenNormal greenGreenGreenOtherEntireOtherErect
186 US TRI 49Ames 5134TricolorUSAUSDAErectAlong the stemGreenNormal greenGreenGreenLanceolateEntireLong branchesErect
187 US TRI 50Ames 25153TricolorUSAUSDAErectAlong the stemPink/GreenBasal areaGreenGreenEllipticalEntireShort branchesErect
188 US TRI 51PI 633591TricolorUnknownUSDAErectAlong the stemPink/GreenBasal areaGreenGreenOvatainateEntireOtherErect
A single plant of each accession was grown under shade-house conditions at University of Nottingham Malaysia (latitude 2.940°N, longitude 101.8740°E), with an average of 36°C daytime temperature, 28°C night temperature and 66% relative humidity. Plants were grown in a 16 x 12.5 x 14.5 cm plastic pot containing 2 kg black compost (Holland peat, Malaysia), irrigated daily to field capacity and at 3 weeks old, 3 g of 15N: 15P: 15K fertilizer was applied once to individual pots. Ten qualitative traits including leaf, petiole and stem pigmentations, growth habit, branching index and, leaf shape and margin were recorded at 7 weeks post-emergence and terminal inflorescence color, shape and attitude were recorded when all accessions had fully set (at 11 weeks post emergence) using AVRDC descriptors (https://avrdc.org/seed/) (Table 1). Young leaf material was collected and snap frozen in liquid N2 and stored at -80°C for DNA analysis.

DNA extraction and DArTSeq genotyping

Total genomic DNA of 188 amaranth accessions was isolated from young leaves using a Qiagen DNeasy plant DNA extraction kit (Qiagen, USA) and DNA quality and quantity was evaluated using a Nanodrop spectrophotometer (Thermo Scientific, USA). The DNA concentration was adjusted within the range of 50-100ng/μl. 2 μg of high molecular weight and good quality DNA per sample was sent to Diversity Arrays Technology Pty Ltd, Canberra, Australia for DArTSeq analysis. In brief, DArTSeq technology relies on the combination of a complexity reduction method to enrich genomic representations, followed by next-generation sequencing by HiSeq2000 (Illumina, USA), as described by Kilian et al. [27]. In this study, a combination of a rare cutting methylation-sensitive restriction enzyme (RE) PstI with secondary frequently cutting RE MseI were selected to optimize the locus coverage, reproducibility and polymorphisms. The PstI-compatible adapter consists of the Illumina flow cell attachment sequence, sequencing primer and a ‘staggered’ of varying length barcode region. The reverse adapter consists of Illumina flow cell attachment region and MseI overhang sequence. The ligated fragments with both a PstI and MseI adapter were amplified via polymerase chain reaction (PCR) with a programme set to an initial denaturation step of 94°C for 1 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 58°C for 30 s and extension at 72°C for 45 s, before a final extension at 72°C for 7 min. Equimolar amounts of PCR products from each sample were combined followed by a single end sequencing of 77 cycles on an Illumina Hiseq2500. Twenty-four DNA samples were also genotyped in two technical replications to obtain the reproducibility of the marker data. The full SNP dataset is shown in S1 Table.

Data analysis

SNP filtering

The SNP data generated from DArTSeq technology were first physically mapped to Amaranthus hypochondriacus genome v2.1 [14] using CLC Genomic Workbench v8 (Qiagen), based on match of aligned sequence tags against the reference genome, with 80% length and similarity fraction [29]. To investigate species-specific SNPs among 12 amaranth species (not including species with one representative), the amaranth species were manually examined for unique SNPs presence in the mapped SNP markers. Six species with the highest species-specific SNPs were subjected to a Venn diagram to visualize the SNP loci shared among the species. The Venn diagram of overlapping SNP loci was generated using the online program Van de Peer Lab (http://bioinformatics.psb.ugent.be/). Genetic diversity and population structure was carried out in two steps. First, all 188 amaranth accessions consisting of 18 species were analyzed together and second, a subset of 120 A. tricolor accessions were analyzed separately, aiming to explore the genetic distances and population structure among the A. tricolor populations, which were of primary interest. In each dataset, the mapped SNP markers were trimmed by removing SNPs with <97% reproducibility, <70% call rate and <0.05 polymorphic information content (PIC) and SNPs located on minor contigs that were not have been annotated. Individual accessions with >30% missing data and SNP loci with >30% missing data were removed. The most informative SNPs with minimum allele frequency (MAF) >0.05 imputed using TASSEL v5.2.52 software [30] were selected for further analysis. Population structure was constructed using the structure-like population genetic analyses using R package LEA [31-33]. The number of populations was determined using cross-entropy criterion, based on the predictions of a fraction of masked genotypes (matrix completion) and on the cross-validation approach, with runs of eight values of K (K = 1:8). A distance matrix was generated using TASSEL v5.2.52 software which was used to conduct principal coordinate analysis (PCoA) and a phylogenetic tree based on UPGMA distance.

Genome-wide association study of morphological traits

GWAS was conducted on the observed ten morphological traits on the same SNP datasets used for genetic diversity analysis. A mixed linear model (MLM) was generated to determine the associations by using the Q-matrix from population structure analysis (R package LEA) and kinship (K) from centered IBS method via TASSEL v5.2.52 and marker trait association (MTA) was determined at P≤0.01 and P≤0.001. The Manhattan plots of–log(p-values) and the quantile-quantile plots (Q-Q) of expected vs observed p-values for SNP based genotype-phenotype associations were generated using TASSEL v5.2.52. The most significant flanking sequences of SNPs associated with the traits (P≤0.001) were queried against JBrowse Phytozome v13 database to obtain the putative biological functions.

Results

SNP marker discovery

DArTSeq generated 74,306 polymorphic SNP reads from the 188 amaranth accessions of 18 species (S1 Table). Of these reads, 63,821 SNPs could be physically mapped to the Amaranthus hypochondriacus genome with an averaged of 100% reproducibility (max = 100%, min = 93%, median = 100%), 74% call rate (max = 100%, min = 19%, median = 74%) and 0.14 PIC (max = 0.50, min = 0, median = 0.09). The majority of the SNPs were an A/G or C/T transition mutation (62%) while the other 38% were A/C, A/T, C/G, and G/T transversion mutation. A Venn diagram of the six largest sets of amaranth species showed species-specific SNP loci, with A. thunbergii showed the highest number of unique SNPs (26,629), followed by A. spinosus (1,008), A. graecizans (1,067), A. tricolor (820), A. hypochondriacus (437) and A. hybridus (296) (Fig 1). There were only 1,394 polymorphic SNP shared by all six species group.
Fig 1

Venn diagram showing the presence, average and overlap of SNPs in the six largest amaranth species sets.

Genetic diversity and population structure of two amaranth sets

First, for all 18 amaranth species, individual genotypes with >30% missing SNP data including A. atropurperus (AV-ATR), A. blitoides (AV-BLITO), and A. spinosus (AV-SPI 1, AV-SPI 5 and AV-SPI 6), A. retroflexus (US-RET 1) and A. hybridus (AV-HYB 3) were removed and a total of 8,668 SNPs remained for 16 amaranth species, comprised of 181 accessions, with an averaged of 100% reproducibility, 91% call rate, 0.28 PIC, 0.16 MAF, and 6.97% averaged missing value in SNP loci and 5% averaged missing value at the individual-level. Second, for 120 A. tricolor accessions, two individual accessions (AV-TRI 20 and AV-TRI 28) which contribute to 30% of the missing values were removed and a total of 9,789 SNPs remained for 118 A. tricolor accessions, with and averaged of 100% reproducibility, 78% call rate, 0.20 PIC, 0.07 MAF, and 2% averaged missing values in SNP loci and at individual-level. Both SNP datasets (from 16 amaranth species [181 accessions] and 118 A. tricolor accessions) shared 1346 SNPs identical markers. Population structure analysis demonstrated that the K-values of the 16 amaranth species dataset and the A. tricolor subset were K = 3 respectively, based on minimal cross-entropy (S1 Fig) and the Q-matrix is displayed in a bar plot representation (Fig 2A and 2B). Each vertical bar represents a single accession, and the length of each bar represents the proportion contributed by each sub-population (admixture) and the grouping of the populations are illustrated in UPGMA phylogenetic tree (Fig 3A and 3B). The PCoA demonstrates the genetic divergence of both marker datasets was consistent with the output of the population structure (Fig 4A and 4B).
Fig 2

Population structure of (A) 16 amaranth species and (B) 118 A. tricolor accessions at K = 3, respectively. Each vertical bar represents a single accession and the length of each bar represents the proportion contributed by each sub-population. The group membership for each population structure is similar to the UPGMA dendogram.

Fig 3

UPGMA phylogenetic tree of (A) 16 amaranth species and (B) 118 A. tricolor accessions. Yellow-dotted accessions were out-grouped A. tricolor and red-colored accessions were amaranth species that are closely related to most A. tricolor. Purple-colored and yellow-colored accessions were positioned in different clades of the second population structure as in (B).

Fig 4

3D-plot principles coordinate analysis of (A) 16 amaranth species and (B) 118 A. tricolor accessions.

Population structure of (A) 16 amaranth species and (B) 118 A. tricolor accessions at K = 3, respectively. Each vertical bar represents a single accession and the length of each bar represents the proportion contributed by each sub-population. The group membership for each population structure is similar to the UPGMA dendogram. UPGMA phylogenetic tree of (A) 16 amaranth species and (B) 118 A. tricolor accessions. Yellow-dotted accessions were out-grouped A. tricolor and red-colored accessions were amaranth species that are closely related to most A. tricolor. Purple-colored and yellow-colored accessions were positioned in different clades of the second population structure as in (B). 3D-plot principles coordinate analysis of (A) 16 amaranth species and (B) 118 A. tricolor accessions. The 16 amaranth species were grouped into three populations. The majority of A. tricolor accessions belonged to Pop 1, with the exception of six A. tricolor accessions which originated from Bangladesh and belonged to POP 2 (brown-colored accessions) while the two out-grouped A. tricolor accessions (AV-TRI 20 and AV-TRI 28) were separated into POP 3 (yellow-dotted colour accessions). The two grain-types amaranth species (A. hypochondriacus and A. cruentus) belonged to Pop 3 together with their putative progenitor (A. hybridus), with the exception of one A. cruentus accession (AV-CRU 5) which belongs to Pop 1. Other cultivated vegetable-type species such as A. blitum, A. graecizan, A. sp and A. thunbergii were closely related to A. tricolor in Pop 1 (red-colored accessions), although several accessions belonged to Pop 2. The weed-type species such as A. retroflexus and A. viridis were diverse between the three populations. The PCoA demonstrated that Pop 1 clustered tightly together depicting that little diversity may exist within the populations and closer to Pop 2 which may explain the inter-specific admixtures. Meanwhile Pop 2 and Pop 3 showed some dispersal and diversity within the populations. The A. tricolor subset demonstrated that accessions were divided into three sub-populations. Sub-pop 1 was made up of 105 accessions from 12 countries of origin, Sub-Pop 2 comprised of seven accessions, of which three accessions were from Papua New Guinea and four accessions from USA, and Sub-pop 3 consists of six Bangladeshi accessions with distinct morphological traits (had branches along the stem, purple-pink stem color, purple leaf and petiole color, red-green inflorescence color and erect terminal inflorescence attitude) (Table 1). In comparison with the 16 amaranth species population structure, A. tricolor accessions that belong to Sub-pop 2 grouped together with the rest of A. tricolor accessions in Pop 1 (brown-colored accessions). Meanwhile, the six distinct Bangladeshi A. tricolor accessions of Sub-pop 3 remained separated from the rest of A. tricolor accessions similar to Pop 2. The PCoA displayed a clear division between the sub-populations and the overall population statistic calculated using a Monte-Carlo test revealed that there is an overall significant difference between the sub-populations (P = 0.002).

SNP associations for morphological traits

GWAS identified 22 significant and “suggestive” MTAs on 16 chromosomes of 16 amaranth species that underline four morphological traits observed in branching index, inflorescence color, leaf shape (P≤0.01, P≤0.001) and leaf pigmentation (P≤0.01) (Table 2; S2 Table). At P≤0.001, four SNP markers were associated with branching index, six SNP markers associated with inflorescence color and two SNP markers associated with leaf shape. Meanwhile, 100 significant MTAs were generated from 118 A. tricolor, distributed among 16 chromosomes that underline four morphological traits observed in inflorescence color, and leaf, petiole and stem pigmentations (P≤0.01, P≤0.001) (Table 3, S2 Table). At P≤0.001, forty-five SNP markers were associated with leaf pigmentation, eight SNP markers associated with petiole pigmentation, four SNP markers associated with inflorescence color and two SNP markers associated with stem pigmentation.
Table 2

12 MTAs (P≤0.001) of three morphological traits; branching index, inflorescence color and leaf shape in 16 amaranth species.

TraitSNP Allele IDChrSNP position (bp)PMarker R2Transcript nameProtein homologs
Branching index33457123|F|0–18:G>A-18:G>A312293250.0000.09588-
33427832|F|0–36:A>T-36:A>T141.1E+070.0010.0854
33440918|F|0–66:G>T-66:G>T584857200.0010.08314
33416254|F|0–26:C>T-26:C>T129883520.0010.08504
Inflorescence color33414045|F|0–22:A>G-22:A>G142E+070.0000.13234-
33416574|F|0–45:G>A-45:G>A111.4E+070.0000.14652-
33420897|F|0–6:A>G-6:A>G1237264570.0000.10482AH018341-RAFTIP1: FT-interacting protein 1 (A. thaliana)
33430487|F|0–18:C>T-18:C>T532818050.0000.18777-
33423475|F|0–8:C>G-8:C>G22.2E+070.0010.08268
33435567|F|0–16:T>C-16:T>C1673109070.0010.08184
Leaf shape33459037|F|0–16:T>C-16:T>C646189290.0010.08407
33443436|F|0–14:G>A-14:G>A643716840.0010.08261
Table 3

58 MTAs (P≤0.001) of four morphological traits; inflorescence color, and leaf, petiole and stem pigmentations in 118 A. tricolor accessions.

TraitSNP Allele IDChrSNP position (bp)PMarker R2Transcript nameProtein homologs
Inflorescence color33421031|F|0–23:A>G-23:A>G1544783220.0000.16AH022173Similar to MIEL1:E3 ubiquitin-protein ligase MIEL1 (A. thaliana)
33435567|F|0–16:T>C-16:T>C1673109070.0000.14
33416574|F|0–45:G>A-45:G>A11136037820.0010.13
33439433|F|0–6:G>T-6:G>T3184205380.0010.12AH006016Similar to MOT 1:MOLYBDATE TRANSPORTER 1 (A. thaliana)
Leaf pigmentation33433818|F|0–31:C>A-31:C>A14193183460.0000.23
33436368|F|0–13:T>C-13:T>C2198850540.0000.21AH003516Similar to ATL: RING-H2 finger protein ATL78 (A. thaliana)
33423765|F|0–13:C>A-13:C>A2199329450.0000.21
33440367|F|0–5:G>A-5:G>A2320785650.0000.20
33405774|F|0–47:T>C-47:T>C2278260390.0000.20AH003908Similar to MRG1: Protein MRG1 (A. thaliana)
33447836|F|0–29:C>T-29:C>T848429220.0000.19
33404573|F|0–8:T>C-8:T>C10217205240.0000.18
33426216|F|0–17:T>C-17:T>C11146545290.0000.18
33437340|F|0–31:A>G-31:A>G10216511220.0000.18
33452287|F|0–24:G>A-24:G>A42618860.0000.17
33415060|F|0–37:A>G-37:A>G342875240.0000.17
33416965|F|0–32:T>C-32:T>C2260273390.0000.16
33448931|F|0–12:C>G-12:C>G2260061460.0000.16
33437546|F|0–67:G>A-67:G>A2284678490.0000.17
33415096|F|0–37:T>C-37:T>C1252180370.0000.16
33415113|F|0–42:G>A-42:G>A2272776610.0000.16
33405454|F|0–57:A>G-57:A>G647906040.0000.16
33445149|F|0–31:G>A-31:G>A9110640670.0000.16
33443618|F|0–25:C>T-25:C>T10221311920.0000.15
33423738|F|0–59:G>A-59:G>A1533818970.0000.15
33439322|F|0–35:C>T-35:C>T11196941420.0000.16
33458033|F|0–44:A>G-44:A>G4250062300.0000.15
33423857|F|0–63:C>T-63:C>T591049860.0000.14
33440367|F|0–41:G>A-41:G>A2320785650.0000.14
33448614|F|0–8:T>C-8:T>C6167030560.0000.14
33427440|F|0–60:T>C-60:T>C825053880.0000.14
33453932|F|0–61:C>T-61:C>T111282990.0000.14
33417247|F|0–61:C>T-61:C>T442751590.0000.14
33403207|F|0–28:A>G-28:A>G5218530560.0000.14
33426523|F|0–61:T>C-61:T>C7115259480.0000.14
33410609|F|0–37:A>G-37:A>G858197310.0000.14
33430995|F|0–34:T>G-34:T>G127071520.0000.14
33414569|F|0–44:A>G-44:A>G13150982940.0000.14
33420689|F|0–33:G>A-33:G>A345159160.0000.14
33436571|F|0–8:A>G-8:A>G4146127330.0000.15
33443221|F|0–41:G>A-41:G>A10219920960.0000.13
33437028|F|0–10:C>T-10:C>T4205571410.0000.13
33455993|F|0–25:T>C-25:T>C4205464810.0000.13
33444523|F|0–46:G>A-46:G>A10220458210.0010.14
33454339|F|0–18:C>T-18:C>T8166785070.0010.13
33439286|F|0–20:G>A-20:G>A1662916350.0010.17
33441539|F|0–51:C>T-51:C>T4277634480.0010.12
33453929|F|0–59:A>C-59:A>C4103495860.0010.12
33422968|F|0–36:C>A-36:C>A2208444220.0010.13
33457404|F|0–39:C>T-39:C>T1327223660.0010.13
Petiole pigmentation33432644|F|0–45:A>C-45:A>C7190472390.0000.21AH011978Similar to DMS3: Protein DEFECTIVE IN MERISTEM SILENCING 3 (A.thaliana)
33456924|F|0–8:T>C-8:T>C7191361160.0000.15
33451216|F|0–61:C>T-61:C>T894102490.0000.14
33422926|F|0–18:G>C-18:G>C10207760620.0010.14AH016449Similar to FPGS1: Folylpolyglutamate synthase (A. thaliana)
33442805|F|0–17:T>C-17:T>C11110117680.0010.12AH017000Similar to HT1:Serine/threonine-protein kinase HT1 (A. thaliana)
33439355|F|0–33:G>A-33:G>A3110595850.0010.12
33415765|F|0–49:T>C-49:T>C7189773960.0010.12
33417264|F|0–60:C>G-60:C>G7189773310.0010.12
Stem pigmentation33424999|F|0–57:T>C-57:T>C385536310.0000.20AH005492Similar to At1g68200: Zinc finger CCCH domain-containing protein 15 (A. thaliana)
Furthermore, the mapping of this amaranth panel with the reference genome, A. hypochondriacus [14] identified twelve putative candidate genes with functional protein. These markers had low phenotypic variation (<20%) evaluated on all respective traits. The Manhattan plots of–log(p)>3 and the Q-Q plots of these traits are presented in Figs 5 and 6.
Fig 5

Manhattan plot and QQ plot for branching index (BI), inflorescence color (IC), leaf pigmentation (LP) and leaf shape (LS) of 16 amaranth species.

Fig 6

Manhattan plot and QQ plot for inflorescence color (IC), leaf pigmentation (LP), petiole pigmentation (PP) and stem pigmentation (SP) of 118 A. tricolor accessions.

Discussion

The evaluation of molecular markers and morphological traits was carried out on single plants to retain homogeneity of germplasm, as morphological variations were observed among amaranth plants within one collection. The evaluation of single plants is necessary as amaranth has high phenotypic plasticity which appears to be heterogamous in field plantings and thus adapts easily to the environmental changes, even though selection within cultivar/landrace has the possibility to be infertile [34]. The capacity of amaranth to have wide genetic variability provides new prospects in the development of new crop varieties. Therefore, the construction of population structure in amaranth through a combination of morphological and molecular data is needed in order to develop a framework for future breeding programmes. GBS data can have a high proportion of missing values [16] and the number of SNPs retained for the analysis depends on the quality control method [35]. In this study, a large number of SNP markers (74,306 SNP) were generated through the DArTSeq method, a non-reference based approach (de novo) using the PstI and MseI endonucleases in the library preparation step. After aligning the sequence tags against the very high quality and full length macromolecules of the A. hypochondriacus reference genome for SNP locations [14], the DArTSeq was able to generate relatively large numbers of SNP marker which could be mapped to the A. hypochondriacus genome (63,821 SNP) and suggests that DArTSeq as a technique should provide for full genome coverage. The number of SNP loci discovered in this study compared favorably with previous GBS studies generated in amaranth species that used ApeKI single enzyme cutting combined with deep reference-based assembly methodsm [17] as well as studies that used two library preparations via reference-based and non-reference based assembly methods [15, 16]. After filtration, the range of polymorphic SNP markers used in this study was comparable with other findings, such as 3,974 DArTSeq SNPs successfully used for population structure of 67 wild Galapagos tomato accessions (Solanum cheesmaniae and S. galapagense) [36] and 3,956 DArTSeq SNPs used in 80 macadamia accessions (Macadamia integrifolia, M. tetraphylla and hybrids) [37]. Population structure analysis on 16 amaranth species generates consistent taxonomic classification of amaranth sub-genera which was previously defined using seeds, inflorescence and floral characteristics [7, 38]. Three amaranth sub-genera Amaranthus Amaranthus, Amaranthus Acnida and Amaranthus Albersia were well defined in this study, consistent with other GBS findings by [15]. Subgenus Amaranthus, comprised of grain amaranth (A. hypochondriacus and A. cruentus) and its weed progenitor (A. hybridus) were distinguished in Pop 3. Subgenus Albersia, which comprised of vegetable amaranth including A. tricolor were distinguished in Pop 1 and Pop 2, together with six out of seven A. blitum accessions, three A. graecizans accessions and four of six A. viridis accessions. Meanwhile, species belonging to subgenus Acnida, which comprised of weedy amaranth, A. spinosus and A. palmeri were diverse between the three sub-populations. Another important finding was A. hybridus that belonged to sub-genus Amaranthus was split into sub-genus Albersia. A. hybridus is the direct ancestor of cultivated grain amaranth species [39, 40], and the split of accessions identity could be due to inter-varietal hybridization. Weedy amaranth, A. spinosus is a cross-pollinated and subsequent gene flow between populations may occur more rapidly than the primarily self-pollinated amaranth species [40]. Lee et al. [47] also have stated that varying amounts of outcrossing and frequent interspecific and inter-varietal hybridization have occurred in amaranth accessions even though it is self-pollinated. Therefore, this could explain the admixture between amaranth species. Besides, this study found that weedy amaranth possessed more unique SNPs per accession than grain amaranth evidently perhaps suggesting that weedy species have had far less selection pressure than the cultivated grain species, which is useful from a breeding perspective There is also genetic differentiation between grain and vegetable amaranth in this study, which has also been observed in many molecular markers studies, including AFLP [41], SSR [23, 42, 43] and GBS [15], although those studies incorporated far fewer A. tricolor accessions. This genetic analysis has not only revealed duplicates and genetically closely related individuals, but also allowed categorization of accessions into the correct species. In this study, two A. tricolor accessions (AV-TRI 20 and AV-TRI 28) from Asia deviated from the A. tricolor clade and were grouped together with sub-genera Amaranthus, which mainly belonged to grain and weed amaranths. There are two assumptions for this finding, either the two amaranths were incorrectly identified as A. tricolor [17] or were originally a landrace that was grown in a region where grain amaranth was traditionally cultivated over a long time through seeds exchange [44-46]. In a previous study, GBS accurately identified A. caudatus accession PI 490752, characterized as A. hypochondriacus by 11 SSR markers [39], but it should be assigned into the A. caudatus group [17]. Therefore, re-analysis should be carried out for these two A. tricolor accessions, with addition of larger morphological dataset, which could correct the possible misclassification. The occurrence of admixed/hybrid genotypes may indicate frequent hybridization or introgression events. An experiment based on SSR markers by [23] revealed that A. tricolor accessions did not correlate between groups which may imply that A. tricolor had larger genetic variation. There was also uncertainty in positioning phylogeny of A. tricolor accessions among amaranth species, although A. tricolor accessions were grouped together in a clade [15]. A. tricolor had by far the largest estimated genome size (782.7Mbp) among 35 amaranth species, and this suggests that polyploidization likely influenced the genome size of this species [15]. In this study, the species groupings were independent of the accession’s geographical origin, contradicting previous GBS findings [15-17]. In previous studies, geographical patterns demonstrate that comprehensive origin sampling can assist in understanding the evolution of the species as shown by a strong split of geographic pattern in A. hybridus between accessions from Central and South America, which later supports the hypothesis that two different lineages were the ancestors of the grain amaranth [15]. In this study, the genetic differentiation between species and geographical origin was weak, although a strong split of geographical pattern was observed in A. hybridus where accessions from America and Africa were divided into two clusters, which may explain the genetic differentiation of hybridus complex [23]. This is probably due to the cosmopolitan nature of the genus, or the results of human activities such as breeding and resource exchange [47]. While the current study used a different restriction endonuclease frequent cutter for construction of the genomic representations sequenced, the biased number of accession per species could contribute to the lack discrimination of geographical origin and species level. This was also observed in 3,431 DArTSeq SNPs used to conduct genetic diversity in 89 safflower accessions (Carthamus tinctorius L.), in which the SNPs showed weak correlation between safflower diversity pattern and origins, when compared with to a larger SNP dataset [48]. However, for a large set of 118 A. tricolor accessions, genetic differentiation of Bangladeshi accessions was clear as they clustered together and had distinct morphological characters. The closely related A. hypochondriacus genome was used as the genome reference for association mapping as no A. tricolor genome is available to date. The utilities of the reference quality genome were demonstrated in two ways, i.e. chromosomal evolution and mapping of genetic locus responsible for stem color, hence ample support to clarify the scientific understanding of a useful agricultural trait in amaranth. The highly significant MTA found in morphological traits in this study illustrate how this DArTSeq data can provide high resolution genome coverage for mapping opportunities. However, the most significant associations detected in the MLM model had a lower threshold (−log(p−value)<4, although the mixed model was superior, it still could be lead to at least one false negative and false positive [49]. This could be due to the use of different amaranth species (A. hypochondriacus) as a reference genome instead of the A. tricolor genome. The difficulty of working with plant genomes is that they are highly repetitive and feature extensive structural variation between members of the same species, mostly attributed to their active transposons [50] and chromosomal rearrangements. For example, in the well-studied species Arabidopsis thaliana, natural accessions are missing 15% of the reference genome, indicating a similar fraction would be absent from the reference, but present in other accessions [51]. Moreover, although A. thaliana has a small (140 Mb) and not very repetitive genome compared to many other plants, SNPs may be assigned to incorrect positions due to sequence similarity shared between unlinked loci [52]. Therefore, more extensive structural variation would be expected in a larger A. tricolor genome, which contain a higher proportion of repeats and has undergone ancient and recent rounds of polyploidization [15].

Conclusions

The findings in this study demonstrated that the DArTSeq SNP data generated from 181 amaranth accessions comprised of 16 species was capable of differentiating vegetable amaranth, A. tricolor from grain and wild amaranth species. The species groupings were independent of accessions’ geographical origin. This is likely a result of germplasm origin being registered as where the seeds were donated from, which may not be the actual origin of the accession or movement of germplasm in recent historical time. For a larger A. tricolor data set, there was likelihood that a good differentiation of A. tricolor could be achieved based on a combined analysis of molecular markers, geographical origin and morphological traits. GWAS used to conduct a pilot genome association for 10 morphological traits demonstrates the potential effectiveness of the amaranth diversity panel for trait dissection. The high degree of morphological variation observed in amaranth may be beneficial in terms of its adaptive capabilities in different climatic conditions.

DArTSeq SNP reads from the 188 amaranth accessions for18 species.

(XLSX) Click here for additional data file.

10 morphological traits of A. tricolor subset observed under shade-house conditions.

(XLSX) Click here for additional data file. Cross-entropy plot for (a) first population structure: 181 amaranth accessions of 16 species and (b) second population structure: 118 A. tricolor accessions. A range of K = 1:8 was tested and K = 3 was chosen as the cross-entropy curve exhibits a plateau in both datasets. (TIF) Click here for additional data file.
  29 in total

Review 1.  Transposable element contributions to plant gene and genome evolution.

Authors:  J L Bennetzen
Journal:  Plant Mol Biol       Date:  2000-01       Impact factor: 4.076

2.  Relative efficiency of morphological characters and molecular markers in the establishment of an apricot core collection.

Authors:  L Krichen; J M Audergon; N Trifi-Farah
Journal:  Hereditas       Date:  2012-09-21       Impact factor: 3.271

3.  Genomic and phenotypic evidence for an incomplete domestication of South American grain amaranth (Amaranthus caudatus).

Authors:  Markus G Stetter; Thomas Müller; Karl J Schmid
Journal:  Mol Ecol       Date:  2017-01-27       Impact factor: 6.185

4.  Parallel Seed Color Adaptation during Multiple Domestication Attempts of an Ancient New World Grain.

Authors:  Markus G Stetter; Mireia Vidal-Villarejo; Karl J Schmid
Journal:  Mol Biol Evol       Date:  2020-05-01       Impact factor: 16.240

5.  A high density GBS map of bread wheat and its application for dissecting complex disease resistance traits.

Authors:  Huihui Li; Prashant Vikram; Ravi Prakash Singh; Andrzej Kilian; Jason Carling; Jie Song; Juan Andres Burgueno-Ferreira; Sridhar Bhavani; Julio Huerta-Espino; Thomas Payne; Deepmala Sehgal; Peter Wenzl; Sukhwinder Singh
Journal:  BMC Genomics       Date:  2015-03-19       Impact factor: 3.969

6.  Diversity in Grain Amaranths and Relatives Distinguished by Genotyping by Sequencing (GBS).

Authors:  Xingbo Wu; Matthew W Blair
Journal:  Front Plant Sci       Date:  2017-11-17       Impact factor: 5.753

7.  Genetic Diversity and Population Structure of Two Tomato Species from the Galapagos Islands.

Authors:  Yveline Pailles; Shwen Ho; Inês S Pires; Mark Tester; Sónia Negrão; Sandra M Schmöckel
Journal:  Front Plant Sci       Date:  2017-02-15       Impact factor: 5.753

8.  Integrating genetic maps in bambara groundnut [Vigna subterranea (L) Verdc.] and their syntenic relationships among closely related legumes.

Authors:  Wai Kuan Ho; Hui Hui Chai; Presidor Kendabie; Nariman Salih Ahmad; Jaeyres Jani; Festo Massawe; Andrzej Kilian; Sean Mayes
Journal:  BMC Genomics       Date:  2017-02-20       Impact factor: 3.969

9.  Crossing Methods and Cultivation Conditions for Rapid Production of Segregating Populations in Three Grain Amaranth Species.

Authors:  Markus G Stetter; Leo Zeitler; Adrian Steinhaus; Karoline Kroener; Michelle Biljecki; Karl J Schmid
Journal:  Front Plant Sci       Date:  2016-06-07       Impact factor: 5.753

10.  1,135 Genomes Reveal the Global Pattern of Polymorphism in Arabidopsis thaliana.

Authors: 
Journal:  Cell       Date:  2016-06-09       Impact factor: 41.582
View more

北京卡尤迪生物科技股份有限公司 © 2022-2023.