Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 1. The genetic map position of the fcl1 locus on chromosome 6. (A) A

linkage map based on the recombination frequency analysis of 254 individuals from an F2

mapping population and twelve SSR markers initially identified to be co-segregated with the

fcl1 locus from a bulked segregant analysis. (B) A fine genetic map based on the recombinant

frequency analysis of 213 fcl1 mutant plants from an F2 mapping population of 787

individuals. The fcl1 locus was linked to the SSR marker AC141436-ssr4.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 2. Confirmation of deletion junctions and expression analysis of genes

deleted in fcl1-1 and fcl1-2 alleles. (A) PCR amplification of deletion junctions in fcl1-1 (lanes

1, 2) and fcl1-2 (lanes 3, 4). A17, wild-type plants; MW, molecular weight ladders. (B) Reverse

transcription (RT)-PCR amplification of transcripts encoded by the twelve predicted ORFs

deleted in the fcl1-1 and fcl1-2 alleles in vegetative shoot apices of WT plants. A high level of

transcripts was detected for ORFs 1, 3, 4, 6, 11 and 12. Primer sequences used are listed in

Supplemental Table 4.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 3. Allelic series tests of fcl1 mutants. (A-E) Four-week-old WT (cv.

Jemalong A17; A), fcl1-2 (B), WT (cv. R108; C), fcl1-3 (D) and an F1 plant from a cross

between fcl1-2 and fcl1-3 (E). (F) The Tnt1 insertion site in fcl1-3 and fcl1-4 mutants. Open

box, untranslated region; solid box, exon; horizontal line, intron and arrowhead, orientation of

the open reading frame. (G) PCR-based genotyping. Lane 1, PCR amplification of the deletion

junction in the fcl1-2 mutant. Lane 2, PCR amplification of the FCL1 gene in WT (cv. Jemlong

A17 or R108). Lane 3, PCR amplification of the Tnt1 insertion site in the fcl1-3 mutant. (H)

Compound leaf phenotype of wild-type (left) and the fcl1-4 mutant (right). (I) Reduced petiole

length of compound leaves from different nodes of the stem in fcl1-4 mutant. Shown were

mean ± s.d., n=3.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 4. Compound leaf phenotypes of fcl1-4 mutant. (A) Three-month-old

fcl1-4 (-/-) homozygous plants (right) exhibited various compound leaf phenotypes, including

leaflet clustering and fusion. fcl1-4 (+/-) heterozygous plants (left) were used as a comparison,

showing wild-type-like trifoliolate compound leaves. (B) Close-up views of compound leaves

of fcl1-4 (+/-) heterozygous plants and fcl1-4 (-/-) homozygous plants as indicated.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 5. Genetic complementation of fcl1-1. (A, B) Transgenic fcl1-1 mutant

harboring a 7.8kb genomic sequence of FCL1 (fcl1-1/FCL1), showing both leaflet fusion and

reduced petiole phenotypes of the mutant were rescued.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 6. Zinc finger domain transcription factor Medtr6g080720.1 did not

rescue fcl1-1 compound leaf defects. (A) Image of a stably transformed fcl1-1 plant. (B) RT-

PCR analysis of gene expression in transgenic fcl1-1 plants. Compared with wild-type

(Jemalong A17) and fcl1-1 mutant, the zinc finger transcription factor (ZnF) was not expressed

in the fcl1-1 mutant but was expressed in transgenic fcl1-1 mutant transformed with the WT

gene similarly to the WT plants. However, the expression of the ZnF gene did not rescue the

mutant compound leaf defects.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

1 aatgttttgagataaatagtatatatgagcagtgttgtagatgtggtatatcaaaggttt

61 ttatttcatctactcaaaaatagttatatggaatctaagATGAATAGTTTTGTTGTTGAT

M N S F V V D

121 CATGAGAAGTACAAACAAGAAGAAAATCAAGAAGAATTGGAATTACAACATAAAGAAGGT

H E K Y K Q E E N Q E E L E L Q H K E G

181 GGTGTTCATGAACATGATCATCAAGAGGAAAATGATGAGATTCTTAAGACAAGAATCTCT

G V H E H D H Q E E N D E I L K T R I S

241 AACCATCCTCTCTATGAACTTCTTGTTCAAGCTCACTTGGATTGTTTGAAGGTTGGTGAC

N H P L Y E L L V Q A H L D C L K V G D

301 ATTTCTAACTTGGAGATAGAGAAATCAGATAAAAAGCAAACACTGAAGAAACAAAACTTG

I S N L E I E K S D K K Q T L K K Q N L

361 GATATGTTAAGCCAATCTGAGCTTGATCTCTTCATGGAAGCATATTGTTTGGCACTTAGT

D M L S Q S E L D L F M E A Y C L A L S

421 AAGCTAAAAGAAGCAATGAAGGAACCACAACAGAATTCAATGGCTTTCATAAACAACATG

K L K E A M K E P Q Q N S M A F I N N M

481 CATTCACAACTTAGGGAACTAACTCAGGCAACTTCATCTTCTAGTGAACCAGATGCTACT

H S Q L R E L T Q A T S S S S E P D A T

541 ACATCTTCAAGTGAGTGCACATTCAGAAGAAATCCATCAATTTAGaggttgatgacttga

T S S S E C T F R R N P S I *

601 ttttcagtagtgtggttgtgcatgtaatatcctaagataggttaattaattaaggtttca

661 attattgtcaaattgtcatgtccatgtttgaaatgttattttatgtgaaattaatgggca

721 atgaatgttgttataaattaattaatctatagtatttccccttatctcattaattaattg

781 cagttgttatctac

Supplemental Figure 7. Full-length FCL1 cDNA and the deduced amino acid sequences.

Lower case letters indicate 5’- and 3’- untranslated regions (UTRs). Capital letters indicate the

coding region. Red and blue arrowheads indicate the Tnt1 insertion sites in fcl1-3 and fcl1-4

mutants, respectively. Red and cyan shaded letters indicate the KNOX1 and KNOX2 domains,

respectively. An asterisk indicates the stop codon.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplement Figure 8. Amino acid sequence alignments of FCL1 and its close homologs.

FCL1 amino acid sequences were used as seeds to BLAST search the NCBI GenBank. The

identified homologous sequences were manually examined to ascertain that full-length

sequences do not contain the homeodomain. Sequences were aligned using Clustal X.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 9. Microarray-based in silico analysis of FCL1 tissue-specific

expression. Tissue-specific expression of FCL1 was analyzed using Affymetrix GeneChip

Medicago Genome Array-based Medicago Gene Expresion Atlas

(http://bioinfo.noble.org/gene-atlas/v2/).

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Figure 10. Quantitative RT-PCR analysis of KNOX2 (A) and KNOX6 (B)

expression in vegetative shoot buds and young leaves in wild-type and fcl1-1 mutant. Shown

were mean ± s.d., n=3.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Table 1. Segregation ratio of fcl1-2 mutants in F2 mapping populations

Population Genetic crosses wild type mutant total 𝑿𝟐 (P value)

1 fcl1-2 x Jemalong A20 175 79 254 5.045 (0.025)

2 fcl1-2 x Jemalong A20 399 134 533 0.006 (0.940)

Total fcl1-2 x Jemalong A20 574 213 787 0.179 (0.181)

𝑋2, Chi square based on 3:1 segregation ratio.

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128 Supplemental Table 2. Recombination frequency and genetic distance between the fcl1-2 locus and molecular markers on chromosome 6

Marker Recombination frequencies (± S.E.) Genetic distance (± S.E.) cM

DK321L 0.021 ± 0.007 2.114 ± 0.007

44D11L 0.005 ± 0.003 0.469 ± 0.003 005D01 0.005 ± 0.003 0.469 ± 0.003 AC141436-ssr4 0 ± 0 0 ± 0 002E12 0.014 ± 0.006 1.409 ± 0.006

002F12 0.021 ± 0.007 2.114 ± 0.007 18A5R 0.026 ± 0.007 2.585 ± 0.008 19O4L 0.026 ± 0.008 2.585 ± 0.008

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Table 3. Genotypes of 20 fcl1-2 mutant plants that carry recombination between fcl1

and closely linked markers

Marker

fc11-2 plant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

DK321L B B B B B B B H B H H H H H B B H H H B

005D01 B B B B B B B B B B B B B B B B H B H B

44D11L B B B B B B B B B B B B B B B B H B H B

AC141436-ssr4 B B B B B B B B B B B B B B B B B B B B

002E 12 B B B H H B H B H B B B B B H H B B B B

002F 12 H H H H H B H B H B B B B B H H B B B B

18A5R H H H H H H H B H B B B B B H H B B B H

19O4L H H H H H H H B H B B B B B H H B B B H

H, heterozygous mutant marker; B, homozygous mutant marker

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Table 5. Arabidopsis thaliana homologs of deleted genes in fcl1 mutants

Deleted gene Encoded

protein

Homolog Annotation Other names

Medtr6g080670.1

(partial)

424 a.a. AT3G17810 dihydropyrimidine

dehydrogenase

PYD1;

MEB5.3;

PYRIMIDINE

1

Medtr6g080680.1 68 a.a. No homolog

Medtr6g080690.1 161 a.a. AT1G14760.2 Class M Knox protein KNATM-B

Medtr6g080700.1 357 a.a. AT2G02650.1 Putative non-LTR

retroelement reverse

transcriptase

Medtr6g080710.1 31 a.a. No homolog

Medtr6g080720.1 1179 a.a. AT1G10170.1 Zinc finger domain

transcription factor

ATNFXL1

Medtr6g080730.1 154 a.a. AT4G33420.1 Putative peroxidase

Medtr6g080740.1 73 a.a. AT4G33420.1 Putative peroxidase

Medtr6g080750.1 60 a.a. No homolog

Medtr6g080760.1 53 a.a. No homolog

Medtr6g080770.1 161 a.a. AT3G05540.1 Translationally

controlled tumor protein

TCTP

Medtr6g080780.1

(partial)

356 a.a. AT1G66200.1 glutamate-ammonia

ligase

ATGSR2

Supplemental Data. Peng et al. (2011). Plant Cell 10.1105/tpc.111.089128

Supplemental Table 6. Double mutant analyses of F2 plants

Genetic cross WT-like sgl1- like fcl1- like plant with novel phenotype

sgl1-1 x fcl1-1 308 69 101 32

Genetic cross WT-like fcl1-like palm1-like plant with novel phenotype

fcl1-2 x palm1-2 400 186 124 0