Characterisation of SSR markers for New Zealand Craspedia and their application in Kahurangi National Park

Figures & data

Table 1 Localities and putative informally named Craspedia species.

Mt Arthur	Mt Mytton	Marino Mountains
‘C. elongata’ (CHR 514388)		‘C. elongata’ (CHR 635113)
‘C. long hairs’ (CHR 514387)	‘C. long hairs’ (limestone: CHR 635118; Thorns Creek: CHR 635119)	‘C. long hairs’ (CHR 635117)
‘C. marble’ (CHR 514389)	‘C. Mytton’ (CHR 514384)	‘C. calcicole’ (CHR 635120)
		‘C. Owen’ (CHR 635116)

Figure 1 Leaves of seven forms of Craspedia. A, ‘C. elongata’; B, ‘C. long hairs’ (Mt Arthur); C, ‘C. marble’; D, ‘C. long hairs’ (Thorns Creek); E, ‘C. calcicole’; F, ‘C. Owen’; G, ‘C. Mytton’.

Figure 2 Sampling locations in Nelson Province.

Table 2 Details of the simple sequence repeat markers characterised in this study.

Locus	Repeat^a	Forward primer	Reverse primer	Annealing temperature (°C)	Length (bp)^b
Cras2	(CTT)10	ACCAGAAACCTTGTCAACCAC	CGTCGATGACCAACTCGAAC	48	412–443
Cras5	(CCT)7N35(CCA)6	ACTGTTTCCGCCGATCCTC	GGTGGCGACTTTATTGGTG	60	207–268
Cras6	(CCA)7	CACATTCTGACCGTGCATC	CTTATAGAAATATGTGATCTC	50	294–309
Cras9	(CTT)7	AACGTGGAATTGTGCAGCG	GCTTCAAGAAAAGTTTCTCC	50	240–306
Cras12	(GTTTTG)6	CCCAAGTTACGTCGCTGAAG	ACCACAGGTTCAACTAACTCG	60	281–323
Cras15	(CCR)6	TCCTATTCACTTTACCAACCGC	GTCGACGGCTTTCTTGGTG	60	215–222
Cras24	(GGT)6	GGATTTCGCAGTCTCGGTG	CGGTTGTTGGAAGTAGGGC	55	436–471
Cras25	(CTTT)5	ATGCTGCTTTACCGCACTC	TGGTTTAATCTGCGCTCGATG	50	302–352
Cras29	(ATC)4	ACCCACCGTCAAACGGAAC	CCGACGAAGAAATTAATCTAGGGC	60	159–180
Cras36	(TGGC)5	CGTCACCGGGATCTCCTAC	TGGATGGAAAGATTAGGGTCTTC	60	306–318
Cras47	(CAC)6N39(CAC)5	CCTACTGAATCCCGGAGC	CGCTCGCCCTCAAACTTAC	60	260–299
Cras48	(TCA)7	CGATCACCACCCTCCATTTC	TCCGCTTCCCAACTTCCAG	60	281–307

^a As originally characterised.

^b Observed fragment length including 18-bp primer tail.

Table 3 Summary statistics for SSR markers characterised in this study.

Marker	Major allele frequency	Genotypes	Obs.	Allele #	Availability	HE	HO	PIC
Cra2	0.42	21	104	10	1	0.71	0.52	0.67
Cra5	0.30	26	102	11	0.98	0.81	0.55	0.79
Cra6b	0.50	11	104	6	1	0.64	0.38	0.58
Cra9b	0.22	37	103	14	0.99	0.86	0.60	0.85
Cra12	0.87	7	100	4	0.96	0.24	0.14	0.22
Cra15	0.95	3	93	2	0.89	0.09	0.01	0.09
Cra36	0.69	8	91	5	0.88	0.47	0.11	0.43
Cra24	0.34	18	103	9	0.99	0.76	0.34	0.72
Cra29	0.66	12	104	7	1	0.52	0.43	0.48
Cra25	0.56	9	102	4	0.98	0.61	0.33	0.56
Cra48	0.24	28	101	10	0.97	0.86	0.85	0.84
Cra47	0.19	42	100	13	0.96	0.88	0.71	0.87
Mean	0.49	18.5	100.58	7.92	0.97	0.62	0.41	0.59

H_E, expected heterozygosity (also called gene diversity); H_O, observed heterozygosity; Obs., number of observations (i.e. the number of individuals for which alleles could be scored); availability, Obs/n; PIC, polymorphism information content (Botstein et al. 1980). Sample size, n = 104 individuals.

Table 4 Values of K (the number of populations) for each structure analysis undertaken as indicated by comparison of ΔK and mean lnPr(X|K).

Samples included	Putative species number	Value K by highest ΔK, mean (var.) lnPr(X|K)	Value K by lnPr(X|K), mean (var.) lnPr(X|K)	Note
Mt Arthur	3	3, –693.31 (0.688719)	3, –693.31 (0.688719)	Values of K with maximum value for both quantities match prediction (K = 3)
Mt Mytton	2	12, −570.18 (4.343526)	3 −534.07 (0.494526)	Prominent peak in ΔK at K = 3; high value of ΔK at K = 12 due to low mean and high variance of lnPr(X|K) for K =13
Marino Mountains	4	2, −1151.85 (0.259272)	5, −1095.42 (23.948639)	Mean probability is only marginally less for K = 3 than K = 4 or K =5
Mt Arthur + Mt Mytton	3	4, −1556.00 (0.584998)	5 −1550.14 (26.401566)	Probability at K = 5 only marginally better than K = 4
All	5–9	6, −2438.41 (1.087760)	6, −2438.41 (1.087760)	Values of K with maximum value for both quantities match prediction (K = 6)

Figure 3 Structure bar-plots based on analyses of 12 SSR markers for putative Craspedia species from Kahurangi National Park, New Zealand. A, Mt Arthur K = 3; B, Mt Mytton with K = 3; C, Mt Arthur and Mt Mytton analysed together with K = 4; D, Marino Mountains with K = 2; E, Marino Mountains with K = 3 showing also collecting locality and field ID. ‘?’ indicates a sample identified as intermediate in morphology in the field; F, all samples analysed together with K = 6.

Figure 4 Neighbour-joining tree for population genetic distances based on 12 SSR markers among populations of Craspedia sampled in this study. Colours of labels match those used in Fig. 3.

Botstein D, White RL, Skolnick M, Davis RW 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics 32: 314–331.

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