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• 1 JN091682 Solanum lycopersicum 684 Present study 2 JN132111 Solanum lycopersicum 684 3 JN132112 Solanum lycopersicum 682 4 JN132113 Solanum lycopersicum 677 5 AB110700 Solanum lycopersicum 559 (31) 6 AY007240 Solanum lycopersicum 684 Xie and Wu (unpublished data) 7 L25128 Solanum lycopersicum 4272 (32) 8 AY129402 Solanum lycopersicum 1670 (33) 9 M15186 Solanum tuberosum 1241 (34) 10 U45450 Solanum tuberosum 2068 (35) 11 X04118 Solanum tuberosum 1914 (36) 12 X78275 Solanum tuberosum (Arran Banner) pin2-CM7 gene 584 (37) 13 Z12753 Solanum tuberosum 1695 (38) 14 Z13992 Solanum tuberosum 2330 Choi et al. (unpublished data) 15 AF330701 Ipomoea batatas 519 (39) 16 AF330702 Ipomoea batatas 524 (39) 17 AY059390 Phaseolus vulgaris 327 Yang et al. (unpublished data) 18 AY204563 Vigna unguiculata subsp. unguiculata 326 Yuan et al. (unpublished data) 19 AY204562 Vigna unguiculata subsp. unguiculata 326 Yuan et al. (unpublished data) 20 AF349441 Populus tremuloides 682 (40) 21 AY749108 Solanum americanum 1943 (41) 22 Z12824 Solanum tuberosum 1573 (38) 23 AM162668 Brassica napus 642 (42) 24 AM162667 Brassica napus 1023 (42) 25 AM162666 Brassica napus 1114 (42) 26 DQ412560 Vigna trilobata 678 Sinha et al. (unpublished data) 27 DQ417203 Vigna radiata 543 Sinha et al. (unpublished data) 28 DQ417204 Vigna unguiculata 663 Sinha et al. (unpublished data) 29 D17331 Solanum tuberosum 1103 (43) 30 D17332 Solanum tuberosum 1036 (43)
• I encodes the rapeseed glutamyl endopeptidase inhibitor. Although these PI genes are from the same plant (Brassica napus), there is a difference in their function and coding products. Genes closely related in terms of function are evolutionarily linked. Genes belonging to the same PI and plant family fall into different clusters on the basis of size difference. From the plant family Fabaceae, 2 PI genes (DQ412560 and DQ417204) fall in cluster I, while 3 others (AY204562, AY204563, and AY059390) are in cluster II. Although these 5 PI genes are trypsin PIs from same plant family
• (Fabaceae), they are in different clusters due to the difference in their sizes (nucleotide length base pairs). The 2 PI genes (DQ412560 and DQ417204) in cluster I are closely related in terms of size at 678 and 663 bp in length, respectively, while 3 other PIs that fall in cluster II
• (AY204562, AY204563, and AY059390) are 326, 326, and 327 bp, respectively. In silico phylogenetic evaluations can lead to important insights in terms of evolutionary affinities among investigated protein genes. Martinez et al. (44) phylogenetically analyzed different plant cystatins from rice, arabidopsis, and barley. In their study, 12 cysteine PI genes from rice, 7 from arabidopsis, and 7 from barley were analyzed in silico by constructing a phylogenetic tree by neighbor-joining method using the amino acid sequences of these 26 cystatin proteins. In an earlier study, molecular and phylogenetic analysis of the wound-inducible PI-I gene was carried out for the 7 direct ancestors of Lycopersicon esculentum: L. pennellii, L. chilense, L. hirsutum, L. parviflorum, L. peruvianum var. humifusum, L. cheesmanii, and L. peruvianum (45).
• In another report, it was observed that HvCPI-4 from Hordeum vulgare and OC-XII protein from Oryza sativa are closely allied with the highest similarity scores, while the Arabidopsis cystatins were found scattered in the resulting tree and appeared to be functionally distant from rice and barley proteins. In yet another study, Martinez   et al. (46) conducted the phylogenetic analysis of 17 cysteine PI proteins from different plants, and it was found that JN132112 Solanum lycopersicum
• AY007240 Solanum lycopersicum
• JN091682 Solanum lycopersicum
• JN132113 Solanum lycopersicum
• JN132111 Solanum lycopersicum Z13992 Solanum tuberosum
• DQ417203 Solanum tuberosum X04118 Solanum tuberosum D17331 Solanum tuberosum
• AF349441 Populus tremuloides AF330701 Ipomoea batatas AF330702 Ipomoea batatas DQ412560 Vigna trilobata
• DQ417204 Vigna unguiculata D17332 Solanum tuberosum Z12753 Solanum tuberosum AM162667 Brassica napus Z12824 Solanum tuberosum U45450 Solanum tuberosum
• AY749108 Solanum americanum
• AB110700 Solanum lycopersicum X78275 Solanum tuberosum
• AY129402 Solanum lycopersicum M15186 Solanum tuberosum
• L25128 Solanum lycopersicum AM162668 Brassica napus AM162666 Brassica napus
• AY204562 Vigna unguiculata
• AY059390 Phaseolus vulgaris
• AY204563 Vigna unguiculata 0.0 0.5 0 5 0 5 0 5 0 5 0 A D C B I II
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