Abstract
Fusarium wilt is a soil borne fungal disease that has devastated banana production in plantations around the world. Most Cavendish-type bananas are susceptible to strains of Fusarium oxysporum f. sp. cubense (Foc) belonging to the Subtropical Race 4 (STR4) and Tropical Race 4 (TR4). The wild banana diploid Musa acuminata ssp. malaccensis (AA, 2n = 22) carries resistance to Foc TR4. A previous study using segregating populations derived from M. acuminata ssp. malaccensis identified a quantitative trait locus (QTL) (12.9 cM) on the distal part of the long arm of chromosome 3, conferring resistance to both Foc TR4 and STR4. An SNP marker, based on the gene Macma4_03_g32560 of the reference genome ‘DH-Pahang’ v4, detected the segregation of resistance to Foc STR4 and TR4 at this locus. Using this marker, we assessed putative TR4 resistance sources in 123 accessions from the breeding program in Brazil, which houses one of the largest germplasm collections of Musa spp. in the world. The resistance marker allele was detected in a number of accessions, including improved diploids and commercial cultivars. Sequencing further confirmed the identity of the SNP at this locus. Results from the marker screening will assist in developing strategies for pre-breeding Foc TR4-resistant bananas. This study represents the first-ever report of marker-assisted screening in a comprehensive collection of banana accessions in South America. Accessions carrying the resistance marker allele will be validated in the field to confirm Foc TR4 resistance.
Keywords: breeding, cleaved amplified polymorphic sequences markers, Fusarium oxysporum sp. cubense, Musa acuminata ssp. malaccensis
1. Introduction
Bananas are among the leading fruits that provide economic sustainability for both small- and large-scale growers, with a worldwide trade value surpassing USD 30 billion per annum [1]. However, banana production has been severely affected by major pests and diseases caused by viruses, bacteria, and fungi. Amongst the fungal diseases affecting bananas, Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is the most economically important.
Fusarium wilt has been an issue in banana monoculture plantation production systems since the 1900s. However, it is the virulent Foc strain known as Tropical Race 4 (TR4) that threatens commercial banana production dominated by Cavendish-type clones, as well as a broad range of other banana varieties [2,3,4]. TR4 was first reported in the regions of southeast Asia and Australia in the 1990s [5]. In recent years, it has made its way into other parts of the world, such as Latin America, reaching the northern region of La Guarija, Colombia, in 2019 [3], the Querecotillo district of Sullana province in the Piura department of Peru in 2021 [2], and more recently, Venezuela [6]. These countries could provide proxies in the potential spread of Foc TR4 across the border to neighboring countries. Thus, pre-emptive breeding to contain and stop the spread of the disease is of utmost importance. Latin America and the Caribbean, which is the world’s largest banana exporting region, is thus endangered [7]. Understanding the epidemiology of Fusarium wilt in bananas is critical for developing control measures, breeding resistant varieties, and formulating effective quarantine strategies to prevent further spread of Foc TR4 in banana-producing regions of the world [8,9]. Foc TR4 remains a critical threat to banana plantations worldwide, partly due to the lack of effective control measures and limited sources of Fusarium wilt resistance within Musa spp. [8,10,11].
Disease symptoms are manifested as vascular discoloration in the corm, while the disease is visually identified on plants displaying leaf yellowing and vascular-induced wilt. Foc, a soilborne pathogen, enters the host plant through the roots and moves through the plant’s vascular system to colonize the whole plant. Its colonization and proliferation in the xylem vessels block water and nutrient supplies to the aerial parts of the plant, leading to wilting of leaves and eventual plant death [8,10]. Internally, Foc-infected corms and pseudo-stems show a reddish-brown discoloration. So far, there are no effective means to control the spread of this disease. Foc can survive in the soil for decades as chlamydospores and cannot be completely eradicated [12]. Therefore, the use of resistant cultivars not only offers the most sustainable and environmentally friendly means of controlling Foc but is perhaps one of the few viable options [13]. This highlights the importance of identifying resistance in wild relatives of cultivated bananas and introducing resistance alleles into commercial cultivars to enhance their resilience against diseases [4,9].
Embrapa Mandioca e Fruticultura (Embrapa Cassava and Fruits, https://www.embrapa.br/mandioca-e-fruticultura, accessed on 21 October 2024) conducts research to improve crop productivity and has an active program for the genetic improvement in bananas since 1976 [14]. It maintains one of the largest banana germplasm collections in South America. This collection includes accessions gathered from various regions worldwide, established through comprehensive collecting expeditions. These expeditions were conducted in the banana center of origin in Southeast Asia and further extended to include accessions from Africa, Central America, and South America. This germplasm collection now has over 400 accessions, aimed at preserving genetic diversity and developing new cultivars suited to both local and global agricultural needs.
Molecular markers based on Single Nucleotide Polymorphisms (SNPs) are used successfully in many crops in the identification of a plant’s resistance to diseases [15,16,17,18]. Previous studies show that lines belonging to the wild diploid banana Musa acuminata subsp. malaccensis (Ma) (AA, 2n = 22) carry resistance to Foc TR4 and STR4 strains [8,10,17,18].
Recently, a detailed study of two Musa acuminata ssp. malaccensis populations segregating for Foc TR4 and STR4 resistance was carried out [18]. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region conferring Foc STR4/TR4 resistance to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of the ‘DH-Pahang’ reference assembly v4 [18].
Within this region, multiple pattern recognition receptors, such as leucine-rich repeats containing receptor-like protein kinases, cysteine-rich cell wall-associated protein kinases, and other genes related to resistance, were identified. To confirm the segregation of single-gene resistance, an inter-cross between the resistant parent ‘Ma850’ and a susceptible line ‘Ma848’ was generated [18]. An informative SNP marker, 29730, based on a single nucleotide change (T544C) in the first intron of a Nuclear transcription factor Y subunit (Macma4_03_g32560, ‘DH-Pahang’ assembly v4.3) was reported as associated with Foc TR4 resistance [18] and allowed the resistance locus to be assessed in a collection of diploid and polyploid banana accessions at Embrapa. The results provide crucial insights into Foc TR4 resistance sources within Embrapa’s banana breeding program, supporting the design of a breeding pipeline geared toward the development of Foc TR4-resistant bananas and as such, mitigate TR4-caused yield losses once TR4 arrives in Brazil. These findings will undoubtedly contribute to the global initiatives aimed at developing Foc TR4-resistant banana varieties.
2. Materials and Methods
M. acuminata-specific primers for the 29730 marker were developed previously, 29730-A-SNP1-F2 5′-GCAATGAGTACCTCTAAGCA-3′ and 29730-A-SNP1-R2 5′-TAAGTTCTAGTATCAAGTACAA-3′, and used to amplify an A-genome specific product of 795 bp. This product was then digested with BcoDI to produce the bi-allelic forms, an undigested dominant band that is putatively associated with resistance, and digested products of 429 bp and 366 bp, linked to susceptibility [18]. Then, 123 accessions from the Embrapa banana germplasm collection (Table 1) were genotyped with this SNP marker, 29730-A-SNP1-F2 and 29730-A-SNP1-R2.
Table 1.
Sample number, accession name and number (Embrapa/ITC), type, genealogy, BcoDI digest, and allele nucleotide of 123 Embrapa accessions genotyped with SNP markers 29730-A-SNP1-F2 and 29730-A-SNP1-R2.
| Sample Number | Accession Name | Accession Number | Type | Pedigree | Marker-Allele Defined by BcoDI Digest | SNP and Allele Zygosity | Predicted Foc TR4 Responses |
|---|---|---|---|---|---|---|---|
| 1 | 042023-03 | 042023-03 | Improved diploid (AA) | M53 × Cultivar sem nome Nº 2 | B | C/C | Sus |
| 2 | 091087-02 | 091087-02 | Improved diploid (AA) | 001016–01 (Borneo × Guyod) × 003038–01 (Calcutta 4 × Heva) | B | C/C | Sus |
| 3 | SH3362R | - | Improved diploid (AA) | - | H | T/T | Res |
| 4 | 003004-02 | 003004-02 | Improved diploid (AA) | Calcutta 4 × Madang | B | C/C | Sus |
| 5 | 013019-01R | 013019-01 | Improved diploid (AA) | Malaccensis × Tjau Lagada | H | T/C | Res |
| 6 | 041054-04 | 041054-04 | Improved diploid (AA) | 003004-01 (Calcutta 4 × Madang) × 001004-01 (Borneo × Madang) | B | - | Sus |
| 7 | 050012-02HR | 050012-02 | Improved diploid (AA) | M61 × Lidi | B | - | Sus |
| 8 | 086094-15 | 086094-15 | Improved diploid (AA) | 03037-02 (Calcutta 4 × Galeo) × SH3263 | H | T/C | Res |
| 9 | 042015-02 | 042015-02 | Improved diploid (AA) | M53 × Madu | B | C/C | Sus |
| 10 | SH3263R | - | Improved diploid (AA) | - | A | T/T | Res |
| 11 | 042085-02 | 042085-02 | Improved diploid (AA) | M53 × 015003-01 (Madu × Calcutta 4) | B | - | Sus |
| 12 | 042079-06 | 042079-06 | Improved diploid (AA) | M53 × 028003-01 (Tuu Gia × Calcutta 4) | B | C/C | Sus |
| 13 | 017041-01 | 017041-01 | Improved diploid (AA) | Jari Buaya × 003004–01 (Calcutta 4 × Madang) | B | C/C | Sus |
| 14 | 042052-03 | 042052-03 | Improved diploid (AA) | M53 × Kumburgh | B | - | Sus |
| 15 | 042049-04 | 042049-04 | Improved diploid (AA) | M53 × M48 | B | C/C | Sus |
| 16 | 003023-03 | 003023-03 | Improved diploid (AA) | Calcutta 4 × S/Nº 2 | B | T/C | Res |
| 17 | 086094-20 | 086094-20 | Improved diploid (AA) | 03037-02 (Calcutta 4 × Galeo) × SH3263 | H | C/C | Sus |
| 18 | 001016-01HR | 001016-01 | Improved diploid (AA) | Borneo × Guyod | H | C/C | Sus |
| 19 | 091094-04 | 091094-04 | Improved diploid (AA) | 001016–01 (Borneo × Guyod) × SH3263 | B | C/C | Sus |
| 20 | TH-0301 | TH-0301 | Improved diploid (AA) | Terrinha × Calcutta 4 | B | - | Sus |
| 21 | 041054-08 | 041054-08 | Improved diploid (AA) | 003004-01 (Calcutta 4 × Madang) × 001004-01 (Borneo × Madang) | B | - | Sus |
| 22 | 058054-03 | 058054-03 | Improved diploid (AA) | 003005–01 (Calcutta 4 × Pahang) × 001004–01 (Borneo × Madang) | B | - | Sus |
| 23 | 089087-01 | 089087-01 | Improved diploid (AA) | 013018-01 (Malaccensis × Sinwobogi) × 003038-01 (Calcutta 4 × Heva) | B | - | Sus |
| 24 | 003037-02 | 003037-02 | Improved diploid (AA) | Calcutta 4 × Galeo | B | C/C | Sus |
| 25 | 086079-09 | 086079-09 | Improved diploid (AA) | 003037–02 (Calcutta 4 × Galeo) × 028003–01 (Tuu Gia × Calcutta 4) | B | - | Sus |
| 26 | 073041-01 | 073041-01 | Improved diploid (AA) | Khai × 003004–01 (Calcutta 4 × Madang) | B | - | Sus |
| 27 | 013004-04 | 013004-04 | Improved diploid (AA) | Malaccensis × Madang | H | C/C | Sus |
| 28 | 013018-01 | 013018-01 | Improved diploid (AA) | Malaccensis × Sinwobogi | B | - | Sus |
| 29 | 028003-01 | 028003-01 | Improved diploid (AA) | Tuu Gia × Calcutta 4 | B | - | Sus |
| 30 | 091079-03 | 091079-03 | Improved diploid (AA) | 01016-01 (Borneo × Guyod) × 028003 (Tuu Gia × Calcutta 4) | B | C/C | Sus |
| 31 | Tropical | - | Silk hybrid (AAAB) | Yangambi no.2 × M53 | B | - | Sus |
| 32 | Belluna | - | Cultivar (AAA) | - | B | - | Sus |
| 33 | Caipira | - | Cultivar (AAA) | - | H | T/C | Res |
| 34 | Prata Graúda | - | Prata hybrid (AAB) | SH3642 | H | T/C | Res |
| 35 | Thap-Maeo | ITC1301 | Cultivar (AAB) | B | - | Sus | |
| 36 | Prata-Anã | ITC0962 | Cultivar (AAB) | B | - | Sus | |
| 37 | BRS Vitória | - | Prata hybrid (AAAB) | Pacovan × M53 | B | - | Sus |
| 38 | BRS Preciosa | - | Prata hybrid (AAAB) | Pacovan × M53 | B | C/C | Sus |
| 39 | BRS Japira | - | Prata hybrid (AAAB) | Pacovan × M53 | B | - | Sus |
| 40 | BRS Garantida | - | Prata hybrid (AAAB) | Pacovan × M53 | H | C/C | Sus |
| 41 | BRS Pacovan-Ken | - | Prata hybrid (AAAB) | Pacovan × M53 | B | - | Sus |
| 42 | Pacovan | - | Prata Type (AAB) | Prata Type triploid | B | - | Sus |
| 43 | Platina | ITC0262 | Prata hybrid (AAAB) | Prata Anã × M53 | B | - | Sus |
| 44 | BRS Caprichosa | - | Prata hybrid (AAAB) | Prata Comum × M53 | B | - | Sus |
| 45 | BRS Pioneira | - | Prata hybrid (AAAB) | Prata São Tomé × M53 | B | - | Sus |
| 46 | BRS Princesa | - | Silk Hybrid (AAAB) | Yangambi no.2 × M53 | B | - | Sus |
| 47 | Pelipita | ITC0095 | Cultivar (ABB) | - | B | - | Sus |
| 48 | Mongolo | - | Plantain (AAB) | - | B | - | Sus |
| 49 | Red Yade | ITC1140 | Plantain (AAB) | - | B | - | Sus |
| 50 | Comprida | - | Plantain (AAB) | - | B | - | Sus |
| 51 | Tipo Velhaca | - | Plantain (AAB) | - | B | - | Sus |
| 52 | Terra Ponta Aparada | - | Plantain (AAB) | - | B | - | Sus |
| 53 | Pinha | - | Plantain (AAB) | - | B | - | Sus |
| 54 | Samura B | - | Plantain (AAB) | - | B | C/C | Sus |
| 55 | Trois Vert | ITC1127 | Plantain (AAB) | - | B | - | Sus |
| 56 | Terra Sem Nome | - | Plantain (AAB) | - | B | - | Sus |
| 57 | FHIA 21 | ITC1306 | Hybrid (AAAB) | - | B | C/C | Sus |
| 58 | Terrinha | - | Plantain (AAB) | - | B | C/C | Sus |
| 59 | Njock Kon | ITC1133 | Plantain (AAB) | - | B | - | Sus |
| 60 | Curare Enano | ITC0559 | Plantain (AAB) | - | B | C/C | Sus |
| 61 | Chifre De Vaca | - | Plantain (AAB) | - | B | - | Sus |
| 62 | Terra Maranhão | - | Plantain (AAB) | - | B | - | Sus |
| 63 | D’Angola | - | Plantain (AAB) | - | B | - | Sus |
| 64 | CNPMF 0557R | 111090-07 | Improved diploid (AA) | [(M61 × ‘Pisang Lilin’)] × [(Malaccensis × Tjau Lagada)] | H | - | Res |
| 65 | CNPMF 0496R | 111040-03 | Improved diploid (AA) | [(M61 × ‘Pisang Lilin’)] × [(Terrinha × Calcutta 4)] | H | - | Res |
| 66 | CNPMF 0513R | 111102-01 | Improved diploid (AA) | [(M61 × ‘Pisang Lilin’)] × [(M53 × Kumburgh) | B | - | Sus |
| 67 | CNPMF 0519 | 116116-01 | Improved diploid (AA) | Self-fertilization (wild diploid Tambi) | B | - | Sus |
| 68 | CNPMF 0536HR | 9041090-02 | Improved diploid (AA) | [(Calcutta 4 × Madang)] × [(Borneo × Guyod)] | H | T/C | Res |
| 69 | CNPMF 0534 | 041090-20 | Improved diploid (AA) | [(Calcutta 4 × Madang)] × [(Borneo × Guyod)] | B | C/C | Sus |
| 70 | CNPMF 0542R | 094089-01 | Improved diploid (AA) | [(SH3263)] × [(Malaccensis × Sinwobogi)] | H | T/C | Res |
| 71 | CNPMF 0565 | 096077-01 | Improved diploid (AA) | [(Calcutta 4 × Pahang) × (Borneo × Madang)] × Khae | B | C/C | Sus |
| 72 | CNPMF 0572 | 106090-01 | Improved diploid (AA) | [(Khai × (Calcutta 4 × Madang)] × [(Calcutta 4 × Madang)] | B | C/C | Sus |
| 73 | CNPMF 0612 | 098094-01 | Improved diploid (AA) | [(M53 × Madu) × Madu)] × SH3263 | H | C/C | Sus |
| 74 | CNPMF 0731R | 088079 | Improved diploid (AA) | [(Malaccensis × Madang)] × [(Tuu Gia × Calcutta 4)] | H | T/C | Res |
| 75 | CNPMF 0767 | 088100 | Improved diploid (AA) | [(Malaccensis × Madang)] × [(Khai × (Calcutta 4 × Madang)] | B | C/C | Sus |
| 76 | CNPMF 0811 | 106096 | Improved diploid (AA) | [(Khai × (Calcutta 4 × Madang)] × [(Calcutta 4 × Pahang) × (Borneo × Madang)] | B | - | Sus |
| 77 | CNPMF 0037 | 098090-01 | Improved diploid (AA) | [(M53 × Madu)] × [(Malaccensis × Tjau Lagada)] | B | - | Sus |
| 78 | CNPMF 0898 | PA × 119 | Improved diploid (AA) | [(Prata Anã)] × [(Malaccensis × Sinwobogi) × (Calcutta 4 × Galeo)] | B | - | Sus |
| 79 | CNPMF 0038 | 098090-01 | Improved diploid (AA) | [(M53 × Madu)] × [(Malaccensis × Tjau Lagada)] | B | - | Sus |
| 80 | CNPMF 1102 | 093117 | Improved diploid (AA) | [(Jari Buaya × (Calcutta 4 × Madang)] × [(Borneo × Guyod) × (Tuu Gia × Calcutta 4)] | B | - | Sus |
| 81 | CNPMF 1171 | 088108 | Improved diploid (AA) | [(Malaccensis × Madang)] × [(M53 × (Tuu Gia × Calcutta 4)] | B | - | Sus |
| 82 | CNPMF 0993R | 117100 | Improved diploid (AA) | [(Borneo × Guyod) × (Tuu Gia × Calcutta 4)] × [(Khai × (Calcutta 4 × Madang)] | B | - | Sus |
| 83 | CNPMF 1323 | 089087 | Improved diploid (AA) | [(Malaccensis × Sinwobogi)] × [(Calcutta 4 × Heva)] | B | - | Sus |
| 84 | CNPMF 1105 | 123097 | Improved diploid (AA) | [(Borneo × Guyod) × (Calcutta 4 × Heva)] × [(Calcutta 4 × Madang)] | B | - | Sus |
| 85 | CNPMF 0998R | 091124 | Improved diploid (AA) | [(Borneo × Guyod)] × [(Borneo × Guyod) × SH3263] | B | - | Sus |
| 86 | CNPMF 0978 | 041040 | Improved diploid (AA) | [(Calcutta 4 × Madang)] × [(Terrinha × Calcutta 4)] | B | C/C | Sus |
| 87 | CNPMF 1272 | 123079 | Improved diploid (AA) | [(Borneo × Guyod) × (Calcutta 4 × Heva)] × [(Tuu gia × Calcutta 4)] | B | C/C | Sus |
| 88 | CNPMF 1286 | 041040 | Improved diploid (AA) | [(Calcutta 4 × Madang)] × [(Terrinha × Calcutta 4)] | B | C/C | Sus |
| 89 | M53R | - | Landrace cultivar (AA) | Malaccensis–Kedah × banksii-Samoa) × (Paka × Banksii-Samoa) | B | C/C | Sus |
| 90 | Pisang Jaran | ITC0678 | Wild diploid (AA) | - | B | C/C | Sus |
| 91 | Malbut | - | Wild diploid (AA) | - | B | C/C | Sus |
| 92 | Calcutta4 | ITC0249 | Wild diploid (AA) | - | B | C/C | Sus |
| 93 | Birmanie | - | Wild diploid (AA) | - | B | C/C | Sus |
| 94 | Akondro Mainty | ITC0281 | Mchare landrace (AA) | - | B | - | Sus |
| 95 | NBA-14 | ITC0267 | Wild diploid (AA) | - | B | C/C | Sus |
| 96 | Khai Nai On | ITC0663 | Wild diploid (AA) | - | B | C/C | Sus |
| 97 | Khai | ITC0532 | Wild diploid (AA) | - | B | C/C | Sus |
| 98 | Pisang Berlin | ITC0611 | Edible diploid (AA) | - | B | T/C | Res |
| 99 | Khi Maeo | - | Wild diploid (AA) | - | B | - | Sus |
| 100 | Borneo | ITC0253 | Wild diploid (AA) | - | B | C/C | Sus |
| 101 | Mambee Thu | ITC0612 | Edible diploid (AA) | - | B | C/C | Sus |
| 102 | Niyarma Yik | ITC0269 | Edible diploid (AA) | - | B | C/C | Sus |
| 103 | M. a. spp. malaccensis | ITC0399 | Wild diploid(AA) | - | H | - | Res |
| 104 | Pisang Tongat | ITC0063 | Edible diploid (AA) | - | B | C/C | Sus |
| 105 | Pa Mysore2 | ITC0668 | Wild diploid (AA) | - | H | C/C | Sus |
| 106 | Pisang Jari Buaya | ITC0690 | Edible diploid (AA) | - | B | C/C | Sus |
| 107 | Tuu Gia | ITC0610 | Edible diploid (AA) | - | B | C/C | Sus |
| 108 | Pisang Nangka | ITC0004 | Edible triploid (AAB) |
- | B | C/C | Sus |
| 109 | Tjau Lagada | ITC0090 | Wild diploid (AA) | - | B | C/C | Sus |
| 110 | Tong Dok Mak | ITC0411 | Wild diploid (AA) | - | H | C/C | Sus |
| 111 | M-61 | - | Improved diploid (AA) | H | C/C | Sus | |
| 112 | Pisang Mas | ITC1403 | Edible diploid (AA) | - | H | T/C | Res |
| 113 | Pisang Cici | ITC0681 | Wild diploid (AA) | - | B | C/C | Sus |
| 114 | Saney | - | Wild diploid (AA) | - | B | C/C | Sus |
| 115 | Yangambi no.2 | ITC1275 | Cultivar (AAB) | - | B | C/C | Sus |
| 116 | Yangambi Km5 | ITC1123 | Cultivar (AAB) | - | H | T/C | Res |
| 117 | Highgate | ITC0263 | Cultivar (AAA) | - | B | - | Sus |
| 118 | BRS Pacoua PV 03-76 | - | Prata hybrid (AAAB) | Pacovan × Calcutta 4 | B | C/C | Sus |
| 119 | PA 42-38 | PA 42-38 | Prata hybrid (AAAB) | Prata Anã × M53 | B | C/C | Sus |
| 120 | PA 42-28 | PA 42-28 | Prata hybrid (AAAB) | Prata Anã × M53 | B | C/C | Sus |
| 121 | PA 42-19 | PA 42-19 | Prata hybrid (AAAB) | Prata Anã × M53 | - | C/C | Sus |
| 122 | M48 | - | Improved diploid (AA) | - | B | C/C | Sus |
| 123 | Ouro | - | Cultivar (AA) | - | H | T/T | Res |
B = Marker homozygous for the susceptible allele, predicted to be Foc TR4-susceptible: H = Marker heterozygous, predicted to be Foc TR4-resistant; A = Marker homozygous for the resistant allele, predicted to be Foc TR4-resistant; Nucleotide ‘T’ corresponds to the resistant allele and nucleotide ‘C’ the susceptible allele; ‘T/C’ and ‘T/T’ are predicted to be resistant whereas ‘C/C’ = is predicted to be susceptible. “HR’ and “R” within the “Accession name” column indicate accessions that were “Highly Resistant” and “Resistant”, respectively, to Foc STR4, as determined in a previous study [19]. Accessions listed in bold within the “Pedigree” column denote parental accessions previously shown to be resistant to Foc TR4. Sanger sequencing was used to determine the SNP identity at this locus. “-” within the “SNP and allele zygosity” column indicates that Sanger sequencing was not performed for these particular accessions.
3. Results and Discussion
The overall results of the marker-assisted screening for all 123 accessions are summarized in Table 1. A restriction digest was performed using BcoDI on PCR amplicons of all 123 Embrapa accessions. A BcoDI cut site within the PCR amplicon allowed bi-allelic forms to be discriminated on an agarose gel (Figure 1). Accessions displaying a dominant resistant band were subsequently analyzed through Sanger sequencing. The sequencing results confirmed the presence of the informative SNP, as identified in the alignment of chromatograms at the expected position (Figure S1).
Figure 1.
BcoDI digest profile of 19 Embrapa accessions PCR-amplified using the SNP marker 29730-A-SNP1-F2 and 29730-A-SNP1-R2. The 795 bp fragment is putatively associated with resistance. Samples are (1) ‘PA Mysore 2’, (2) ‘Pisang Jari Buaya’, (3) ‘Tuu Gia’, (4) ‘Pisang Nangka’, (5) ‘Tjau Lagada’, (6) ‘Tong Dok Mak’, (7) ‘M-61’, (8) ‘Pisang Mas’, (9) ‘Pisang Cici’, (10) ‘Saney’, (11) ‘Yangambi no.2’, (12) ‘Yangambi Km5’, (13) ‘Highgate’, (14) ‘Pacoua PV03-76’, (15) ‘PA 42-38’, (16) ‘Pa 42-28’, (17) ‘PA 42-19’, (18) ‘M48’, and (19) ‘Ouro’. M = molecular ladder 1 kb (N3232S, New England Biolabs, VIC, Australia), (-): Non-amplified sample. The alternatively cut allele (429 bp and 366 bp) may indicate the presence of the Foc TR4-susceptible allele. Accessions heterozygous for the marker locus are predicted for resistance to Foc STR4 and Foc TR4 given the complete dominance of resistance over susceptibility at this resistance locus. Asterisk = putative Foc TR4/STR4-resistant genotypes.
The SNP marker was screened in all putative genotypes for resistance. PCR products of selected accessions were Sanger-sequenced in both directions by ACTGene Company, using the AB3500 sequencing platform (ACTGene, Porto Alegre, RS, Brazil). Of the retrieved sequences, these were analyzed using the multiple-sequence alignment software MAFFT v 7.490 [20] in Geneious Prime v 2024.0.7 (Biomatter Pty. Ltd., Auckland, New Zealand). Overall, 14 genotypes, out of 15, showed the presence of the resistance allele (Supplementary Figure S1). Of these, three improved diploids have Malaccensis in their genetic background, namely ‘013019’, ‘CNPMF 0542’, and ‘CNPMF 0731’. Given that the breeding program at Embrapa focuses on a preventive breeding strategy to combat the disease, these improved diploids will play a key role in the development of Foc TR4-resistant cultivars aimed at enhancing resistance to Fusarium wilt in the field.
Among the 15 accessions previously identified as highly resistant or resistant to Foc STR4 [19], 10 were associated with the resistance marker band. These included the improved diploids CNPMF- ‘0496’, ‘0534’, ‘0536’, ‘0542’, ‘0557’, ‘0731’, ‘013019-01’, ‘050012-02’, ‘SH3263’, and ‘SH3362’ (Table 1). However, five accessions, including CNPMF- ‘1323’, ‘0513’, ‘0993’, ‘0998’, ‘001016-01’, and ‘M53’, did not exhibit the resistance marker allele despite being resistant to Foc STR4 [19] (Table 1). These findings indicate a 62.5% detection rate of resistant genotypes using the marker, suggesting that while the marker is a useful tool for identifying resistance, it may not capture all resistant genotypes due to linkage disequilibrium and resistance sources controlled by other unlinked loci.
Improved diploids are developed by crossing wild diploids with the objective of obtaining hybrids with important agronomical characteristics such as disease resistance. There are three types of improved diploids: (1) first generation − wild diploid × wild diploid, (2) second generation − Improved diploid × wild diploid, and (3) third generation − improved diploid × improved diploid. The third generation may have many wild diploids in its genealogy, whereas the hybrid combines important agronomical characteristics that were distributed among the wild diploids [14].
The screening results suggest that the marker did not detect the resistant allele in diploids including ‘Calcutta 4’, ‘Pisang Lilin’, ‘Tuu Gia’, and ‘Borneo’ despite having good or intermediate levels of resistance/tolerance to Foc race 1 and Foc TR4 [11,21]. The negative results in marker screening highlight the limitations of this marker in detecting resistance traits at loci other than the target or in genotypes lacking Musa acuminata ssp. malaccensis origin. This suggests that additional markers may be needed to identify resistance in a broader range of banana genotypes and as well in Musa acuminata ssp. malaccensis-derived bananas such as ‘Pisang Lilin’. These genotypes are scheduled for field evaluation to assess their resistance to Foc TR4, adhering to quarantine protocols in Australia and Colombia. Additionally, we plan to use the putative Foc TR4-resistant improved diploids in breeding efforts aimed at introducing sources of Foc TR4-resistance into dessert bananas including ‘Prata’, ‘Silk’, and ‘Cavendish’.
This work highlights the importance of improved diploid genotypes, particularly ‘CNPMF 512’ and ‘CNPMF 1323’, which carry resistance to other pests and diseases. These genotypes also possess desirable agronomic traits, including short plant height, early flowering time, and good tillering ability [22]. The genotypes will be field-tested for resistance to Foc TR4 in Australia and Colombia, in collaboration with the Department of Agriculture, Fisheries, and Forestry and Agrosavia, to further validate the marker.
This study represents the first report of screening an SNP marker across such an extensive number of banana accessions in Latin America. Given that Foc TR4 is already present in Latin America, the urgent need for validation of this SNP marker in genotypes is underscored by its potential impact. Our findings provide valuable insights to expedite resistance breeding in other banana programs worldwide, such as those conducted by IITA for cooking and plantain bananas, and by CIRAD [7,23,24,25].
Acknowledgments
The authors would like to thank the National Council of Scientific and Technological Development–CNPq for research productivity grants of CFF and EPA and Embrapa Mandioca e Fruticultura for the technical support.
Supplementary Materials
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof10120839/s1: Supplementary Figure S1: Sequencing chromatograms for the Macma4_03_g32560 gene were generated using both forward and reverse primers.
Author Contributions
Conceptualization, C.F.F., A.C., E.A.B.A. and E.P.A.; methodology, C.F.F., A.C., E.A.B.A., A.d.J.R. and J.M.d.S.S.; software, C.F.F. and A.C.; validation, C.F.F., A.C. and A.P.d.S.R.; formal analysis, C.F.F., A.C., E.A.B.A., R.S. and B.U.; investigation, C.F.F., A.C., E.P.A., R.S. and B.U.; resources, C.F.F., A.C. and E.A.B.A.; data curation, C.F.F. and A.C.; writing—original draft preparation, C.F.F., A.C., E.A.B.A. and E.P.A.; writing—review and editing, C.F.F., A.C., E.A.B.A., E.P.A., R.S. and B.U.; visualization, C.F.F., A.C., E.A.B.A., E.P.A., R.S. and B.U.; supervision, C.F.F., A.C., E.A.B.A. and E.P.A.; project administration, A.C., E.A.B.A. and E.P.A.; funding acquisition, A.C., E.A.B.A. and E.P.A. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The authors will provide data upon request.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research was funded by The Bill and Melinda Gates Foundation (Project Grant ID: OPP1093845) through its grant to the International Institute of Tropical Agriculture (IITA) under the project Accelerated Breeding of Better Bananas, grant number IITA 20600.15/0008-8—Phase II. Andrew Chen and Elizabeth Aitken were also funded by Hort Innovation Australia through grant ‘BA17006’.
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The authors will provide data upon request.