A Training Manual

on

Cytoplasmic Male Sterility (CMS) Breeding

in Chilli Pepper

Date: 6-8 November 2017

Organized by the World Vegetable Center, Bangladesh

Facilitated by Bangladesh Agricultural Research Institute (BARI)

Supported by the Swisscontact-Katalyst

Published in October 2017

Venue: Spices Research Center (SRC), Shibgonj, Bogra

Cytoplasmic Male Sterility (CMS) Breeding in Chilli Pepper

Susan Lin, Vivian Wang, Derek W. Barchenger, and Sanjeet Kumar World Vegetable Center, Shanhua, Tainan, Taiwan

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1. BACKGROUND INFORMATIONNuclear male sterility, also known as genetic male sterility (GMS), and cytoplasmic male sterility (CMS) are the most commonly used systems for cost effective F1 hybrid seed production. These systems eliminate the need for laborious hand emasculation in numerous crops, including peppers (Capsicum annuum L.). In chilli pepper, the cost of hybrid pepper seed production can be reduced by up to 40% with the use of the CMS system.

This brochure provides a general outline of the origin and genetics of CMS and examples to be used for scheme for CMS breeding. Practical approaches to develop CMS systems in pepper and their utilization in experimental and commercial hybrid seed production have been demonstrated with appropriate photographs.

2. ORIGIN OF CYTOPLASMIC MALE STERILITY (CMS)Cytoplasmic male sterility is known in more than 150 plant species including pepper. Generally, CMS is caused by mutations in the mitochondrial genome, resulting in novel chimeric mitochondrial open reading frames (ORF), which interfere with mitochondrial function and pollen development. In pepper, CMS has been found to be associated with two different ORFs, orf507 and ψatp6-2. These ORFs potentially impair ATP synthesis activity in the cytoplasm, resulting in non-functional pollen.

Cytoplasmic male sterility can originate from natural or artificial intergeneric or interspecific hybridizations (alloplastic cytoplasm), or artificially induced through mutagenesis or antibiotic effects on cytoplasmic genes. Cytoplasmic male sterile plants have also been developed in several vegetables through protoplast fusion. In the near future, genetically engineered cytoplasmic male sterility may be available - once transformation techniques for mt-genome are standardized.

In pepper, interspecific hybridization between C. annuum and C. frutescens has resulted in male sterility. Interestingly, the plasmatype of this induced interspecific hybrid was found to be identical to the original naturally occurring source of CMS in pepper, identified from an accession collected in India. Additionally, CMS in pepper has been induced through C. chacoense by C. annuum hybridizations, with C. chacoense used as the female parent all progeny are male-sterile.

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3. MODES OF INHERITANCE AND MOLECULAR MARKERS Cytoplasmic male sterility is a maternally inherited trait, because the chimeric ORFs responsible for the expression of male (pollen) sterility are located in the mt-genome, and he mitochondria are usually excluded from the pollen during fertilization. The expression of male sterility in sterile (S) cytoplasm is suppressed by nuclear encoded Restorer-of-fertility (Rf) gene(s), which usually have dominant gene action. These Rf genes generally encode pentatricopeptide repeat (PPR) proteins, which are 35 amino acid sequence motifs that act to mask the effects of CMS ORFs through translation inhibition. Hence, in the CMS system, although S-cytoplasm causes male sterility, the expression of male sterility is dependent on nuclear constitution at Rf loci. In peppers, a number of Rf genes and modifiers have been identified. It has also been reported that distribution of Rf alleles is more common in chili peppers, while the rf (maintainer) allele is more frequently distributed in sweet peppers.

Several molecular markers have been de-veloped for marker assisted selection of CMS, each with varying levels of applica-bility. The SCAR130 marker has been widely identified as being the most applicable molecular marker for accurate selection of S-cytoplasm in most species of pepper. Similarly, there are many molecular markers that have been found to be associated with restoration of fertility in pepper;

however, as of 2017, there are no mark-ers will 100% selection accuracy. Although recently developed markers are becoming increasingly more applicable. One reason for the lack of a widely applicable Rf mark-ers might be because pepper has been found to have a high number of Rf-like (RFL) genes clusters on the distal end of chromosome 6. It has been hypothesized that in conjunction with gene duplication, that the generation of new RFL genes and subsequent loss of non-functional RFLs is relatively rapid, and keeps pace with the generation of novel CMS sources. Despite the lack of a single marker for selection of Rf genes, program- and line-specific molecular markers are useful tools for marker assisted selection in the CMS-Rf breeding system.

4. DEVELOPMENT AND USE OF PEPPER CMSAn A-line can be developed by backcross-ing a selected B-line with an already avail-able A-line for six to seven generations. This generates a pair of A- and B-lines in the new genetic background “(detailed in next section 4.1)”. The Rf allele is either introgressed into an identified male par-ent (e.g. sweet pepper) or a male parent is directly used for hybrid seed produc-tion with a CMS line, if Rf allele in the homozygous state is available in the male parent (e.g. chilli pepper). Cytoplasmic male sterility without an Rf gene cannot be utilized in vegetables where the fruit is harvest, such as in tomato, chilli, and melon but it can be utilized in vegetables where vegetative part is of economic value, such as onion, carrot, radish, and leafy vegetables.

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4.1. BREEDING NEW PAIRS OF CMS (A) AND MAINTAINER (B) LINE The first perquisites of CMS line (A) development is the avail-ability of a male sterile, A-line (S-rfrf) and its maintainer male fertile, B-line (N-rfrf). Additionally, access to a greenhouse to carry out rapid backcrossing and generation advancement is important. New CMS (A) line is developed by backcrossing of selected B-line on to an already available A-line for six to seven generations. This generates a pair of A- and B-lines in the new genetic background.

A breeder should select several potential open pollinated (OP) varieties, ideally with very good combining ability and resistance to abiotic and biotic stresses, for use in test crosses. These well adapted OP lines are hybridized as male parents with male sterile (CMS) plants. These OP varieties will become the maintainer lines (B-line) to maintain the viable seed of the A-line. The further steps along with figures are described below:

Step 1. Sow F1 seeds of the test cross hybridizations (A-lines x OP varieties), A- and B-lines. Transplant F1 progeny of each test cross along with the parents in the greenhouse (Fig. 1).

Step 2. After flowering, test each F1 plant from test crosses for the presence/absence of pollen. For rapid evaluation, mature anthers can be tapped onto a fingernail (Fig. 2) or other surface to evaluate for pollen production. Cytoplasmic male sterility results in the failure to produce pollen, there-fore any F1 lines with viable pollen can be discarded at this point (Fig. 3)

Step 3. Select a single unopened flower bud, that is white and nearly at the stage of anthesis (Fig. 4) from each male sterile plants. These unopened flower buds will be used for confirmation of pollen sterility (unstainable pollen).

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Step 4. Prepare 1.5% acetiocarmine stains, using 70 ml al-cohol and 30 ml acetic acid and add 1.5 g carmine powder (Fig. 5). Arrange glass slides, cover slips, dissecting needle and tweezers and disinfect all supplies using 70% “ethyl alcohol” (Fig. 6).

Step 5. Using the dissecting needle, crush the anthers of the selected unopened flower bud onto a grass slide (Figs. 7 & 8).

Step 6. Place a drop of acetocarmine stain (made in step 4) on pollen that was released from crushed anther and cover the solution with a cover slip and use a needle to tap the cover slip (Figs. 9 - 11).

Step 7. Observe slides under 100x magnification “Figs. 11-15” and select those F1 plants that have no visable stained pollen (Fig. 13). Discard the plants that have any stainable pollen (Fig. 14) or mostly stainable pollen (Fig. 15), as these lines will not have stable sterility.

Step 8. Select the best sterile F1 lines and hybridize these with their respective B-lines. The offspring from this hybrid-ization will constitute the BC1F1 generation (Figs. 16-18).

Step 9. For each subsequent generation, repeat the steps 1 through 8 until the BC7F1 generation is developed. At the same time, use single seed decent to purify the B-lines, resulting in new CMS A-lines and the maintainer B-lines.

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Schematic of the genetic mechanisms required to maintain the A- (S-rfrf) and B-lines (N-rfrf) (left) and the development of an F1 hybrid cultivar using the A-line and a C-line (N/S-Rf) (right).

4.2. MAINTENANCE OF A-, B-, AND C-LINES

AND HYBRID DEVELOPMENT

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Schematic of the genetic mechanisms required to maintain the A- (S-rfrf) and B-lines (N-rfrf) (left) and the development of an F1 hybrid cultivar using the A-line and a C-line (N/S-Rf) (right).

4.3. USE OF CMS LINE IN DEVELOPING POTENTIAL CROSSES Generally, hybrid breeding involving CMS line is termed as “Three Line Hybrid Breeding”, which involves an A-line (CMS; S-rfrf), B-line (maintainer; N-rfrf) and C- or R-line (restorer; S- or N-RfRf). For F1 hybrid cultivar development in chili pepper, CMS plants can be used as the female parent and no manual emasculation is needed, because the flowers are genetically male sterile. In contrast, if normal male fertile plants are used for hybrid cultivar development, flowers are manually emasculated after flowering and emasculated flow-ers are pollinated manually.

Using CMS lines, a large number of test crosses can be made and evaluated for their economic potential. This is usually done by growing A- and C/R-lines in nethouses (Fig. 19), which excludes pollinators and eliminates the possibility for unde-sirable cross-pollination. The collected pollen from C-lines (Fig. 20) is used to pollinate the A-lines (Fig. 21). The cross-pollinated flowers are labeled using a ring or thread (Fig. 22).

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World Vegetable Center - South Asia ICRISAT Campus, Patancheru 502 324 Hyderabad, Telangana, India. Tel: +91-40-30713755 Fax: +91-40-30713074 / 75 info-southasia(at)worldveg.org Website: worldveg.org 8

4.4 USE OF CMS LINE TO PRODUCE COMMERCIAL HYBRID SEEDS

For commercial hybrid cultivar develop-ment, the parental A- and C/R-lines are grown in separate nethouses. During flow-ering, the anthers/pollen are collected from the C/R-lines (A) and used to pol-linate A-line flowers (B).