**3. Content and description of Australian cotton germplasm collections**

Initially, Australian plant breeders acquired and maintained their own germplasm collections. However, in the early 1980s the Commonwealth and State governments established a series of genetic resource centres to conserve national germplasm collections. The Australian Tropical Crops and Forages Genetic Resource Centre (ATCF) Biloela, Qld is the centre that included cotton. Currently, the only other two cotton collections in Australia reside at CSIRO Plant Industry in Narrabri, NSW and Canberra, ACT.

Unfortunately, the ATCF cotton collection is, at present, only funded to maintain germplasm under long-term storage, so the collection is not actively maintained or expanded. However, germplasm can still be requested from this collection. The CSIRO collection at Narrabri is maintained and funded by the dedicated breeding program. The CSIRO collection in Canberra is largely dedicated to the long-term storage of indigenous Australian species that have been collected through germplasm explorations [38] over the last few decades. Table 1 describes the number of cultivated tetraploid accessions amalgamated across the ATCF and CSIRO Narrabri collections. This includes both cultivars as well as locally developed and introduced germ‐ plasm. Table 2 describes the wild accessions held in all three collections.


**Table 1.** Number of cultivated tetraploid accessions in Australian collections.


a limited water supply, so increases in water-use efficiency (WUE) are being sought through management and breeding. In addition, the energy consumed and the emissions produced by a cotton crop are also being closely examined. Due to the heavy influence that yield has in the WUE calculation, this must continue to be the major focus of breeding programs. The efficient use of other resources, such as nitrogen fertiliser, will significantly influence the emissions from a crop [36, 37], so breeding programs also have responsibilities to continue research in

**3. Content and description of Australian cotton germplasm collections**

Plant Industry in Narrabri, NSW and Canberra, ACT.

plasm. Table 2 describes the wild accessions held in all three collections.

**Species Description Number of accessions**

Cultivars 611 Landraces 10 *Total 1573*

Cultivars 45 Landraces 3 *Total 99*

*G. hirsutum* Germplasm 952

*G. barbadense* Germplasm 51

Other hybrids/genetic stocks 12

**Table 1.** Number of cultivated tetraploid accessions in Australian collections.

Initially, Australian plant breeders acquired and maintained their own germplasm collections. However, in the early 1980s the Commonwealth and State governments established a series of genetic resource centres to conserve national germplasm collections. The Australian Tropical Crops and Forages Genetic Resource Centre (ATCF) Biloela, Qld is the centre that included cotton. Currently, the only other two cotton collections in Australia reside at CSIRO

Unfortunately, the ATCF cotton collection is, at present, only funded to maintain germplasm under long-term storage, so the collection is not actively maintained or expanded. However, germplasm can still be requested from this collection. The CSIRO collection at Narrabri is maintained and funded by the dedicated breeding program. The CSIRO collection in Canberra is largely dedicated to the long-term storage of indigenous Australian species that have been collected through germplasm explorations [38] over the last few decades. Table 1 describes the number of cultivated tetraploid accessions amalgamated across the ATCF and CSIRO Narrabri collections. This includes both cultivars as well as locally developed and introduced germ‐

this area.

8 World Cotton Germplasm Resources

**Table 2.** Number of wild primary, secondary and tertiary tetraploid and diploid accessions in Australian collections.

#### **3.1. Australian native** *Gossypium*

Australia is home to 17 native *Gossypium* species that are found exclusively in Australia and are distantly related to cultivated cotton. These contain the C, G and K genome species. The distribution and characterisation of these diploid species has been reasonably well document‐ ed [40-43]. The potential for transferring traits from these species, together with techniques for the production of fertile hybrids has also been reported [44, 45]. There is interest in a number of traits from these species, however, research has largely focused on disease resistance [46, 47], glandless, and seed-glanded plants [45, 48]. Despite this research, there are no documented cases of successfully transferring economically important traits from the wild Australian *Gossypium* species to cultivated cotton. There still remains interest in this area and improve‐ ments in molecular techniques may facilitate the successful introgression of traits.

identified, various databases were searched and institutions contacted in an attempt to find a source for the material. Although germplasm was sourced from a number of countries, the USDA collection in TX proved to be an extremely valuable resource for resistance to Australian isolates of Fusarium. This example highlights the need to be aware of what is available, to seek

Australian Cotton Germplasm Resources http://dx.doi.org/10.5772/58414 11

Although the ATCF collection is acknowledged as a valuable national resource, funding of this collection remains uncertain. On the other hand, the CSIRO collection at Narrabri is a live collection associated with an active breeding program. Although not an extensive collection by world standards, containing around 1000 introduced cultivars and germplasm, it is nonetheless a critical component of the CSIRO breeding program. The collection is continually being added to when new material is identified which may contain diversity for traits of interest. Many of these relate to insect and disease resistance, but yield, fibre quality and

The ATCF collection is maintained under long-term conditions in which germplasm is preserved as fuzzy seed that is dried to 6% moisture content at 15°C. Once the seed is dry, it is sealed in laminated aluminium bags and stored at -20°C. Under these conditions, it is expected that viability will be maintained for at least 50 years. Previously, when funding was available, all accessions were routinely monitored for germination every 10 years and regen‐ erated if germination fell below 60% of the starting germination. Seed was either regenerated in the field or greenhouse with controlled pollination. Germplasm was extensively character‐ ised in the past, but this is no longer undertaken. The collection maintains an in-house seed management database that manages germplasm passport, collection, characterization, inventory, seed health, regeneration and distribution data. The passport and characterisation data are freely available via the Australian Plant Genetic Resources Information System Web site (http://www2.dpi.qld.gov.au/extra/asp/auspgris) [verified December 2013]. A national review of Plant Genetic Resource Centres in Australia has determined that this website will be replaced with GRIN-Global. However, a deadline for this transition has not been finalised.

The CSIRO Narrabri collection is maintained in short-term storage as delinted seed at 7°C and 50% relative humidity. Around 1 kg of each accession are stored in cotton bags. A 10g backup collection is also maintained in sealed laminated aluminium bags at 4°C in an additional facility on the same site. Each accession is regenerated in the field every 10 years, or when stocks have been depleted to less than 200g. During regeneration, accessions are inspected and compared to known visual characteristics and off types are removed. Accessions that are photoperiod sensitive are grown in the greenhouse during the winter when the short days promote flowering. In addition, those accessions in which purity is considered vital to maintain are also grown in the greenhouse. The CSIRO breeding program maintains an internal database where

accession data is recorded. This database is not publicly available.

out that germplasm and request it under as favourable terms as possible.

**3.5. Current status of collections**

maturity are also targeted.

**3.6. Storage and renewal of germplasm collections**

#### **3.2. Plant quarantine requirements**

Australia has strict plant quarantine regulations which aim to safeguard Australia's plant health status to maintain overseas markets and protect the economy from the impact of exotic pests and diseases. This is administered by the Commonwealth (Federal) Department of Agriculture, Fisheries and Forestry (DAFF) Biosecurity based on policy determined in consultation with industry stakeholders (http://www.daff.gov.au/bsg) [verified December 2013]. For cotton, these requirements are quite onerous and costly, which restricts capacity for importing cotton seed into Australia.

#### **3.3. Focus of the collections**

Both the ATCF and the CSIRO Canberra collections have actively collected wild *Gossypium* accessions. As can be seen in Table 2, this has largely focused on *G. sturtianum, G. australe, G. nelsonii* and *G. bickii.* However, the major focus of the CSIRO Narrabri and ATCF collections have been the collecting of overseas cultivars, predominantly *G. hirsutum*, for use in the Australian cotton breeding programs. Over time, these programs have had a range of objec‐ tives as mentioned previously which is reflected in the material accumulated. Major areas of interest have been general genetic diversity, targeted host-plant resistance to insects and diseases, crop maturity, fibre quality and yield.

#### **3.4. Key contributors to collections**

Over time, germplasm representing most cotton producing countries has been sourced. This has either come directly from breeders (through exchange), acquisition of commercially released cultivars, or requests to centralised cotton collections. Initially, germplasm was sourced in larger numbers to rapidly develop the collections, however, as time has gone on, the requests for germplasm have been much more focused, as for example when Fusarium wilt was discovered in the early 1990s in Australia. Although this is a locally evolved strain that is unique to Australia, significant efforts went into searching for germplasm that had improved levels of resistance. Extensive literature searches were undertaken to identify potential germplasm from other countries to evaluate for resistance. Once a genotype had been identified, various databases were searched and institutions contacted in an attempt to find a source for the material. Although germplasm was sourced from a number of countries, the USDA collection in TX proved to be an extremely valuable resource for resistance to Australian isolates of Fusarium. This example highlights the need to be aware of what is available, to seek out that germplasm and request it under as favourable terms as possible.

#### **3.5. Current status of collections**

**3.1. Australian native** *Gossypium*

10 World Cotton Germplasm Resources

**3.2. Plant quarantine requirements**

importing cotton seed into Australia.

diseases, crop maturity, fibre quality and yield.

**3.4. Key contributors to collections**

**3.3. Focus of the collections**

Australia is home to 17 native *Gossypium* species that are found exclusively in Australia and are distantly related to cultivated cotton. These contain the C, G and K genome species. The distribution and characterisation of these diploid species has been reasonably well document‐ ed [40-43]. The potential for transferring traits from these species, together with techniques for the production of fertile hybrids has also been reported [44, 45]. There is interest in a number of traits from these species, however, research has largely focused on disease resistance [46, 47], glandless, and seed-glanded plants [45, 48]. Despite this research, there are no documented cases of successfully transferring economically important traits from the wild Australian *Gossypium* species to cultivated cotton. There still remains interest in this area and improve‐

Australia has strict plant quarantine regulations which aim to safeguard Australia's plant health status to maintain overseas markets and protect the economy from the impact of exotic pests and diseases. This is administered by the Commonwealth (Federal) Department of Agriculture, Fisheries and Forestry (DAFF) Biosecurity based on policy determined in consultation with industry stakeholders (http://www.daff.gov.au/bsg) [verified December 2013]. For cotton, these requirements are quite onerous and costly, which restricts capacity for

Both the ATCF and the CSIRO Canberra collections have actively collected wild *Gossypium* accessions. As can be seen in Table 2, this has largely focused on *G. sturtianum, G. australe, G. nelsonii* and *G. bickii.* However, the major focus of the CSIRO Narrabri and ATCF collections have been the collecting of overseas cultivars, predominantly *G. hirsutum*, for use in the Australian cotton breeding programs. Over time, these programs have had a range of objec‐ tives as mentioned previously which is reflected in the material accumulated. Major areas of interest have been general genetic diversity, targeted host-plant resistance to insects and

Over time, germplasm representing most cotton producing countries has been sourced. This has either come directly from breeders (through exchange), acquisition of commercially released cultivars, or requests to centralised cotton collections. Initially, germplasm was sourced in larger numbers to rapidly develop the collections, however, as time has gone on, the requests for germplasm have been much more focused, as for example when Fusarium wilt was discovered in the early 1990s in Australia. Although this is a locally evolved strain that is unique to Australia, significant efforts went into searching for germplasm that had improved levels of resistance. Extensive literature searches were undertaken to identify potential germplasm from other countries to evaluate for resistance. Once a genotype had been

ments in molecular techniques may facilitate the successful introgression of traits.

Although the ATCF collection is acknowledged as a valuable national resource, funding of this collection remains uncertain. On the other hand, the CSIRO collection at Narrabri is a live collection associated with an active breeding program. Although not an extensive collection by world standards, containing around 1000 introduced cultivars and germplasm, it is nonetheless a critical component of the CSIRO breeding program. The collection is continually being added to when new material is identified which may contain diversity for traits of interest. Many of these relate to insect and disease resistance, but yield, fibre quality and maturity are also targeted.

#### **3.6. Storage and renewal of germplasm collections**

The ATCF collection is maintained under long-term conditions in which germplasm is preserved as fuzzy seed that is dried to 6% moisture content at 15°C. Once the seed is dry, it is sealed in laminated aluminium bags and stored at -20°C. Under these conditions, it is expected that viability will be maintained for at least 50 years. Previously, when funding was available, all accessions were routinely monitored for germination every 10 years and regen‐ erated if germination fell below 60% of the starting germination. Seed was either regenerated in the field or greenhouse with controlled pollination. Germplasm was extensively character‐ ised in the past, but this is no longer undertaken. The collection maintains an in-house seed management database that manages germplasm passport, collection, characterization, inventory, seed health, regeneration and distribution data. The passport and characterisation data are freely available via the Australian Plant Genetic Resources Information System Web site (http://www2.dpi.qld.gov.au/extra/asp/auspgris) [verified December 2013]. A national review of Plant Genetic Resource Centres in Australia has determined that this website will be replaced with GRIN-Global. However, a deadline for this transition has not been finalised.

The CSIRO Narrabri collection is maintained in short-term storage as delinted seed at 7°C and 50% relative humidity. Around 1 kg of each accession are stored in cotton bags. A 10g backup collection is also maintained in sealed laminated aluminium bags at 4°C in an additional facility on the same site. Each accession is regenerated in the field every 10 years, or when stocks have been depleted to less than 200g. During regeneration, accessions are inspected and compared to known visual characteristics and off types are removed. Accessions that are photoperiod sensitive are grown in the greenhouse during the winter when the short days promote flowering. In addition, those accessions in which purity is considered vital to maintain are also grown in the greenhouse. The CSIRO breeding program maintains an internal database where accession data is recorded. This database is not publicly available.

The CSIRO Canberra collection is in long-term storage at -20°C. This collection provides a resource, but is not currently funded or maintained. Many of its accessions are also available from major US germplasm collections, as US researchers were often involved in the initial exploration. As the majority of these lines have been collected during genetic explorations, an internal database contains details of the collector, location and date. This database is not publicly available. There has been a decline in funding in CSIRO over the last few decades for extending germplasm collections for native *Gossypium* and other crop relatives like *Glycine*, as more and more biodiversity research shifts to consolidation of national collections, electronic archiving of data and making them more broadly accessible on-line.

available, but historically, genuine exchange with other breeding programs has been practised. In recent years however, commercial agreements and patents have made this exchange difficult. This has restricted the amount of new material added to the collection. However, it is imperative that exchange of germplasm continues. Many of the patent and commercial 'in confidence' issues can be addressed with specific (though often restrictive) material transfer agreements. This can allow new germplasm to be evaluated prior to negotiating further

Australian Cotton Germplasm Resources http://dx.doi.org/10.5772/58414 13

Improving disease resistance against indigenous Australian strains of Fusarium has been a high priority in the Australian breeding program and a number of new sources of resistance have been identified through the screening of introductions (mostly) from the US, China and India where Fusarium is also endemic*.* To investigate the genetic basis of the Fusarium wilt resistance in the Indian *G. hirsutum* cultivar MCU-5, a bi-parental cross was made between MCU-5 and Siokra 1-4; a *Fusarium* susceptible Australian okra leaf *G. hirsutum* cultivar [51]. An F3 population consisting of 244 lines was developed from this cross, and from single seed decent from each F3 line, 244 F4 lines subsequently produced. The F3 and F4 populations were assayed for Fusarium wilt resistance using a glasshouse bioassay [52] and genotyped with 151 markers (95 SSR and 56 AFLP). QTL analysis revealed the presence of multiple regions that were associated with resistance that provide targets for introgression into elite cultivars to improve Fusarium wilt resistance [51]. Subsequently, it was found that MCU-5 which was derived from a multi-line cross between Indian Cambodia-type cultivars (MCU-1 and MCU-2) and cultivars from East Africa, the West Indies and the US, including some contribution from *G. barbadense*, also possesses significant resistance to non-defoliating strains of *Verticillium*

In an international collaboration with CIRAD (France), CSIRO obtained a 140 RIL inter-specific population ranging from the F6 to F9 stages of selfing through single seed descent. The two parents; Guazuncho 2 (*G. hirsutum*), and VH8-4602 (*G. barbadense*), were chosen for their agronomic performance (Guazuncho 2) and superior fibre quality parameters (VH8-4602) [53]. This population was grown in several countries including Australia in the glasshouse and field and analysed for many different traits (including fibre, leaf shape hairiness, boll size and number and earliness) and genotypes consisting of 1,745 loci derived from 597 SSR and 763 ALFP markers [54-56]. Not all of the lines performed well under Australian conditions with only about 55 flowering within an acceptable timeframe for harvesting and thus seed stocks are severely restricted for most of the RILs. The QTLs associated with fibre traits identified in Australia may help generate markers for selection for these traits in breeding populations. The population also represents a resource for linking markers with mite resistance, as the parent

freedoms to utilise germplasm in a breeding program.

**4.1. Mapping populations**

**4. Other cotton germplasm resources beyond seed collections**

*dahliae,* as well as possessing significantly longer fibre than Siokra 1-4.

lines show differentiation in resistance.

There are increasing challenges associated with maintenance and regeneration of these collections. Ensuring genetic purity of accessions has always been difficult when regeneration is conducted in the field. However, the GM era has added another level of complexity to the problem. Even though Australia does not have large numbers of insects that will cause cross pollination in cotton (only the European honey bee), measurable out-crossing can occur in some seasons and some locations [49, 50]. Care must be taken to locate regeneration blocks away from known sources of bee activity as well as isolated from commercial GM cotton crops. CSIRO also has substantial activities in areas of biotech research in addition to the traits sourced from third parties and some of these activities are discussed in a later section. This research is geographically isolated (in Canberra) from the breeding program and only traits that have reached the advanced field evaluation stage are grown at Narrabri. As CSIRO is developing cultivars for release commercially, stewardship of germplasm and GM traits is extremely important and internal quality assurance protocols are rigorously adhered to. These protocols dictate that all material needs to be tested via protein or DNA analysis prior to handover to commercial partners for seed increase and sale.

#### **3.7. Germplasm passport data**

The current status of the accession descriptions varies depending on when and who imported them, as well as the donor. As many characteristics are either invisible or not able to be determined except in specific environments (e.g., disease resistance), the data provided by the donor is often critical. However, in many cases, particularly for older material, this information is simply not available for many accessions that may have been obtained via an intermediary collection or supplier. When it was actively funded, the ATCF collection generated substantial passport data on all lines including morphological characteristics, boll size, lint percent and fibre quality parameters. This is invaluable information for a breeder searching for specific characteristics. Descriptions in the CSIRO collection are more *ad-hoc*. This is largely a function of the collection not being 'public', and simply a resource for the breeding program that operates it. The data that does exist largely relate to the traits that were initially identified as being of interest *i.e*., HPR to insects and disease or fibre quality.

#### **3.8. Sharing and exchange**

The ATCF collection is based on a national collection model with small quantities (20-30 seeds) of germplasm freely available to researchers worldwide. The CSIRO collection is not publicly available, but historically, genuine exchange with other breeding programs has been practised. In recent years however, commercial agreements and patents have made this exchange difficult. This has restricted the amount of new material added to the collection. However, it is imperative that exchange of germplasm continues. Many of the patent and commercial 'in confidence' issues can be addressed with specific (though often restrictive) material transfer agreements. This can allow new germplasm to be evaluated prior to negotiating further freedoms to utilise germplasm in a breeding program.
