This project is:
identifying and developing effective screening/evaluation protocols for each key trait,
identifying appropriate variability for key traits through use of screening protocols,
enhancing germplasm in all countries for key traits through selection and breeding,
identifying heritability of key traits, genetic distance and heterotic pools (agronomic analysis, molecular analysis) by undertaking genetic variability/distance studies on germplasm from all countries, and
developing and providing appropriate information on improved germplasm and disease epidemiology for incorporation into existing technology transfer protocols.
Oilseed brassicas are an extremely important crop in China and India. More than 6 million hectares are planted to B. napus (rapeseed) in China and B. juncea (Indian mustard) in India. Achieving canola quality oils (low in erucic acid and glucosinolates) is an aim for both countries. B napus varieties grown in Australia, Europe and Canada all achieve canola quality. Of the B napus types grown, those planted in Australia are best suited to Chinese and Indian growing conditions. Germplasm with improved traits for both B napus and B juncea will be tested to improve canola quality oilseed production in China, India and Australia.
Brassica production in all three countries is limited by a number of key diseases and environmental stresses. Sclerotinia and white rust resistant traits are needed to reduce the losses these diseases cause. Agronomic traits such as drought tolerance and quality will also boost yields and oil quality. Molecular genetic and quality analysis can be used to determine key traits including quality, disease resistance and drought tolerance.
Objective 1. Appropriate and effective screening/evaluation protocols identified for each key character listed.
During 2004 protocols to be used by all the collaborators for measuring agronomic and disease traits in the agronomy field trials were discussed and a protocol document was circulated for all collaborators to use.
The plant pathologist position at the University of Western Australia to develop screening/evaluation protocols for key disease characters was advertised in late 2004. The position has been filled and the research will be initiated in late February 2005. During 2004 isolates of Sclerotinia were collected in Western Australia.
A plant pathologist was also appointed on the project at the University of Melbourne during 2004 to investigate Australian white rust isolates. During 2004 Australian isolates of the white rust pathogen, Albugo candida, were collected and screening protocols for white rust resistance were initiated using the Australian isolates.
Disease resistance screening was also initiated in India.
Objective 3. Enhanced oilseed Brassica germplasm in all collaborating countries though germplasm exchange, crossing and selection.
The enhancement of Brassica germplasm in all countries for the key characters was initiated through the exchange of germplasm among the three countries. Twenty five Australian B. napus cultivars, 12 Australian B. juncea lines 20 Chinese B. napus cultivars were distributed to the collaborators in each country in late 2004. The seed arrived too late for sowing in the 2004/2005 season but the material was multiplied in India and China to provide a larger quantity of seed for the coming season’s trials. In addition, some field observations were taken in India and analysis of fatty acid profile and oil content was undertaken.
Efforts were initiated to obtain government approvals for the distribution of the remaining lines (10 B. juncea lines from China and 22 B. juncea and 3 B. napus lines from India). A list of the characteristics of each of the lines was also distributed to all the collaborators. In total, the exchange of germplasm will result in field trials with 48 B. napus and 44 B. juncea lines. It is expected that all of the germplasm will be sown in each of the collaborating countries in the 2005/06 season.
Objective 4. Heterotic pools for production of elite germplasm or F1 hybrids identified from genetic distance studies on germplasm from all three collaborating countries.
The research associate position at UWA to study genetic variability/distance among the germplasm was advertised in late 2004 and the research will be initiated in 2005.
Objective 6. Scientific skills of Chinese and Indian scientists enhanced through scientific exchanges and training.
Indian scientists from each of the collaborating institutes visited Australia during the period 22nd Sep to 7th Oct. The visit included two meetings to discuss project plans, including exchange of seed. In addition, the visit included tours of canola field trials in Western Australia and NSW, visits to molecular and chemistry laboratories and meetings with key Brassica breeding and research personnel. This scientific exchange and training visit also involved the Indian collaborators participating in the 4th International Crop Science Congress in Brisbane and the Australian Oilseeds Federation Annual Conference in Melbourne.
Preparations for Dr Phil Salisbury and Assoc Prof Martin Barbetti’s trip to India in January 2005 were undertaken during the visit of the Indian scientists.
Significant progress was made during 2005 to identify variability for key agronomic, quality and disease resistance characters through the use of screening protocols. The first exchange of germplasm of B. napus and B. juncea between Indian, China and Australia was completed by mid 2005 to allow sowing of field trials in the 2005/06 season. The material to be assessed in each country for the key characters consists of, B. napus: 25 Australian, 20 Chinese and 3 Indian lines and B. juncea: 22 Indian, 12 Australian and 10 Chinese lines. In Australia the field trials were sown in Vic, NSW, SA and WA and were harvested at the end of 2005. The data from these trials will be analysed in early 2006. In China the germplasm was sown at two sites in Wuhan in Sep/Oct 2005 and the key characters are currently being assessed. In India the germplasm was sown in Oct/Nov 2005 and to date emergence, early vigour and initiation of flowering data have been recorded. Results from a preliminary screening of Australian and Indian germplasm in India in the 2004/05 season indicated that there is substantial variability in key characters including date of emergence, seedling vigour, white rust incidence, oil and glucosinolate content and fatty acid composition. Screening and characterisation of B. juncea and B. napus for seedling stage thermotolerance and terminal stage heat tolerance is underway in the field and laboratory at HAU Hisar and PAU Ludhiana, India. Drought tolerance screening is also underway at HAU. Screening of germplasm for Sclerotinia was also initiated in 2005 at HAU, India and UWA, Australia. In Australia it was observed that most B. juncea germplasm was highly susceptible to Sclerotinia, based on the length of stem lesions, although there were some more resistant lines from Australia and China. Significant differences for white rust resistance between Australian B. juncea lines were also observed in WA.
During 2005 activities were initiated to enhance the germplasm in all countries for key characters through selection and breeding. At PAU, India interspecific hybridisation between B. napus and B. carinata / B. juncea was undertaken and desirable F2/BC1 plants from both the crosses will be backcrossed with selected Australian B. napus lines. At HAU, India 40 crosses have been made between Indian and Australian lines of B. juncea, and 60 crosses between Australian and Indian lines of B. napus are also being attempted this season. At TERI, India shattering tolerant B. napus lines (developed at TERI) have been sown and will be crossed with the Australian lines to transfer shatter tolerance. In NSW, Australia, 21 crosses were made between Sclerotinia tolerant Chinese lines and elite Australian lines and further crossing will be done in 2006.
Progress was made in 2005 towards the identification of genetic distance and heterotic pools. At UWA, useful SSR markers were identified on all chromosomes for estimation of genetic distance among B. napus lines from Australia, China and India, and selfing and purification of these lines began for future genetic studies. At HAU, China a preliminary experiment was conducted to assess marker techniques for genetic diversity analysis. Results indicated that SRAP (sequence-related amplified polymorphism) are distributed on all 19 chromosomes of B. napus more equally than AFLP and the technique is more efficient than SSR, so SRAP and SSR markers will be used to evaluate the genetic diversity of all B. napus and B. juncea lines. At PAU, India DNA isolation from all the B. juncea genotypes has also been completed.
During 2005 significant progress was made towards the objective of increasing the scientific skills of scientists collaborating in the project through scientific exchanges. Dr Abha Agnihotri, Dr Dhiraj Singh and Dr Surinder Banga visited Australia in 2005 for a scientific interaction/study program. The program included 3 days of NIR training at Wagga Wagga Research Institute, visits to Brassica trials in NSW, Victoria and SA, meetings with scientists at CSIRO, Canberra, a meeting with John Cullen and attendance at the Australian Research Assembly on Brassicas (Port Lincoln, SA). Dr Maharaj Singh (NRCRM, Bharatpur) also visited Australia from Sep to Dec 2005 for training. Dr Singh conducted a drought tolerance project supervised by Dr Rob Norton (University of Melbourne) at DPI Horsham. Dr Singh learned to use a range of equipment, visited scientists at CSIRO and ANU Canberra to discuss drought screening and also attended the Australian Research Assembly on Brassicas. Assoc Prof Phil Salisbury and Assoc Prof Martin Barbetti visited India in February 2005 for discussions with project collaborators. In addition, Dr Martin Barbetti and Dr Caixia Li attended the International Sclerotinia Workshop in California in June 2005. Following this workshop, Dr Li visited Huazhong Agricultural University and Wuhan Oil Crops Research Institute, China, where she met with oilseed Brassica scientists working on this ACIAR project, and, in particular, had the opportunity to assess and develop common approaches on study of Scelorotinia disease.
Advances were made in 2006 in the development of screening protocols for sclerotinia and white rust resistance. Severity of Sclerotinia infection was found to be related to stem diameter and stem lesion length 3 weeks after inoculation was significantly and positively correlated with the percentage of plant death at maturity, which indicated the value of the stem inoculation method for the effective identification of Sclerotinia resistance under field conditions (UWA-B). Assessment of white rust screening protocols demonstrated that controlled environmental conditions are suitable for rapid identification of resistant genotypes and that genotypes with high levels of resistance can be reliably identified at either the cotyledonary, seedling, or flowering stages (UWA-B). Pathotyping experiments of the white rust pathogen have identified only race 2 pathotype 2A isolates in Australia to date (UM). Further surveying is underway to determine if the virulent pathotype (2V) is present in Australia.
During 2006 considerable progress was made in the identification of variability for key agronomic, quality and disease resistance characters in the Indian, Chinese and Australian B. napus and B. juncea germplasm in each country. In addition, breeding programs to enhance the germplasm in all countries for shatter resistance, disease resistance, agronomy and quality and drought tolerance characters progressed. Crossing programs, which were initiated in 2005, have advanced significantly, and the first generation of populations developed from the crosses have been sown and are currently being screened.
Some key characters that were identified in the germplasm screening that will be beneficial to the breeding of improved lines for each country included: white rust resistance in Australian and Chinese B. juncea lines; low erucic acid and low glucosinolate levels in Chinese and Australian B. napus and B. juncea lines; drought tolerance in Australian B. juncea lines; variation for Sclerotinia tolerance among all B. napus lines (varied among locations); potential blackleg resistance in Chinese and Indian B. juncea germplasm in Australia
Cluster analysis of Australian and Indian B. napus and B. juncea varieties by HAU and PAU showed no association between geographical distance and divergence based on morpho-physiological traits as genotypes from different locations fall in the same group and vice versa, which may be due to continuous exchange of germplasm.
Extensive work was undertaken in China (HZAU, IOCR, XAAS), India (HAU, PAU) and Australia (UWA-C) during 2006 to identify genetic distance, heterotic pools and heritability of key traits in the germplasm. Dendrograms showing the relationships among the 48 B. napus lines have been prepared at UWA-C and HZAU. Clustering in the HZAU dendrogram indicated that the Chinese B. napus lines were relatively more genetically diverse. Clustering in the UWA-C diagram showed the clear distinction of Indian B. napus to accessions from other countries, and a higher proportion of “private alleles” in Indian and some Chinese accessions. Genetic distance analysis of B. juncea is also underway at UWA-C.
Crosses have been made for hybrid vigour studies and at most institutes the F1 generations have been sown or will be sown soon. At UWA-C, selections were made for F1 hybrid evaluation based on the most homozygous selfed plants from each accession. Results from India (PAU), indicate that hybrids involving Australian B. napus germplasm and Indian non canola types were most productive. In China, (HZAU), examination of B. napus hybrids at the vegetative stage found positive mid-parent heterosis. Analysis of hybrid vigour in B. juncea was undertaken in China (XAAS) with diallel crosses using five Australian and five Chinese lines. The productivity of most of the F1s was higher than their parents and although the maturity of the varieties from both Australia and China was similar, the maturity of F1 was later than their parents.
Preliminary data has been generated in China to study the relationship between hybrid performance of B. napus and genetic distance. Results from IOCR indicate the performance of hybrids for yield traits was not consistent with genetic distances, although the genetic distances did show correlation with hybrid performance to a certain degree. At HZAU, neither a positive nor negative relationship was observed between molecular genetic distance and mid-parent heterosis for seedling traits of the F1 hybrids. Further analysis is underway to determine whether or not correlations exist between F1 seedling characters and F1 yield and yield-related characters, and between F1 yield and yield-related characters and their parents’ molecular genetic distance.
During 2006 significant progress was also made towards the objective of increasing the scientific skills of scientists collaborating in the project through scientific exchanges. Mr Wan Zhengjie (PhD student), Huazhong Agricultural University, Wuhan, began 5 months of molecular biology training at UWA in Assoc Prof Wallace Cowling’s laboratory on 30 October 2006. Mr Wan Zhengjie will work on two projects at UWA: (i) identification of a gene for male sterility in B. juncea, and (ii) genetic distance studies on B. juncea in the ACIAR project collection. Mr Mei Desheng, Oil Crops Research Institute, Wuhan, began 6 months of molecular biology training at CSIRO Plant Industry with Dr Allan Green in mid November 2006. Mr Mei Desheng’s project will involve cloning of a range of fatty acid biosynthesis genes from the Crambe abyssinica oilseed species.
During 2007 the second round of screening of the series I B. napus and B. juncea germplasm from India, China and Australia was completed. Useful variation for several key agronomic, quality and disease resistance characters in the germplasm from each country was confirmed. The best performing lines have been used in breeding programs to enhance the germplasm in all countries for shatter resistance, disease resistance, agronomic and quality traits and drought tolerance. The second series of germplasm exchange was also undertaken in 2007, with 58 B. napus lines (25 Chinese, 2 Indian and 31 Australian) and 60 B. juncea lines (20 Chinese, 23 Indian and 17 Australian) exchanged.
Some key traits were identified in the series II germplasm screening that will be beneficial to the breeding of improved lines for each country. These included white rust resistance in Australian B. juncea lines, low erucic acid and low glucosinolate levels in Chinese and Australian B. napus and B. juncea lines, Sclerotinia resistance in Chinese and Australian lines, terminal stage thermotolerance in Australian B. napus and Chinese B. juncea lines and seedling stage thermotolerance in Indian B. juncea lines, blackleg resistance in Australian B. juncea lines and shatter resistance in Indian and Australian B. napus lines.
In 2007, advances were made in verifying shatter resistance screening techniques. A very high correlation between the results of visual shatter observations and percentage of pod shattering on the main stem of Brassica lines that were left standing in the field 4 weeks post maturity was observed in India, indicating that either method is valuable for estimating shatter resistance.
The genetic distance analyses of the series I B. napus and B. juncea germplasm, using the SSR technique, was completed in 2007. Cluster analysis of the data showed abundant genetic diversity among the lines of both species, and will assist breeders in their selection of the most diverse lines to widen their genepools.
The first F1 hybrid field trials of 13 B. napus parents and 84 F1 hybrids were conducted in Australia (WA, NSW, Vic), China (2 sites in Wuhan) and India (Punjab) in 2007-08. The F1 hybrid seed produced by hand-crossing in 2006 was distributed in 2007 together with seed of pure breeding lines of the parents. The parallel trials were designed with a special spatial randomisation program with 1-3 replicates per entry. The first data were received from Australian sites at the end of 2007. Seven agronomic traits were measured (vegetative vigour, date of 50% flowering, height of first branch, height of first pod, mature height, seed yield and 1000-seed weight) and data analysis is underway using a multi-environment trials analysis. Heterosis measured in F1 hybrids and combining ability measured in diallel crosses of pure lines and the relationship between molecular genetic distance and heterosis will be reported.
F1 seed produced from the second series of exchanged germplasm has been or will be sent to collaborators in 2008 for F1 hybrid trials in Australia (WA, NSW, Vic), China (1 site in Wuhan) and India (Punjab, Harayana) in 2008-09.
Analysis of the relationship between molecular genetic distance and heterosis was also carried out in an additional experiment in China using 12 parents and 36 hybrids. The lines were planted in two Chinese locations and 11 traits were recorded. Positive mid-parent heterosis and positive high-parent heterosis for seed yield were observed. Most of the correlation coefficients between molecular genetic distance and mid-parent heterosis were positive, but few items were at a significant level. However, when dividing hybrids into intra- and inter-regional hybrids, correlations between genetic distance and mid-parent heterosis increased among intra-regional hybrids for most traits especially siliquas per plant and seed yield per plant. The results indicated that it may be possible to predict heterosis by molecular markers among intra-regional hybrids.
During 2007 significant progress was also made towards the objective of increasing the scientific skills of scientists collaborating in the project through scientific exchanges. Mr Wan Zhengjie (PhD student), Huazhong Agricultural University, Wuhan, completed his 5 months of molecular biology training at UWA in Assoc Prof Wallace Cowling’s laboratory. Likewise, Dr Mei Desheng, Oil Crops Research Institute, Wuhan, completed 6 months of molecular biology training at CSIRO Plant Industry with Dr Allan Green. In addition, Dr Chirantan Chattopadhyay (NRCRM, Bharatpur, India) undertook a 3 week training program in Australia in September 2007. Dr Chattopadhyay presented a paper at the Australian Research Assembly on Brassicas, participated in the pre-conference tour, met with project collaborators in WA, NSW and VIC, toured disease nurseries and attended a scientific writing workshop at the Australasian Plant Pathology conference.
In April 2007 a mid-term project progress meeting was held in Wuhan, China, and was attended by collaborators from each institute. The 3 day meeting consisted of presentations, trait group discussions and tours of laboratories and field trials.
In early 2008 (Jan 28 to Feb 3), a progress meeting was held in India. The meeting comprised a one day workshop, followed by visits to each of the collaborating institutes to inspect the exchanged lines in the field.