Chromosome elimination and in vivo haploid product

Authors: Zili Zhang Fazhan Qiu Yongzhong Liu Kejun Ma Zaiyun Li Shangzhong Xu

Publish Date: 2008/09/20

Volume: 27, Issue: 12, Pages: 1851-1860

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Abstract

In vivo haploid production induced by inducer lines derived from Stock 6 is widely used in breeding program of maize Zea mays L but the mechanisms behind have not yet been fully understood In this study average frequency of haploid induction in four inbred lines by Stock 6derived inducer line HZI1 was above 10 About 02 kernels from the cross Hua24 × HZI1 had mosaic endosperm showing yellow shrunken parts from Hua24 to normal parts with purple aleurone from HZI1 Individual lagged chromosomes and micronuclei were observed in mitotic cells of ovules pollinated by HZI1 Above 564 of the radicles from the kernels with purple aleurone and colorless embryos were mixoploid 2n = 9–21 and more than 4522 cells were haploid cells 2n = 10 in three crosses More than 625 of the radicles from the kernels with purple aleurone and purple embryos were mixoploid 2n = 9–21 having 5427 cells with 2n = 20 SSR analysis showed that all haploids from the cross Hua24 × HZI1 shared the same genomic compositions as Hua24 except for plants Nos 862 and 857 with some polymorphic DNA bands The results revealed that chromosome elimination after fertilization caused the haploid production in maizeIt takes 7–8 generations to obtain a maize inbred line and the plants were still not 100 pure in traditional breeding Doubledhaploid DH technology improves breeding efficiency by generating inbred lines with 100 purity and genetic uniformity in just two generations DH lines make it easy to carry genetic studies and shorten the breeding time significantly Maize Zea mays L is a typical diploid plant 2n = 20 but haploid individuals 2n = 10 occur naturally at a rate of one per 1000 kernels Chase 1949 Maize haploid can be derived either through tissue culture technique in vitro or through genetic induction in vivo Anther culture in maize is a highly complex and expensive method with low plantlet regeneration rate which is dependent on genetic background Beckert 1994 Shatskaya et al 1994 and is greatly limited for the application in breeding programs Nowadays in vivo haploid induction by inducer lines is widely used by most breeders for its high frequency simple operation and inexpensivenessThere are two modes of in vivo haploid induction in maize leading to maternal and paternal haploids respectively The genomes of maternal haploids originate from the maternal parent and those of paternal haploids come from the paternal parent Coe 1959 discovered a haploid inducer Stock 6 and 1–2 of the progeny was maternal haploids when it was used as pollinator Kermicle 1969 obtained 0–2 paternal haploids when the W23 indeterminate gametophyte ig line was used as female parent However the rate of haploids obtained with Stock 6 and W23 ig was still low and depended on the genotypes Lashermes and Beckert 1988 The haploidinducing capacity of the inducer can be increased by selection Sarkar et al 1972 To date a number of new inducers with higher haploidinduction rate have been created Eder and Chalyk 2002 such as WS14 Lashermes et al 1988 ZMS Chalyk 1994 KMS Tyrnov and Zavalishina 1984 MHI Eder and Chalyk 2002 and RWS Röber et al 2005The mechanism of in vivo haploid induction has not yet been full understood until now Eder and Chalyk 2002 Röber et al 2005 Several hypotheses have been put forward to explain the haploid formation through in vivo haploid induction in maize Firstly the irregularities during microsporogenesis and fertilization may be involved in haploid induction Hu 1990 found the difference between the transmission velocities of two sperms in one microspore and the sperm with high velocity fertilized normally while the one with low velocity missed the fertilization which broke the normal double fertilization and developed the kernels with a haploid embryo Enaleeva et al 1996 Chang 1992 suggested two irregular cases occurring in the maize double fertilization Primarily the polar nucleus is fertilized while the egg cell remains unfertilized and develops into embryos with the cell division of the fertilized polar nucleus Alternatively the egg cell is destroyed when the pollen tube is entering into embryo sacs one of the sperms fertilizes the polar nucleus and other develops into haploid embryos Bylich and Chalyk 1996 detected pollen grains with a pair of morphologically different sperm nuclei in ZMS inducer line as a result one of the sperms can fertilize normally but another does not Rotarenco and Eder 2003 detected a more than three time higher rate of heterofertilization when haploid inducer was used as pollinator compared to a normal inbred line Chalyk et al 2003 observed above 15 aneuploidy microsporocyte in the inducer MHI and only about 1 in two inbred lines used as checks Briefly the abnormality in microsporogenesis and fertilization may be the reasons of haploid induction Coe and Neuffer 2005 also supported this hypothesis Secondly chromosome elimination after fertilization might be the major mechanism in maize in vivo haploid induction Wedzony et al 2002 studied ovaries of inducer line RWS during the first 20 days after selfpollination In about 10 of the embryos micronuclei of variable size were found in the cytoplasm of every cell of the shoot primordium Such micronuclei generally were the symbol of chromosome elimination from the cell in subsequent divisions Kasha and Kao 1970 Fischer 2004 showed that a small proportion 1–2 of haploids obtained from the cross between the inducer line and a broadbased sample of breeding materials carried one seldom several paternal chromosome segments through SSR markers analysis It indicated that a small fragment from the inducer genome can be transferred into maternal genome of the haploids Röber et al 2005In present study we find that chromosome elimination after fertilization leads to in vivo maize haploid production induced by inducer line HZI1 derived from Stock 6 Cells with variable chromosome numbers appear in the radicles of the F1 kernels from crosses with inducer HZI1 and individual lagged chromosomes and micronuclei are observed in the pollinated ovules by HZI1 The F1 kernels with mosaic endosperm consisting of normal part with purple aleurone and sweet shrunken part without purple aleurone are also observedAn inducer line HZI1 derived from Stock 6 with Rnj Rnj Rnavajo a domiant gene on chromosome 10 for purple aleurone and purple embryo and Sh2 the dominant gene of sh2 on chromosome 3 for normal endosperm was used as male parent and it has normal endosperm with purple aleurone and purple embryo Three normal inbred lines were used as female parents HZ124b HZ85 HZ141 with rr recessive gene of Rnj and Sh2 and with the normal endosperm with colorless aleurone and colorless embryo as well as a sweet corn inbred line Hua24 with r r recessive gene of Rnj and sh2sh2 shrunken2 showing shrunken endosperm and with shrunken endosperm with colorless aleurone and colorless embryoThe crosses between the inducer HZI1 and above inbred lines were made in field respectively and all F1 kernels were harvested by single ear and analyzed separately We identified haploid kernels by using the system of dominant anthocyanin marker genes Chase 1969 Nanda and Chase 1966 In this system the expression of the Rnj gene provided an anthocyanin pigmentation of the embryo and the endosperm Kernels with purple aleurone and colorless embryo were putative haploids and the kernels with purple aleurone and purple embryo were hybrid kernels The kernels with colorless aleurone and purple embryo were defect kernels and the kernels with colorless aleurone and colorless embryo were from pollen contamination

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